WorldWideScience

Sample records for antifreeze protein nmr

  1. Antifreeze Proteins of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030 ...

  2. Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance {sup 13}C NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daley, Margaret E.; Sykes, Brian D. [University of Alberta, Department of Biochemistry, CIHR Group in Protein Structure and Function and Protein Engineering Network of Centres of Excellence (Canada)

    2004-06-15

    The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance {sup 13}C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the {sup 1}H-{sup 13}C NOE were determined in this study. The C{alpha}H relaxation measurements were compared to the previously measured {sup 15}N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the {chi}{sub 1} dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than {+-}25 deg.

  3. Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

    International Nuclear Information System (INIS)

    Daley, Margaret E.; Sykes, Brian D.

    2004-01-01

    The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance 13 C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the 1 H- 13 C NOE were determined in this study. The CαH relaxation measurements were compared to the previously measured 15 N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the χ 1 dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than ±25 deg

  4. Antivirulence Properties of an Antifreeze Protein

    Directory of Open Access Journals (Sweden)

    Martin Heisig

    2014-10-01

    Full Text Available As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

  5. Tailoring Peptidomimetics Antifreeze Protein from Exotic Antarctic Marine

    International Nuclear Information System (INIS)

    Mohd Basyaruddin Abdul Rahman; Azren Aida Asmawi; Emilia Abdulmalek

    2016-01-01

    Antifreeze proteins (AFPs) are synthesized by various cold-adapted organisms to enable them to survive in subzero environment. The unique role of AFPs recently attracted enormous interest to develop them as commercial products. In this work, we have studied the antifreeze activity of short helical protein fragments (peptides) instead of the entire antifreeze protein of Antarctic yeast Glaciozyma antarctica. Several short peptide segments were designed according to amino acid sequence of helical region of AFP-1 G.antarctica, which are assumed to be involved in its antifreeze activity. We have demonstrated that short peptide segments derived from yeast AFP possess antifreeze activity and result in modification of the ice crystals growth rates and habits. This strategy has enabled the preparation of short AFP with high antifreeze activity in large amount of quantities at a low cost further opens the chance of developing the commercial potentials of AFPs.(author)

  6. Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Kristiansen, Erlend; von Solms, Nicolas

    2014-01-01

    The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding fac...

  7. Studies on new antifreeze protein from the psychrophilic diatom ...

    African Journals Online (AJOL)

    Studies on new antifreeze protein from the psychrophilic diatom, Fragilariopsis cylindrus. ... African Journal of Biotechnology. Journal Home · ABOUT THIS ... The predicted gene product, AfpA, had a molecular mass of 27 kDa. Expression of ...

  8. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    structure. SOSUI server predicts one transmembrane region in winter flounder fish and atlantic cod and ..... result in a better interaction with water. The secon- ... structure of antifreeze protein P05140 (using PDB template 2AFP_A). The 10 ...

  9. Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available Antifreeze proteins (AFPs enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.

  10. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    2014-10-20

    Oct 20, 2014 ... Recent studies have shown that plant AFPs bind to both prism planes and basal ... Abbreviations used: AFP, antifreeze protein; ECP, extra-cellular protein; IAC, ice adsorption ...... This work was partially supported by a grant (BT/PR10799/ ... ity in Ammopiptanthus mongolicus (in Chinese with English.

  11. In silico Characterization of Plant and Microbial Antifreeze Proteins

    Directory of Open Access Journals (Sweden)

    Abdul Mohin Sajib

    2012-12-01

    Full Text Available Antifreeze proteins (AFPs are class of proteins that protect organisms from the damage caused by freezing through their ability to inhibit ice growth and effectively lower the temperature at which water freezes. In this study, a total of 25 antifreeze proteins were selected from four different sources (plant, bacteria and fungus where they represent distinct physicochemical and structural features. Several Physico-chemical properties such as grand average hydropathy (GRAVY, aliphatic index (AI, extinction coefficient (EC, isolelectric point (pI, and instability index (II were computed. S-S bridges and secondary structures were analyzed using CYS_REC and SOPMA programs respectively. The three dimensional structure of Antifreeze proteins is predicted by using three homology modelling server Geno3D, Swiss-model and CPHmodels. These models were evaluated with PROCHECK, What If, and ProSA programs. Model visualization and analysis was done with Pymol. These structures will provide a good foundation for functional analysis of experimentally derived crystal structures.

  12. Solution Structure of an Antifreeze Protein CfAFP-501 from Choristoneura fumiferana

    International Nuclear Information System (INIS)

    Li Congmin; Guo Xianrong; Jia Zongchao; Xia Bin; Jin Changwen

    2005-01-01

    Antifreeze proteins (AFPs) are widely employed by various organisms as part of their overwintering survival strategy. AFPs have the unique ability to suppress the freezing point of aqueous solution and inhibit ice recrystallization through binding to the ice seed crystals and restricting their growth. The solution structure of CfAFP-501 from spruce budworm has been determined by NMR spectroscopy. Our result demonstrates that CfAFP-501 retains its rigid and highly regular structure in solution. Overall, the solution structure is similar to the crystal structure except the N- and C-terminal regions. NMR spin-relaxation experiments further indicate the overall rigidity of the protein and identify a collection of residues with greater flexibilities. Furthermore, Pro91 shows a cis conformation in solution instead of the trans conformation determined in the crystal structure

  13. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

    NARCIS (Netherlands)

    Olijve, L.L.C.; Meister, K.; DeVries, A.L.; Duman, J.G.; Guo, S.; Bakker, H.J.; Voets, I.K.

    2016-01-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of

  14. Plant ice-binding (antifreeze) proteins

    Science.gov (United States)

    Proteins that determine the temperature at which ice crystals will form in water-based solutions in cells and tissues, that bind to growing ice crystals, thus affecting their size, and that impact ice re-crystallization have been widely-documented and studied in many plant, bacterial, fungal, insect...

  15. Janus effect of antifreeze proteins on ice nucleation.

    Science.gov (United States)

    Liu, Kai; Wang, Chunlei; Ma, Ji; Shi, Guosheng; Yao, Xi; Fang, Haiping; Song, Yanlin; Wang, Jianjun

    2016-12-20

    The mechanism of ice nucleation at the molecular level remains largely unknown. Nature endows antifreeze proteins (AFPs) with the unique capability of controlling ice formation. However, the effect of AFPs on ice nucleation has been under debate. Here we report the observation of both depression and promotion effects of AFPs on ice nucleation via selectively binding the ice-binding face (IBF) and the non-ice-binding face (NIBF) of AFPs to solid substrates. Freezing temperature and delay time assays show that ice nucleation is depressed with the NIBF exposed to liquid water, whereas ice nucleation is facilitated with the IBF exposed to liquid water. The generality of this Janus effect is verified by investigating three representative AFPs. Molecular dynamics simulation analysis shows that the Janus effect can be established by the distinct structures of the hydration layer around IBF and NIBF. Our work greatly enhances the understanding of the mechanism of AFPs at the molecular level and brings insights to the fundamentals of heterogeneous ice nucleation.

  16. Hyperactive antifreeze proteins from longhorn beetles: some structural insights.

    Science.gov (United States)

    Kristiansen, Erlend; Wilkens, Casper; Vincents, Bjarne; Friis, Dennis; Lorentzen, Anders Blomkild; Jenssen, Håvard; Løbner-Olesen, Anders; Ramløv, Hans

    2012-11-01

    This study reports on structural characteristics of hyperactive antifreeze proteins (AFPs) from two species of longhorn beetles. In Rhagium mordax, eight unique mRNAs coding for five different mature AFPs were identified from cold-hardy individuals. These AFPs are apparently homologues to a previously characterized AFP from the closely related species Rhagium inquisitor, and consist of six identifiable repeats of a putative ice binding motif TxTxTxT spaced irregularly apart by segments varying in length from 13 to 20 residues. Circular dichroism spectra show that the AFPs from both species have a high content of β-sheet and low levels of α-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these β-sheets within the putative ice-binding motifs and the central parts of the segments separating them, consistent with an overall β-helical structure with the ice-binding motifs stacked in a β-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a β-helical conformation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    Science.gov (United States)

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  18. Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.

    Science.gov (United States)

    Scotter, Andrew J; Kuntz, Douglas A; Saul, Michelle; Graham, Laurie A; Davies, Peter L; Rose, David R

    2006-06-01

    We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.

  19. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Unknown

    2005-01-03

    Jan 3, 2005 ... covering all the systems, so far discovered.5,7,8,12. With the increasing ... Structural investigations on proteins by NMR are, currently ... rapid analysis of unfolded proteins. ...... and hence help in design of drugs against them.

  20. Cloning and expression of a novel antifreeze protein AFP72 from the beetle Tenebrio molitor.

    Science.gov (United States)

    Yan, Qing-Hua; Yang, Li; Wang, Qing; Zhang, Hui-Rong; Shao, Qiang

    2012-01-01

    A novel antifreeze protein AFP72 cDNA (GenBbank accession No. AY929389) was obtained by RT-PCR from Tenebrio molitor. The 216 bp fragment encodes a protein of 72 amino acid residues. Sequence analysis revealed that the cDNA displays a high degree of homology with T. molitor antifreeze proteins, ranging up to 90.78%. Recombinant plasmids pMAL-p2X-afp72 and pMAL-c2X-afp72 were transferred into E. coil TBI to induce a MBP fusion protein by IPTG. The target fusion protein was released from the periplasm and cytoplasm by the cold osmotic shock procedure and sonication respectively. The content of the fusion protein came up to 38.9 and 41.5% of the total dissolved protein, respectively. The fusion protein was purified through an amylose affinity column, and incised by factor Xa. Molecular sieve chromatography was used to achieve a high state of purity of the target protein. The purified target protein displayed a single band in SDS-PAGE. The fusion protein was shown to increase resistance to low temperatures in bacteria. This finding could help in further investigations of the properties and function of antifreeze proteins.

  1. Purification, composition, and physical properties of a thermal hysteresis "antifreeze" protein from larvae of the beetle, Tenebrio molitor.

    Science.gov (United States)

    Tomchaney, A P; Morris, J P; Kang, S H; Duman, J G

    1982-02-16

    Proteins which produce a thermal hysteresis (difference between the freezing and melting points) in aqueous solution are well-known for their antifreeze activity in polar marine fishes. Much less is known about the biology and biochemistry of similar antifreeze proteins found in certain insects. A thermal hysteresis protein was purified from cold acclimated larvae of the beetle, Tenebrio molitor, by using ethanol fractionation, DEAE ion-exchange chromatography, gel filtration, and high-pressure liquid chromatography. The purified protein had a molecular mass of 17 000 daltons and its N terminus was lysine. The amino acid composition of the antifreeze protein contained more hydrophilic amino acids than the fish antifreezes. This is consistent with the compositions of previously purified insect thermal hysteresis proteins. However, the percentage of hydrophilic amino acids in this Tenebrio antifreeze protein was considerably less than that of other insect thermal hysteresis proteins. The freezing point depressing activity of the Tenebrio antifreeze was less than that of fish proteins and glycoproteins at low protein concentrations but was greater at high protein concentrations.

  2. Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

    Science.gov (United States)

    Ramya, L; Ramakrishnan, Vigneshwar

    2016-07-01

    Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. HEAT INDUCIBLE EXPRESSION OF ANTIFREEZE PROTEIN GENES FROM THE BEETLES Tenebrio molitor AND Microdera punctipennis.

    Science.gov (United States)

    Li, Jieqiong; Ma, Wenjing; Ma, Ji

    2016-01-01

    Antifreeze proteins (AFPs) play important roles in protecting poikilothermic organisms from cold damage. The expression of AFP genes (afps) is induced by low temperature. However, it is reported that heat can influence the expression of afps in the desert beetle Microdera punctipennis. To further detect whether heat also induce the expression of afps in other insects, and to determine the expression profiling of insect afps at different temperatures. The expression of antifreeze protein genes in the two beetles, Microdera punctipennis and Tenebrio molitor that have quite different living environment, under different temperatures were studied by using real-time quantitative PCR. Mild low temperatures (5~15 degree C), high temperature (38~47 degree C for M. punctipennis, or 37~42 degree C for T. molitor) and temperature difference (10~30 degree C) all stimulated strongly to the expression of AFP genes (Mpafps) in M. punctipennis which lives in the wild filed in desert. The mRNA level of Mpafps after M. punctipennis were exposed to these temperatures for 1h~5h was at least 30-fold of the control at 25 degree C. For T. molitor which is breeding in door with wheat bran all these temperatures stimulated significantly to the expression of Tmafps, while the extent and degree of the temperature stimulation on Tmafps expression were much lower than on Mpafps. After T. molitor were exposed to 5 degree C and 15 degree C for 1h~5h, the mRNA level of Tmafps was over 6-fold and 45-fold of the control at 25 degree C. High temperature (37~42 degree C) for 1h~3h treatments increased Tmafps mRNA level 4.8-fold of the control. Temperature difference of 10 degree C was effective in stimulating Tmafps expression. The expression of insect antifreeze protein genes both in M. punctipennis and T. molitor was induced by heat, suggesting that this phenomenon may be common in insects; the extent and degree of the influence differ in species that have different living conditions. The heat

  4. Ice Shaping Properties, Similar to That of Antifreeze Proteins, of a Zirconium Acetate Complex

    Science.gov (United States)

    Deville, Sylvain; Viazzi, Céline; Leloup, Jérôme; Lasalle, Audrey; Guizard, Christian; Maire, Eric; Adrien, Jérôme; Gremillard, Laurent

    2011-01-01

    The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances. PMID:22028886

  5. Ice shaping properties, similar to that of antifreeze proteins, of a zirconium acetate complex.

    Directory of Open Access Journals (Sweden)

    Sylvain Deville

    Full Text Available The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances.

  6. Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

    DEFF Research Database (Denmark)

    Kristiansen, E; Ramløv, Hans; Højrup, Peter

    2011-01-01

    Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn...... of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs....... beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs...

  7. Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers.

    Science.gov (United States)

    Graham, Ben; Bailey, Trisha L; Healey, Joseph R J; Marcellini, Moreno; Deville, Sylvain; Gibson, Matthew I

    2017-12-11

    Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent "antifreezes". Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid-phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function.

    Science.gov (United States)

    Duman, John G

    2015-06-01

    Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants. © 2015. Published by The Company of Biologists Ltd.

  9. Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein

    International Nuclear Information System (INIS)

    Petit-Haertlein, Isabelle; Blakeley, Matthew P.; Howard, Eduardo; Hazemann, Isabelle; Mitschler, Andre; Haertlein, Michael; Podjarny, Alberto

    2009-01-01

    Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm 3 crystal. The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D 2 O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a ‘radically small’ perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm 3

  10. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-08

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  11. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

  12. Studies on new antifreeze protein from the psychrophilic diatom ...

    African Journals Online (AJOL)

    Administrator

    2011-09-12

    Sep 12, 2011 ... 27-kDa protein modified with His-tag. According to bioinformatics data, a comparison ... for plant biotechnology application on tomato. MATERIALS AND METHODS. The ESTs library was ... specific expression in tomato as previously described (Deng, 2003). Sequence analysis and prediction of protein ...

  13. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    GRAVY, Grand Average Hydropathy. structure of protein (3D coordinates data). The 3D structure of AFPs Q01758 and P05140 were gener- ated by homology modelling using Esypred34 server. The similar 3D structures (for the AFPs Q01758 and. P05140 sequences) in the Protein Data bank. (www.rscb.org) were identified ...

  14. Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

    Directory of Open Access Journals (Sweden)

    Srisulak Dheeranupattana

    2005-07-01

    Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

  15. Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein

    International Nuclear Information System (INIS)

    Hakim, Aaron; Thakral, Durga; Zhu, Darren F.; Nguyen, Jennifer B.

    2012-01-01

    A novel hyperactive antifreeze protein from R. inquisitor (RiAFP) has been overexpressed, purified and crystallized. A complete native X-ray diffraction data set was recorded to 1.3 Å resolution. Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 Å resolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3 1 21 (or P3 2 21), with unit-cell parameters a = b = 46.46, c = 193.21 Å

  16. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    Science.gov (United States)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  17. Hybridization assay of insect antifreezing protein gene by novel multilayered porous silicon nucleic acid biosensor.

    Science.gov (United States)

    Lv, Xiaoyi; Chen, Liangliang; Zhang, Hongyan; Mo, Jiaqing; Zhong, Furu; Lv, Changwu; Ma, Ji; Jia, Zhenhong

    2013-01-15

    A fabrication of a novel simple porous silicon polybasic photonic crystal with symmetrical structure has been reported as a nucleic acid biosensor for detecting antifreeze protein gene in insects (Microdera puntipennis dzhungarica), which would be helpful in the development of some new transgenic plants with tolerance of freezing stress. Compared to various porous silicon-based photonic configurations, porous silicon polytype layered structure is quite easy to prepare and shows more stability; moreover, polybasic photonic crystals with symmetrical structure exhibit interesting optical properties with a sharp resonance in the reflectance spectrum, giving a higher Q factor which causes higher sensitivity for sensing performance. In this experiment, DNA oligonucleotides were immobilized into the porous silicon pores using a standard crosslink chemistry method. The porous silicon polybasic symmetrical structure sensor possesses high specificity in performing controlled experiments with non-complementary DNA. The detection limit was found to be 21.3nM for DNA oligonucleotides. The fabricated multilayered porous silicon-based DNA biosensor has potential commercial applications in clinical chemistry for determination of an antifreeze protein gene or other genes. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. An Effective Antifreeze Protein Predictor with Ensemble Classifiers and Comprehensive Sequence Descriptors

    Directory of Open Access Journals (Sweden)

    Runtao Yang

    2015-09-01

    Full Text Available Antifreeze proteins (AFPs play a pivotal role in the antifreeze effect of overwintering organisms. They have a wide range of applications in numerous fields, such as improving the production of crops and the quality of frozen foods. Accurate identification of AFPs may provide important clues to decipher the underlying mechanisms of AFPs in ice-binding and to facilitate the selection of the most appropriate AFPs for several applications. Based on an ensemble learning technique, this study proposes an AFP identification system called AFP-Ensemble. In this system, random forest classifiers are trained by different training subsets and then aggregated into a consensus classifier by majority voting. The resulting predictor yields a sensitivity of 0.892, a specificity of 0.940, an accuracy of 0.938 and a balanced accuracy of 0.916 on an independent dataset, which are far better than the results obtained by previous methods. These results reveal that AFP-Ensemble is an effective and promising predictor for large-scale determination of AFPs. The detailed feature analysis in this study may give useful insights into the molecular mechanisms of AFP-ice interactions and provide guidance for the related experimental validation. A web server has been designed to implement the proposed method.

  19. The mysteries of memory effect and its elimination with antifreeze proteins

    Energy Technology Data Exchange (ETDEWEB)

    Walker, V.; Gordienko, R.; Kuiper, M.; Huva, E.; Wu, Z. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology; Zeng, H.; Ripmeester, J. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology]|[National Research Council of Canada, Ottawa, ON (Canada). Steacie Inst. for Molecular Sciences

    2008-07-01

    With the decline in easily accessible and conventional hydrocarbon supplies, exploration will focus on hydrocarbons in deep offshore waters, in permafrost or in crystalline water as gas hydrates. Crystallization of water or water-encaged gas molecules takes place when nuclei reach a critical size, but the crystal growth may be inhibited by certain antifreeze proteins (AFPs). In this study, the authors hypothesized that the crystal lattice of gas hydrates may act as an alternative for substrate antifreeze proteins (AFPs). AFP-mediated inhibition of ice and clathrate hydrate crystallization was examined. Since the AFPs had a notable ability to eliminate the memory effect (ME) or the faster reformation of clathrate hydrates after melting, the authors were prompted to examine heterogeneous nucleation. Silica, served as a model nucleator hydrophilic surface. Quartz crystal microbalance-dissipation (QCM-D) experiments showed that an active AFP was tightly adsorbed to the silica surface. However, polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap), 2 commercial hydrate kinetic inhibitors that do not eliminate ME, were not as tightly adsorbed. A mutant AFP inhibited tetrahydrofuran clathrate hydrate growth, but not ME. QCM-D analysis showed that adsorption of the mutant AFP was more similar to PVCap than the active AFP. It was concluded that although there is no evidence for memory in ice reformation, the crystallization of ice and hydrates, and the elimination of the more rapid recrystallization of hydrates, can be mediated by the same proteins. The properties of adsorbed layers can be effectively monitored by QCM-D. These study results provided useful information about the inhibition mechanism of heterogeneous nucleation of clathrate hydrate. The technique facilitates the screening of potential low dose hydrate inhibitors and residues in AFPs that are involved in silica adsorption. 24 refs., 1 tab., 4 figs.

  20. Fundamentals of Protein NMR Spectroscopy

    CERN Document Server

    Rule, Gordon S

    2006-01-01

    NMR spectroscopy has proven to be a powerful technique to study the structure and dynamics of biological macromolecules. Fundamentals of Protein NMR Spectroscopy is a comprehensive textbook that guides the reader from a basic understanding of the phenomenological properties of magnetic resonance to the application and interpretation of modern multi-dimensional NMR experiments on 15N/13C-labeled proteins. Beginning with elementary quantum mechanics, a set of practical rules is presented and used to describe many commonly employed multi-dimensional, multi-nuclear NMR pulse sequences. A modular analysis of NMR pulse sequence building blocks also provides a basis for understanding and developing novel pulse programs. This text not only covers topics from chemical shift assignment to protein structure refinement, as well as the analysis of protein dynamics and chemical kinetics, but also provides a practical guide to many aspects of modern spectrometer hardware, sample preparation, experimental set-up, and data pr...

  1. Conformation of antifreeze glycoproteins as determined from conformational energy calculations and fully assigned proton NMR spectra

    International Nuclear Information System (INIS)

    Bush, C.A.; Rao, B.N.N.

    1986-01-01

    The 1 H NMR spectra of AFGP's ranging in molecular weight from 2600 to 30,000 Daltons isolated from several different species of polar fish have been measured. The spectrum of AFGP 1-4 from Pagothenia borchgrevinki with an average of 30 repeating subunits has a single resonance for each proton of the glycotripeptide repeating unit, (ala-[gal-(β-1→3) galNAc-(α--O-]thr-ala)/sub n/. Its 1 H NMR spectrum including resonances of the amide protons has been completely assigned. Coupling constants and nuclear Overhauser enhancements (n.O.e.) between protons on distant residues imply conformational order. The 2600 dalton molecular weight glycopeptides (AFGP-8) have pro in place of ala at certain specific points in the sequence and AFGP-8R of Eleginus gracilis has arg in place of one thr. The resonances of pro and arg were assigned by decoupling. The resonances of the carboxy and amino terminals have distinct chemical shifts and were assigned in AFGP-8 of Boreogadus saida by titration. n.O.e. between α--protons and amide protons of the adjacent residue (sequential n.O.e.) were used in assignments of additional resonances and to assign the distinctive resonances of thr followed by pro. Conformational energy calculations on the repeating glycotripeptide subunit of AFGP show that the α--glucosidic linkage has a fixed conformation while the β--linkage is less rigid. A conformational model for AFGP 1-4, which is based on the calculations has the peptide in an extended left-handed helix with three residues per turn similar to polyproline II. The model is consistent with CD data, amide proton coupling constants, temperature dependence of amide proton chemical shifts

  2. The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

    Science.gov (United States)

    Daley, Margaret E.; Sykes, Brian D.

    2003-01-01

    Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H-1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand. PMID:12824479

  3. Hydration behavior at the ice-binding surface of the Tenebrio molitor antifreeze protein.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2014-05-08

    Molecular dynamics (MD) simulations have been carried out at two different temperatures (300 and 220 K) to study the conformational rigidity of the hyperactive Tenebrio molitor antifreeze protein (TmAFP) in aqueous medium and the structural arrangements of water molecules hydrating its surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its nonice-binding surface (NIBS). The presence of a set of regularly arranged internally bound water molecules is found to play an important role in maintaining the flat rigid nature of the IBS. Importantly, the calculations reveal that the strategically located hydroxyl oxygens of the threonine (Thr) residues in the IBS influence the arrangements of five sets of ordered waters around it on two parallel planes that closely resemble the basal plane of ice. As a result, these waters can register well with the ice basal plane, thereby allowing the IBS to preferentially bind at the ice interface and inhibit its growth. This provides a possible molecular reason behind the ice-binding activity of TmAFP at the basal plane of ice.

  4. ANTIFREEZE PROTEINS IN PLANTS: AN OVERVIEW WITH AN INSIGHT INTO THE DETECTION TECHNIQUES INCLUDING NANOBIOTECHNOLOGY

    Directory of Open Access Journals (Sweden)

    Bhavana Sharma

    2014-08-01

    Full Text Available Antifreeze proteins (AFPs are a class of polypeptides which enables various organisms to survive subzero temperatures and have been found in vertebrates, invertebrates, plants, fungi and lichens. AFPs possess the characteristic thermal hysteresis (TH and ice recrystallization inhibition (IRI properties which allow them to adsorb the surface of ice crystals and inhibit their growth and recrystallization. AFPs are also known as ice restructuring proteins due to their ability to modify ice crystal morphology which leads to formation of hexagonal shape ice crystals in the presence of AFPs and disc shape AFPs in its absence. AFPs have various applications in medical, agricultural, industrial and biotechnological field. This review provides an overview of the AFPs, their TH and IRI properties and potential biotechnological applications of AFPs. Various conventional detection methods like Capillary assay and Differential Scanning Calorimetry (DSC with their advantages and disadvantages are discussed in detail along with the commonly used Splat assay and Nanoliter osmometer. Moreover, a novel, high-throughput and efficient nanobiotechnological method for AFP detection is also discussed. The method is based on colorimetric detection of freeze-labile gold nanoparticles and can provide an alternative to overcome the limitations of conventional methods by providing quick and easy way to screen AFPs in multiple systems simultaneously

  5. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Johnsen, Johannes Lørup; Kristiansen, Erlend

    2014-01-01

    The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect......, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show...

  6. Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein.

    Science.gov (United States)

    Ideta, Atsushi; Aoyagi, Yoshito; Tsuchiya, Kanami; Nakamura, Yuuki; Hayama, Kou; Shirasawa, Atsushi; Sakaguchi, Kenichiro; Tominaga, Naomi; Nishimiya, Yoshiyuki; Tsuda, Sakae

    2015-01-01

    Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.

  7. Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions.

    Science.gov (United States)

    Hansen, T N; Carpenter, J F

    1993-01-01

    Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP. Images FIGURE 8 PMID:7690257

  8. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    Science.gov (United States)

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  9. Expression, purification and activity determination of the beetle tenebrio molitor antifreeze protein afp84c in escherichia coli

    International Nuclear Information System (INIS)

    Yang, Q.; Feng, H.

    2013-01-01

    Summary: A cDNA encoding antifreeze protein (AFP84c) was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 252 bp encodes a protein of 84 amino acid residues and was fused to the expression vectors pMAL-c2X and pMAL-p2X. The expression plasmids pMAL-c2X-afp84c and pMAL-p2X-afp84c were constructed and transformed into Escherischia coli strains TBI, respectively. Strategy of optimization of induction conditions were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in pMALTM expression system. The target fusion protein was released from the cytoplasm and periplasm by sonication and cold osmotic shock procedure respectively. Recombinant AFP84c was purified by amylose affinity column. The purified target protein displayed a single band in SDS-PAGE. Expressed AFP84c exhibits to increase low temperature resistance of bacteria. (author)

  10. Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

    Science.gov (United States)

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  11. De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS

    Directory of Open Access Journals (Sweden)

    Ricardo Vera Bravo

    2011-12-01

    Full Text Available A new strategy is presented for the designand synthesis of peptides that exhibitice-binding and antifreeze activity. Apennant-type dendrimer polypeptidescaffold combining an α-helical backbonewith four short β-strand branches wassynthesized in solid phase using Fmocchemistry in a divergent approach. The51-residue dendrimer was characterizedby reverse phase high performance liquidchromatography, mass spectrometry andcircular dichroism. Each β-strand branchcontained three overlapping TXT aminoacid repeats, an ice-binding motif foundin the ice-binding face of the sprucebudworm (Choristoneura fumiferanaand beetle (Tenebrio molitor antifreezeproteins. Ice crystals in the presence ofthe polypeptide monomer displayed flat,hexagonal plate morphology, similar tothat produced by weakly active antifreezeproteins. An oxidized dimeric form of thedendrimer polypeptide also produced flathexagonal ice crystals and was capableof inhibiting ice crystal growth upontemperature reduction, a phenomenontermed thermal hysteresis, a definingproperty of antifreeze proteins. Linkageof the pennant-type dendrimer to a trifunctionalcascade-type polypeptideproduced a trimeric macromolecule thatgave flat hexagonal ice crystals withhigher thermal hysteresis activity thanthe dimer or monomer and an ice crystal burst pattern similar to that producedby samples containing insect antifreezeproteins. This macromolecule was alsocapable of inhibiting ice recrystallization.

  12. Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

    Directory of Open Access Journals (Sweden)

    Jaewang Lee

    Full Text Available Ovarian tissue (OT cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs on mouse ovarian tissue cryopreservation and transplantation.Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III and concentration (0.1, 1, 10 mg/mL used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs and repair (DDR, respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control. Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL

  13. Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

    DEFF Research Database (Denmark)

    Wilkens, Casper; Poulsen, Jens-Christian Navarro; Ramløv, Hans

    2014-01-01

    Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform......, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18...... equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge...

  14. Solution NMR structure determination of proteins revisited

    International Nuclear Information System (INIS)

    Billeter, Martin; Wagner, Gerhard; Wuethrich, Kurt

    2008-01-01

    This 'Perspective' bears on the present state of protein structure determination by NMR in solution. The focus is on a comparison of the infrastructure available for NMR structure determination when compared to protein crystal structure determination by X-ray diffraction. The main conclusion emerges that the unique potential of NMR to generate high resolution data also on dynamics, interactions and conformational equilibria has contributed to a lack of standard procedures for structure determination which would be readily amenable to improved efficiency by automation. To spark renewed discussion on the topic of NMR structure determination of proteins, procedural steps with high potential for improvement are identified

  15. A microscale protein NMR sample screening pipeline

    Energy Technology Data Exchange (ETDEWEB)

    Rossi, Paolo; Swapna, G. V. T.; Huang, Yuanpeng J.; Aramini, James M. [State University of New Jersey, Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers (United States); Anklin, Clemens [Bruker Biospin Corporation (United States); Conover, Kenith; Hamilton, Keith; Xiao, Rong; Acton, Thomas B.; Ertekin, Asli; Everett, John K.; Montelione, Gaetano T., E-mail: guy@cabm.rutgers.ed [State University of New Jersey, Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers (United States)

    2010-01-15

    As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 {mu}g in 8-35 {mu}l volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.

  16. MAS NMR of HIV-1 protein assemblies

    Science.gov (United States)

    Suiter, Christopher L.; Quinn, Caitlin M.; Lu, Manman; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

    2015-04-01

    The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

  17. Seasonal changes in antifreeze protein gene transcription and water content of beetle Microdera punctipennis (Coleoptera, Tenebrionidae) from Gurbantonggut desert in Central Asia.

    Science.gov (United States)

    Hou, F; Ma, J; Liu, X; Wang, Y; Liu, X N; Zhang, F C

    2010-01-01

    Desert beetle Microdera punctipennis (Coleoptera: Tenebriondae) is a special species in Gurbantonggut Desert in Central Asia. To investigate the possible strategy it employs for cold survival, seasonal changes in supercooling point (SCP), body water content, haemolymph osmolality and antifreeze protein gene (Mpafp) expression were measured over 13 months. Our results show SCPs in M. punctipennis adults changed from -8.0°C in summer to -18.7°C in winter. During winter, adults endured modest water loss; total water decreased from 65.4 percent in summer to 55.9% in winter. Mpafp mRNAs level increased by 13.1 fold from summer to early winter, and haemolymph osmolality increased accordingly from 550 mOsm to 1486 mOsm. Correlation coefficient of Mpafp mRNAs level and SCP indicates that Mpafp mRNA explained 65.3 percent of the variation in SCPs. The correlation between Mpafp mRNA level and total water reflected an indirect influence of antifreeze protein on water content via reducing SCP.

  18. Automated protein structure calculation from NMR data

    International Nuclear Information System (INIS)

    Williamson, Mike P.; Craven, C. Jeremy

    2009-01-01

    Current software is almost at the stage to permit completely automatic structure determination of small proteins of <15 kDa, from NMR spectra to structure validation with minimal user interaction. This goal is welcome, as it makes structure calculation more objective and therefore more easily validated, without any loss in the quality of the structures generated. Moreover, it releases expert spectroscopists to carry out research that cannot be automated. It should not take much further effort to extend automation to ca 20 kDa. However, there are technological barriers to further automation, of which the biggest are identified as: routines for peak picking; adoption and sharing of a common framework for structure calculation, including the assembly of an automated and trusted package for structure validation; and sample preparation, particularly for larger proteins. These barriers should be the main target for development of methodology for protein structure determination, particularly by structural genomics consortia

  19. NMR Studies of Protein Hydration and Protein-Ligand Interactions

    Science.gov (United States)

    Chong, Yuan

    Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

  20. (S)Pinning down protein interactions by NMR

    DEFF Research Database (Denmark)

    Teilum, Kaare; Kunze, Micha Ben Achim; Erlendsson, Simon

    2017-01-01

    Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions...... all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative...... descriptions of protein interactions. In this review we provide an easy-access approach to NMR for the non-NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background...

  1. Unraveling the meaning of chemical shifts in protein NMR.

    Science.gov (United States)

    Berjanskii, Mark V; Wishart, David S

    2017-11-01

    Chemical shifts are among the most informative parameters in protein NMR. They provide wealth of information about protein secondary and tertiary structure, protein flexibility, and protein-ligand binding. In this report, we review the progress in interpreting and utilizing protein chemical shifts that has occurred over the past 25years, with a particular focus on the large body of work arising from our group and other Canadian NMR laboratories. More specifically, this review focuses on describing, assessing, and providing some historical context for various chemical shift-based methods to: (1) determine protein secondary and super-secondary structure; (2) derive protein torsion angles; (3) assess protein flexibility; (4) predict residue accessible surface area; (5) refine 3D protein structures; (6) determine 3D protein structures and (7) characterize intrinsically disordered proteins. This review also briefly covers some of the methods that we previously developed to predict chemical shifts from 3D protein structures and/or protein sequence data. It is hoped that this review will help to increase awareness of the considerable utility of NMR chemical shifts in structural biology and facilitate more widespread adoption of chemical-shift based methods by the NMR spectroscopists, structural biologists, protein biophysicists, and biochemists worldwide. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Fast mapping of global protein folding states by multivariate NMR:

    DEFF Research Database (Denmark)

    Malmendal, Anders; Underhaug, Jarl; Otzen, Daniel

    2010-01-01

    To obtain insight into the functions of proteins and their specific roles, it is important to establish efficient procedures for exploring the states that encapsulate their conformational space. Global Protein folding State mapping by multivariate NMR (GPS NMR) is a powerful high-throughput method......-lactalbumin in the presence of the anionic surfactant sodium dodecyl sulfate, SDS, and compare these with other surfactants, acid, denaturants and heat....

  3. Cell signaling, post-translational protein modifications and NMR spectroscopy

    International Nuclear Information System (INIS)

    Theillet, Francois-Xavier; Smet-Nocca, Caroline; Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas; Yoon, Mi-Kyung; Kriwacki, Richard W.; Landrieu, Isabelle; Lippens, Guy; Selenko, Philipp

    2012-01-01

    Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

  4. Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy.

    Science.gov (United States)

    Konuma, Tsuyoshi; Harada, Erisa; Sugase, Kenji

    2015-12-01

    Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

  5. Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Konuma, Tsuyoshi [Icahn School of Medicine at Mount Sinai, Department of Structural and Chemical Biology (United States); Harada, Erisa [Suntory Foundation for Life Sciences, Bioorganic Research Institute (Japan); Sugase, Kenji, E-mail: sugase@sunbor.or.jp, E-mail: sugase@moleng.kyoto-u.ac.jp [Kyoto University, Department of Molecular Engineering, Graduate School of Engineering (Japan)

    2015-12-15

    Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

  6. Quantitative analysis of protein-ligand interactions by NMR.

    Science.gov (United States)

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  7. De novo protein structure determination using sparse NMR data

    International Nuclear Information System (INIS)

    Bowers, Peter M.; Strauss, Charlie E.M.; Baker, David

    2000-01-01

    We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with ∼1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined

  8. NMR structural studies of peptides and proteins in membranes

    Energy Technology Data Exchange (ETDEWEB)

    Opella, S J [Pennsylvania Univ., Philadelphia, PA (United States). Dept. of Chemistry

    1994-12-31

    The use of NMR methodology in structural studies is described as applicable to larger proteins, considering that the majority of membrane proteins is constructed from a limited repertoire of structural and dynamic elements. The membrane associated domains of these proteins are made up of long hydrophobic membrane spanning helices, shorter amphipathic bridging helices in the plane of the bilayer, connecting loops with varying degrees of mobility, and mobile N- and C- terminal sections. NMR studies have been successful in identifying all of these elements and their orientations relative to each other and the membrane bilayer 19 refs., 9 figs.

  9. Protein folding on the ribosome studied using NMR spectroscopy

    Science.gov (United States)

    Waudby, Christopher A.; Launay, Hélène; Cabrita, Lisa D.; Christodoulou, John

    2013-01-01

    NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome–nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity. PMID:24083462

  10. Hydration layer dynamics and association mechanisms of food and antifreeze proteins : A Molecular Dynamics and Transition Path Sampling study

    NARCIS (Netherlands)

    Brotzakis, Z.F.

    2017-01-01

    By the time the reader reads this line, billions of protein association events just occurred in our body, such as the ones regulating cell communication, signaling pathways, or in initiating a self-assembly processes, such as tissue fabrication, etc. The timescale of such transitions is slow,

  11. Application of Solution NMR Spectroscopy to Study Protein Dynamics

    Directory of Open Access Journals (Sweden)

    Christoph Göbl

    2012-03-01

    Full Text Available Recent advances in spectroscopic methods allow the identification of minute fluctuations in a protein structure. These dynamic properties have been identified as keys to some biological processes. The consequences of this structural flexibility can be far‑reaching and they add a new dimension to the structure-function relationship of biomolecules. Nuclear Magnetic Resonance (NMR spectroscopy allows the study of structure as well as dynamics of biomolecules in a very broad range of timescales at atomic level. A number of new NMR methods have been developed recently to allow the measurements of time scales and spatial fluctuations, which in turn provide the thermodynamics associated with the biological processes. Since NMR parameters reflect ensemble measurements, structural ensemble approaches in analyzing NMR data have also been developed. These new methods in some instances can even highlight previously hidden conformational features of the biomolecules. In this review we describe several solution NMR methods to study protein dynamics and discuss their impact on important biological processes.

  12. NMR spectroscopy of muscle proteins; Spektroskopia MRJ bialek miesniowych

    Energy Technology Data Exchange (ETDEWEB)

    Slosarek, G. [Inst. Fizyki, Univ. A. Mickiewicza, Poznan (Poland)

    1995-12-31

    Author reviews various experimental techniques used for study of the structure of muscle proteins. Difficulties of application of NMR are described. Studies of the influence of Ca{sup 2+} on flexibility of actin polymer are presented. 11 refs, 3 figs.

  13. Protein structure estimation from NMR data by matrix completion.

    Science.gov (United States)

    Li, Zhicheng; Li, Yang; Lei, Qiang; Zhao, Qing

    2017-09-01

    Knowledge of protein structures is very important to understand their corresponding physical and chemical properties. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the main methods to measure protein structure. In this paper, we propose a two-stage approach to calculate the structure of a protein from a highly incomplete distance matrix, where most data are obtained from NMR. We first randomly "guess" a small part of unobservable distances by utilizing the triangle inequality, which is crucial for the second stage. Then we use matrix completion to calculate the protein structure from the obtained incomplete distance matrix. We apply the accelerated proximal gradient algorithm to solve the corresponding optimization problem. Furthermore, the recovery error of our method is analyzed, and its efficiency is demonstrated by several practical examples.

  14. High Resolution NMR Studies of Encapsulated Proteins In Liquid Ethane

    Science.gov (United States)

    Peterson, Ronald W.; Lefebvre, Brian G.; Wand, A. Joshua

    2005-01-01

    Many of the difficulties presented by large, aggregation-prone, and membrane proteins to modern solution NMR spectroscopy can be alleviated by actively seeking to increase the effective rate of molecular reorientation. An emerging approach involves encapsulating the protein of interest within the protective shell of a reverse micelle, and dissolving the resulting particle in a low viscosity fluid, such as the short chain alkanes. Here we present the encapsulation of proteins with high structural fidelity within reverse micelles dissolved in liquid ethane. The addition of appropriate co-surfactants can significantly reduce the pressure required for successful encapsulation. At these reduced pressures, the viscosity of the ethane solution is low enough to provide sufficiently rapid molecular reorientation to significantly lengthen the spin-spin NMR relaxation times of the encapsulated protein. PMID:16028922

  15. DNA nanotubes for NMR structure determination of membrane proteins.

    Science.gov (United States)

    Bellot, Gaëtan; McClintock, Mark A; Chou, James J; Shih, William M

    2013-04-01

    Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.

  16. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

    2015-06-15

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

  17. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    International Nuclear Information System (INIS)

    Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

    2015-01-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

  18. Antifreeze life cycle assessment (LCA

    Directory of Open Access Journals (Sweden)

    Kesić Jelena

    2005-01-01

    Full Text Available Antifreeze based on ethylene glycol is a commonly used commercial product The classification of ethylene glycol as a toxic material increased the disposal costs for used antifreeze and life cycle assessment became a necessity. Life Cycle Assessment (LCA considers the identification and quantification of raw materials and energy inputs and waste outputs during the whole life cycle of the analyzed product. The objectives of LCA are the evaluation of impacts on the environment and improvements of processes in order to reduce and/or eliminate waste. LCA is conducted through a mathematical model derived from mass and energy balances of all the processes included in the life cycle. In all energy processes the part of energy that can be transformed into some other kind of energy is called exergy. The concept of exergy considers the quality of different types of energy and the quality of different materials. It is also a connection between energy and mass transformations. The whole life cycle can be described by the value of the total loss of exergy. The physical meaning of this value is the loss of material and energy that can be used. The results of LCA are very useful for the analyzed products and processes and for the determined conditions under which the analysis was conducted. The results of this study indicate that recycling is the most satisfactory solution for the treatment of used antifreeze regarding material and energy consumption but the re-use of antifreeze should not be neglected as a solution.

  19. Structure and Dynamic Properties of Membrane Proteins using NMR

    DEFF Research Database (Denmark)

    Rösner, Heike; Kragelund, Birthe

    2012-01-01

    conformational changes. Their structural and functional decoding is challenging and has imposed demanding experimental development. Solution nuclear magnetic resonance (NMR) spectroscopy is one of the techniques providing the capacity to make a significant difference in the deciphering of the membrane protein...... structure-function paradigm. The method has evolved dramatically during the last decade resulting in a plethora of new experiments leading to a significant increase in the scientific repertoire for studying membrane proteins. Besides solving the three-dimensional structures using state-of-the-art approaches......-populated states, this review seeks to introduce the vast possibilities solution NMR can offer to the study of membrane protein structure-function analyses with special focus on applicability. © 2012 American Physiological Society. Compr Physiol 2:1491-1539, 2012....

  20. Covariance NMR Processing and Analysis for Protein Assignment.

    Science.gov (United States)

    Harden, Bradley J; Frueh, Dominique P

    2018-01-01

    During NMR resonance assignment it is often necessary to relate nuclei to one another indirectly, through their common correlations to other nuclei. Covariance NMR has emerged as a powerful technique to correlate such nuclei without relying on error-prone peak peaking. However, false-positive artifacts in covariance spectra have impeded a general application to proteins. We recently introduced pre- and postprocessing steps to reduce the prevalence of artifacts in covariance spectra, allowing for the calculation of a variety of 4D covariance maps obtained from diverse combinations of pairs of 3D spectra, and we have employed them to assign backbone and sidechain resonances in two large and challenging proteins. In this chapter, we present a detailed protocol describing how to (1) properly prepare existing 3D spectra for covariance, (2) understand and apply our processing script, and (3) navigate and interpret the resulting 4D spectra. We also provide solutions to a number of errors that may occur when using our script, and we offer practical advice when assigning difficult signals. We believe such 4D spectra, and covariance NMR in general, can play an integral role in the assignment of NMR signals.

  1. Contemporary NMR Studies of Protein Electrostatics.

    Science.gov (United States)

    Hass, Mathias A S; Mulder, Frans A A

    2015-01-01

    Electrostatics play an important role in many aspects of protein chemistry. However, the accurate determination of side chain proton affinity in proteins by experiment and theory remains challenging. In recent years the field of nuclear magnetic resonance spectroscopy has advanced the way that protonation states are measured, allowing researchers to examine electrostatic interactions at an unprecedented level of detail and accuracy. Experiments are now in place that follow pH-dependent (13)C and (15)N chemical shifts as spatially close as possible to the sites of protonation, allowing all titratable amino acid side chains to be probed sequence specifically. The strong and telling response of carefully selected reporter nuclei allows individual titration events to be monitored. At the same time, improved frameworks allow researchers to model multiple coupled protonation equilibria and to identify the underlying pH-dependent contributions to the chemical shifts.

  2. Peakr: simulating solid-state NMR spectra of proteins

    International Nuclear Information System (INIS)

    Schneider, Robert; Odronitz, Florian; Hammesfahr, Bjorn; Hellkamp, Marcel; Kollmar, Martin

    2013-01-01

    When analyzing solid-state nuclear magnetic resonance (NMR) spectra of proteins, assignment of resonances to nuclei and derivation of restraints for 3D structure calculations are challenging and time-consuming processes. Simulated spectra that have been calculated based on, for example, chemical shift predictions and structural models can be of considerable help. Existing solutions are typically limited in the type of experiment they can consider and difficult to adapt to different settings. Here, we present Peakr, a software to simulate solid-state NMR spectra of proteins. It can generate simulated spectra based on numerous common types of internuclear correlations relevant for assignment and structure elucidation, can compare simulated and experimental spectra and produces lists and visualizations useful for analyzing measured spectra. Compared with other solutions, it is fast, versatile and user friendly. (authors)

  3. NMR

    International Nuclear Information System (INIS)

    Kneeland, J.B.; Lee, B.C.P.; Whalen, J.P.; Knowles, R.J.R.; Cahill, P.T.

    1984-01-01

    Although still quite new, NMR imaging has already emerged as a safe, noninvasive, painless, and effective diagnostic modality requiring no ionizing radiation. Also, NMR appears already to have established itself as the method of choice for the examination of the brain spinal cord (excluding herniated disks). Another area in which NMR excels is in the examination of the pelvis. The use of surface coils offers the promise of visualizing structures with resolution unobtainable by any other means. In addition, NMR, with its superb visualization of vascular structures and potential ability to measure flow, may soon revolutionize the diagnosis of cardiovascular disease. Finally, NMR, through biochemically and physiologically based T/sub 1/ and T/sub 2/ indices or through spectroscopy, may provide a means of monitoring therapeutic response so as to permit tailoring of treatment to the individual patient. In short, NMR is today probably at the same stage as the x-ray was in Roentgen's day

  4. Systematic comparison of crystal and NMR protein structures deposited in the protein data bank.

    Science.gov (United States)

    Sikic, Kresimir; Tomic, Sanja; Carugo, Oliviero

    2010-09-03

    Nearly all the macromolecular three-dimensional structures deposited in Protein Data Bank were determined by either crystallographic (X-ray) or Nuclear Magnetic Resonance (NMR) spectroscopic methods. This paper reports a systematic comparison of the crystallographic and NMR results deposited in the files of the Protein Data Bank, in order to find out to which extent these information can be aggregated in bioinformatics. A non-redundant data set containing 109 NMR - X-ray structure pairs of nearly identical proteins was derived from the Protein Data Bank. A series of comparisons were performed by focusing the attention towards both global features and local details. It was observed that: (1) the RMDS values between NMR and crystal structures range from about 1.5 Å to about 2.5 Å; (2) the correlation between conformational deviations and residue type reveals that hydrophobic amino acids are more similar in crystal and NMR structures than hydrophilic amino acids; (3) the correlation between solvent accessibility of the residues and their conformational variability in solid state and in solution is relatively modest (correlation coefficient = 0.462); (4) beta strands on average match better between NMR and crystal structures than helices and loops; (5) conformational differences between loops are independent of crystal packing interactions in the solid state; (6) very seldom, side chains buried in the protein interior are observed to adopt different orientations in the solid state and in solution.

  5. Optimization of protein samples for NMR using thermal shift assays

    International Nuclear Information System (INIS)

    Kozak, Sandra; Lercher, Lukas; Karanth, Megha N.; Meijers, Rob; Carlomagno, Teresa; Boivin, Stephane

    2016-01-01

    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor"® provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

  6. Optimization of protein samples for NMR using thermal shift assays

    Energy Technology Data Exchange (ETDEWEB)

    Kozak, Sandra [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Lercher, Lukas; Karanth, Megha N. [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Meijers, Rob [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Carlomagno, Teresa, E-mail: teresa.carlomagno@oci.uni-hannover.de [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Boivin, Stephane, E-mail: sboivin77@hotmail.com, E-mail: s.boivin@embl-hamburg.de [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany)

    2016-04-15

    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor{sup ®} provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

  7. Exploring translocation of proteins on DNA by NMR

    International Nuclear Information System (INIS)

    Marius Clore, G.

    2011-01-01

    While an extensive body of knowledge has accumulated on the structures of transcription factors, DNA and their complexes from both NMR and crystallography, much less is known at a molecular level regarding the mechanisms whereby transcription factors locate their specific DNA target site within an overwhelming sea of non-specific DNA sites. Indirect kinetic data suggested that three processes are involved in the search procedure: jumping by dissociation of the protein from the DNA followed by re-association at another site, direct transfer from one DNA molecule or segment to another, and one-dimensional sliding. In this brief perspective I summarize recent NMR developments from our laboratory that have permitted direct characterization of the species and molecular mechanisms involved in the target search process, including the detection of highly transient sparsely-populated states. The main tool in these studies involves the application of paramagnetic relaxation enhancement, supplemented by z-exchange spectroscopy, lineshape analysis and residual dipolar couplings. These studies led to the first direct demonstration of rotation-coupled sliding of a protein along the DNA and the direct transfer of a protein from one DNA molecule to another without dissociating into free solution.

  8. Probabilistic validation of protein NMR chemical shift assignments

    International Nuclear Information System (INIS)

    Dashti, Hesam; Tonelli, Marco; Lee, Woonghee; Westler, William M.; Cornilescu, Gabriel; Ulrich, Eldon L.; Markley, John L.

    2016-01-01

    Data validation plays an important role in ensuring the reliability and reproducibility of studies. NMR investigations of the functional properties, dynamics, chemical kinetics, and structures of proteins depend critically on the correctness of chemical shift assignments. We present a novel probabilistic method named ARECA for validating chemical shift assignments that relies on the nuclear Overhauser effect data. ARECA has been evaluated through its application to 26 case studies and has been shown to be complementary to, and usually more reliable than, approaches based on chemical shift databases. ARECA is available online at http://areca.nmrfam.wisc.edu/ http://areca.nmrfam.wisc.edu/

  9. Probabilistic validation of protein NMR chemical shift assignments

    Energy Technology Data Exchange (ETDEWEB)

    Dashti, Hesam [University of Wisconsin-Madison, Graduate Program in Biophysics, Biochemistry Department (United States); Tonelli, Marco; Lee, Woonghee; Westler, William M.; Cornilescu, Gabriel [University of Wisconsin-Madison, Biochemistry Department, National Magnetic Resonance Facility at Madison (United States); Ulrich, Eldon L. [University of Wisconsin-Madison, BioMagResBank, Biochemistry Department (United States); Markley, John L., E-mail: markley@nmrfam.wisc.edu, E-mail: jmarkley@wisc.edu [University of Wisconsin-Madison, Biochemistry Department, National Magnetic Resonance Facility at Madison (United States)

    2016-01-15

    Data validation plays an important role in ensuring the reliability and reproducibility of studies. NMR investigations of the functional properties, dynamics, chemical kinetics, and structures of proteins depend critically on the correctness of chemical shift assignments. We present a novel probabilistic method named ARECA for validating chemical shift assignments that relies on the nuclear Overhauser effect data. ARECA has been evaluated through its application to 26 case studies and has been shown to be complementary to, and usually more reliable than, approaches based on chemical shift databases. ARECA is available online at http://areca.nmrfam.wisc.edu/ http://areca.nmrfam.wisc.edu/.

  10. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    International Nuclear Information System (INIS)

    Hayashi, Kokoro; Kojima, Chojiro

    2010-01-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in 1 H- 15 N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  11. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Kokoro [Fujifilm Corporation, Analysis Technology Center (Japan); Kojima, Chojiro, E-mail: kojima@protein.osaka-u.ac.j [Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences (Japan)

    2010-11-15

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in {sup 1}H-{sup 15}N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  12. [Non-invasive analysis of proteins in living cells using NMR spectroscopy].

    Science.gov (United States)

    Tochio, Hidehito; Murayama, Shuhei; Inomata, Kohsuke; Morimoto, Daichi; Ohno, Ayako; Shirakawa, Masahiro

    2015-01-01

    NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.

  13. A probabilistic approach for validating protein NMR chemical shift assignments

    International Nuclear Information System (INIS)

    Wang Bowei; Wang, Yunjun; Wishart, David S.

    2010-01-01

    It has been estimated that more than 20% of the proteins in the BMRB are improperly referenced and that about 1% of all chemical shift assignments are mis-assigned. These statistics also reflect the likelihood that any newly assigned protein will have shift assignment or shift referencing errors. The relatively high frequency of these errors continues to be a concern for the biomolecular NMR community. While several programs do exist to detect and/or correct chemical shift mis-referencing or chemical shift mis-assignments, most can only do one, or the other. The one program (SHIFTCOR) that is capable of handling both chemical shift mis-referencing and mis-assignments, requires the 3D structure coordinates of the target protein. Given that chemical shift mis-assignments and chemical shift re-referencing issues should ideally be addressed prior to 3D structure determination, there is a clear need to develop a structure-independent approach. Here, we present a new structure-independent protocol, which is based on using residue-specific and secondary structure-specific chemical shift distributions calculated over small (3-6 residue) fragments to identify mis-assigned resonances. The method is also able to identify and re-reference mis-referenced chemical shift assignments. Comparisons against existing re-referencing or mis-assignment detection programs show that the method is as good or superior to existing approaches. The protocol described here has been implemented into a freely available Java program called 'Probabilistic Approach for protein Nmr Assignment Validation (PANAV)' and as a web server (http://redpoll.pharmacy.ualberta.ca/PANAVhttp://redpoll.pharmacy.ualberta.ca/PANAV) which can be used to validate and/or correct as well as re-reference assigned protein chemical shifts.

  14. Effects of NMR spectral resolution on protein structure calculation.

    Directory of Open Access Journals (Sweden)

    Suhas Tikole

    Full Text Available Adequate digital resolution and signal sensitivity are two critical factors for protein structure determinations by solution NMR spectroscopy. The prime objective for obtaining high digital resolution is to resolve peak overlap, especially in NOESY spectra with thousands of signals where the signal analysis needs to be performed on a large scale. Achieving maximum digital resolution is usually limited by the practically available measurement time. We developed a method utilizing non-uniform sampling for balancing digital resolution and signal sensitivity, and performed a large-scale analysis of the effect of the digital resolution on the accuracy of the resulting protein structures. Structure calculations were performed as a function of digital resolution for about 400 proteins with molecular sizes ranging between 5 and 33 kDa. The structural accuracy was assessed by atomic coordinate RMSD values from the reference structures of the proteins. In addition, we monitored also the number of assigned NOESY cross peaks, the average signal sensitivity, and the chemical shift spectral overlap. We show that high resolution is equally important for proteins of every molecular size. The chemical shift spectral overlap depends strongly on the corresponding spectral digital resolution. Thus, knowing the extent of overlap can be a predictor of the resulting structural accuracy. Our results show that for every molecular size a minimal digital resolution, corresponding to the natural linewidth, needs to be achieved for obtaining the highest accuracy possible for the given protein size using state-of-the-art automated NOESY assignment and structure calculation methods.

  15. Temperature-induced transitions in disordered proteins probed by NMR spectroscopy

    DEFF Research Database (Denmark)

    Kjærgaard, Magnus; Poulsen, Flemming Martin; Kragelund, Birthe Brandt

    2012-01-01

    Intrinsically disordered proteins are abundant in nature and perform many important physiological functions. Multidimensional NMR spectroscopy has been crucial for the understanding of the conformational properties of disordered proteins and is increasingly used to probe their conformational...... ensembles. Compared to folded proteins, disordered proteins are more malleable and more easily perturbed by environmental factors. Accordingly, the experimental conditions and especially the temperature modify the structural and functional properties of disordered proteins. NMR spectroscopy allows analysis...... of temperature-induced structural changes at residue resolution using secondary chemical shift analysis, paramagnetic relaxation enhancement, and residual dipolar couplings. This chapter discusses practical aspects of NMR studies of temperature-induced structural changes in disordered proteins....

  16. Metal binding proteins and antifreeze proteins in the beetle Tenebrio molitor: a study on possible competition for the semi-essential amino acid cysteine

    OpenAIRE

    Pedersen, Sindre Andre

    2007-01-01

    In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are ...

  17. NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

    Directory of Open Access Journals (Sweden)

    Lola A. Brown

    2015-04-01

    Full Text Available Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2 is mediated by Gag’s N-terminally myristylated matrix (MA domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV, a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S. These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  18. NMR structure of the myristylated feline immunodeficiency virus matrix protein.

    Science.gov (United States)

    Brown, Lola A; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G; Kuo, Lillian; Freed, Eric O; Summers, Michael F

    2015-04-30

    Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag's N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  19. An open source cryostage and software analysis method for detection of antifreeze activity

    DEFF Research Database (Denmark)

    Lørup Buch, Johannes; Ramløv, H

    2016-01-01

    AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage.......The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method...

  20. PDBStat: a universal restraint converter and restraint analysis software package for protein NMR

    International Nuclear Information System (INIS)

    Tejero, Roberto; Snyder, David; Mao, Binchen; Aramini, James M.; Montelione, Gaetano T.

    2013-01-01

    The heterogeneous array of software tools used in the process of protein NMR structure determination presents organizational challenges in the structure determination and validation processes, and creates a learning curve that limits the broader use of protein NMR in biology. These challenges, including accurate use of data in different data formats required by software carrying out similar tasks, continue to confound the efforts of novices and experts alike. These important issues need to be addressed robustly in order to standardize protein NMR structure determination and validation. PDBStat is a C/C++ computer program originally developed as a universal coordinate and protein NMR restraint converter. Its primary function is to provide a user-friendly tool for interconverting between protein coordinate and protein NMR restraint data formats. It also provides an integrated set of computational methods for protein NMR restraint analysis and structure quality assessment, relabeling of prochiral atoms with correct IUPAC names, as well as multiple methods for analysis of the consistency of atomic positions indicated by their convergence across a protein NMR ensemble. In this paper we provide a detailed description of the PDBStat software, and highlight some of its valuable computational capabilities. As an example, we demonstrate the use of the PDBStat restraint converter for restrained CS-Rosetta structure generation calculations, and compare the resulting protein NMR structure models with those generated from the same NMR restraint data using more traditional structure determination methods. These results demonstrate the value of a universal restraint converter in allowing the use of multiple structure generation methods with the same restraint data for consensus analysis of protein NMR structures and the underlying restraint data

  1. PDBStat: a universal restraint converter and restraint analysis software package for protein NMR

    Energy Technology Data Exchange (ETDEWEB)

    Tejero, Roberto [Rutgers, The State University of New Jersey, Center for Advanced Biotechnology and Medicine (United States); Snyder, David [William Paterson University, Department of Chemistry (United States); Mao, Binchen; Aramini, James M.; Montelione, Gaetano T., E-mail: guy@cabm.rutgers.edu [Rutgers, The State University of New Jersey, Center for Advanced Biotechnology and Medicine (United States)

    2013-08-15

    The heterogeneous array of software tools used in the process of protein NMR structure determination presents organizational challenges in the structure determination and validation processes, and creates a learning curve that limits the broader use of protein NMR in biology. These challenges, including accurate use of data in different data formats required by software carrying out similar tasks, continue to confound the efforts of novices and experts alike. These important issues need to be addressed robustly in order to standardize protein NMR structure determination and validation. PDBStat is a C/C++ computer program originally developed as a universal coordinate and protein NMR restraint converter. Its primary function is to provide a user-friendly tool for interconverting between protein coordinate and protein NMR restraint data formats. It also provides an integrated set of computational methods for protein NMR restraint analysis and structure quality assessment, relabeling of prochiral atoms with correct IUPAC names, as well as multiple methods for analysis of the consistency of atomic positions indicated by their convergence across a protein NMR ensemble. In this paper we provide a detailed description of the PDBStat software, and highlight some of its valuable computational capabilities. As an example, we demonstrate the use of the PDBStat restraint converter for restrained CS-Rosetta structure generation calculations, and compare the resulting protein NMR structure models with those generated from the same NMR restraint data using more traditional structure determination methods. These results demonstrate the value of a universal restraint converter in allowing the use of multiple structure generation methods with the same restraint data for consensus analysis of protein NMR structures and the underlying restraint data.

  2. Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

    International Nuclear Information System (INIS)

    Zhou, Donghua H.; Nieuwkoop, Andrew J.; Berthold, Deborah A.; Comellas, Gemma; Sperling, Lindsay J.; Tang, Ming; Shah, Gautam J.; Brea, Elliott J.; Lemkau, Luisel R.; Rienstra, Chad M.

    2012-01-01

    Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.

  3. Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques

    DEFF Research Database (Denmark)

    Calcutta, Antonello; Jessen, Christian M; Behrens, Manja Annette

    2012-01-01

    induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl ß-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010......)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows...

  4. (3,2)D GFT-NMR experiments for fast data collection from proteins

    International Nuclear Information System (INIS)

    Xia Youlin; Zhu Guang; Veeraraghavan, Sudha; Gao Xiaolian

    2004-01-01

    High throughput structure determination of proteins will contribute to the success of proteomics investigations. The G-Matrix Fourier Transformation NMR (GFT-NMR) method significantly shortens experimental time by reducing the number of the dimensions of data acquisition for isotopically labeled proteins (Kim, S. and Szyperski, T. (2003) J. Am. Chem. Soc.125, 1385). We demonstrate herein a suite of ten 3D → 2D or (3,2)D GFT-NMR experiments using 13 C/ 15 N-labeled ubiquitin. These experiments were completed within 18 hours, representing a 4- to 18-fold reduction in data acquisition time compared to the corresponding conventional 3D experiments. A subset of the GFT-NMR experiments, (3,2)D HNCO, HNCACB, HN(CO)CACB, and 2D 1 H- 15 N HSQC, which are necessary for backbone assignments, were carried out within 6 hours. To facilitate the analysis of the GFT-NMR spectra, we developed automated procedures for viewing and analyzing the GFT-NMR spectra. Our overall strategy allows (3,2)D GFT-NMR experiments to be readily performed and analyzed. Nevertheless, the increase in spectral overlap and the reduction in signal sensitivity in these fast NMR experiments presently limit their application to relatively small proteins

  5. In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

    International Nuclear Information System (INIS)

    Sakai, Tomomi; Tochio, Hidehito; Tenno, Takeshi; Ito, Yutaka; Kokubo, Tetsuro; Hiroaki, Hidekazu; Shirakawa, Masahiro

    2006-01-01

    In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. 15 N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional 1 H- 15 N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein-protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions

  6. The eigenmode perspective of NMR spin relaxation in proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shapiro, Yury E., E-mail: shapiro@nmrsgi4.ls.biu.ac.il, E-mail: eva.meirovitch@biu.ac.il; Meirovitch, Eva, E-mail: shapiro@nmrsgi4.ls.biu.ac.il, E-mail: eva.meirovitch@biu.ac.il [The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900-02 (Israel)

    2013-12-14

    We developed in recent years the two-body (protein and probe) coupled-rotator slowly relaxing local structure (SRLS) approach for elucidating protein dynamics from NMR spin relaxation. So far we used as descriptors the set of physical parameters that enter the SRLS model. They include the global (protein-related) diffusion tensor, D{sub 1}, the local (probe-related) diffusion tensor, D{sub 2}, and the local coupling/ordering potential, u. As common in analyzes based on mesoscopic dynamic models, these parameters have been determined with data-fitting techniques. In this study, we describe structural dynamics in terms of the eigenmodes comprising the SRLS time correlation functions (TCFs) generated by using the best-fit parameters as input to the Smoluchowski equation. An eigenmode is a weighted exponential with decay constant given by an eigenvalue of the Smoluchowski operator, and weighting factor determined by the corresponding eigenvector. Obviously, both quantities depend on the SRLS parameters as determined by the SRLS model. Unlike the set of best-fit parameters, the eigenmodes represent patterns of motion of the probe-protein system. The following new information is obtained for the typical probe, the {sup 15}N−{sup 1}H bond. Two eigenmodes, associated with the protein and the probe, dominate when the time scale separation is large (i.e., D{sub 2} ≫ D{sub 1}), the tensorial properties are simple, and the local potential is either very strong or very weak. When the potential exceeds these limits while the remaining conditions are preserved, new eigenmodes arise. The multi-exponentiality of the TCFs is associated in this case with the restricted nature of the local motion. When the time scale separation is no longer large, the rotational degrees of freedom of the protein and the probe become statistically dependent (coupled dynamically). The multi-exponentiality of the TCFs is associated in this case with the restricted nature of both the local and the

  7. Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a

    International Nuclear Information System (INIS)

    Zuiderweg, E.R.P.; Fesik, S.W.

    1989-01-01

    The utility of three-dimensional heteronuclear NMR spectroscopy for the assignment of 1 H and 15 N resonances of the inflammatory protein C5a (MW 8500), uniformly labeled with 15 N, is demonstrated at a protein concentration of 0.7 mM. It is shown that dramatic simplification of the 2D nuclear Overhauser effect spectrum (NOESY) is obtained by editing with respect to the frequency of the 15 N heteronucleus in a third dimension. The improved resolution in the 3D experiment largely facilitates the assignment of protein NMR spectra and allows for the determination of distance constraints from otherwise overlapping NOE cross peaks for purposes of 3D structure determination. The results show that 15 N heteronuclear 3D NMR can facilitate the structure determination of small proteins and promises to be a useful tool for the study of larger systems that cannot be studied by conventional 2D NMR techniques

  8. Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a

    Energy Technology Data Exchange (ETDEWEB)

    Zuiderweg, E.R.P.; Fesik, S.W. (Abbott Laboratories, Abbott Park, IL (USA))

    1989-03-21

    The utility of three-dimensional heteronuclear NMR spectroscopy for the assignment of {sup 1}H and {sup 15}N resonances of the inflammatory protein C5a (MW 8500), uniformly labeled with {sup 15}N, is demonstrated at a protein concentration of 0.7 mM. It is shown that dramatic simplification of the 2D nuclear Overhauser effect spectrum (NOESY) is obtained by editing with respect to the frequency of the {sup 15}N heteronucleus in a third dimension. The improved resolution in the 3D experiment largely facilitates the assignment of protein NMR spectra and allows for the determination of distance constraints from otherwise overlapping NOE cross peaks for purposes of 3D structure determination. The results show that {sup 15}N heteronuclear 3D NMR can facilitate the structure determination of small proteins and promises to be a useful tool for the study of larger systems that cannot be studied by conventional 2D NMR techniques.

  9. Non-Uniform Sampling and J-UNIO Automation for Efficient Protein NMR Structure Determination.

    Science.gov (United States)

    Didenko, Tatiana; Proudfoot, Andrew; Dutta, Samit Kumar; Serrano, Pedro; Wüthrich, Kurt

    2015-08-24

    High-resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non-uniform sampling (NUS) of 3D heteronuclear-resolved [(1)H,(1)H]-NOESY data yielded two- to three-fold savings of instrument time for structure determinations of soluble proteins. With the 152-residue protein NP_372339.1 from Staphylococcus aureus and the 71-residue protein NP_346341.1 from Streptococcus pneumonia we show that high-quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. NMR studies of a new family of DNA binding proteins: the THAP proteins

    International Nuclear Information System (INIS)

    Gervais, Virginie; Campagne, Sébastien; Durand, Jade; Muller, Isabelle; Milon, Alain

    2013-01-01

    The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

  11. NMR studies of a new family of DNA binding proteins: the THAP proteins

    Energy Technology Data Exchange (ETDEWEB)

    Gervais, Virginie, E-mail: virginie.gervais@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France); Campagne, Sebastien [ETH Zurich (Switzerland); Durand, Jade; Muller, Isabelle; Milon, Alain, E-mail: alain.milon@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France)

    2013-05-15

    The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

  12. Uniform and selective deuteration in two-dimensional NMR of proteins

    International Nuclear Information System (INIS)

    LeMaster, D.M.

    1990-01-01

    This paper reports on the practicality of isotopic labeling, particularly deuteration, that has received considerable impetus from advances in molecular biology, which have allowed ready production of NMR quantities of labeled proteins. Protein expression in Escherichia coli allows use of the considerable metabolic genetics known for the organism in shaping the biosynthetic process to meet the labeling demands of the NMR experiments. In addition to deuteration's common use in spectral assignment problems, it also offers considerable potential for enhancing the quality of the nuclear Overhauser effect (NOE) distance and dihedral angle constraints used for solution structural analysis of proteins. Recent reviews emphasize the sample preparation and spectral benefits of protein deuteration

  13. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

    International Nuclear Information System (INIS)

    Clark, Lindsay; Zahm, Jacob A.; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M.; Gardner, Kevin H.; Rosen, Michael K.; Rosenbaum, Daniel M.

    2015-01-01

    13 C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13 C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13 C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets

  14. Efficiency of Composite Binders with Antifreezing Agents

    Science.gov (United States)

    Ogurtsova, Y. N.; Zhernovsky, I. V.; Botsman, L. N.

    2017-11-01

    One of the non-heating methods of cold-weather concreting is using concretes hardening at negative temperatures. This method consists in using chemical additives which reduce the freezing temperature of the liquid phase and provide for concrete hardening at negative temperatures. The non-heating cold-weather concreting, due to antifreezing agents, allows saving heat and electric energy at the more flexible work performance technology. At selecting the antifreezing components, the possibility of concreting at temperatures up to minus 20 °C and combination with a plasticizer contained in the composite binder were taken into account. The optimal proportions of antifreezing and complex agents produced by MC-Bauchemie Russia for fine-grained concretes were determined. So, the introduction of antifreezing and complex agents allows obtaining a structure of composite characteristic for cement stone in the conditions of below zero temperatures at using different binders; the hydration of such composite proceeded naturally. Low-water-demand binders (LWDB) based composites are characterized by a higher density and homogeneity due to a high dispersity of a binder and its complicated surface providing for a lot of crystallization centers. LWDB contains small pores keeping water in a liquid form and promoting a more complete hydration process.

  15. Modified Antifreeze Liquids for Use on Surfaces

    Science.gov (United States)

    Lynn, R. O.

    1983-01-01

    Report presents results of evaluation of two antifreeze liquids, dimethyl sulfoxide and ethylene glycol and five viscosity modifiers: gelatin, gum tragacanth, starch, agarose powder and citrus pectin. Purpose of evaluation to find best way of dealing with frost formation on Space Shuttle.

  16. Introduction to the conformational investigation of peptides and proteins by using two-dimensional proton NMR experiments

    International Nuclear Information System (INIS)

    Neumann, J.M.; Macquaire, F.

    1991-01-01

    This report presents the elementary bases for an initiation to the conformational study of peptides and proteins by using two-dimensional proton NMR experiments. First, some general features of protein structures are summarized. A second chapter is devoted to the basic NMR experiments and to the spectral parameters which provide a structural information. This description is illustrated by NMR spectra of peptides. The third chapter concerns the most standard two-dimensional proton NMR experiments and their use for a conformational study of peptides and proteins. Lastly, an example of NMR structural investigation of a peptide is reported [fr

  17. Structures of larger proteins in solution: Three- and four-dimensional heteronuclear NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gronenborn, A.M.; Clore, G.M. [National Institutes of Health, Bethesda, MD (United States)

    1994-12-01

    Complete understanding of a protein`s function and mechanism of action can only be achieved with a knowledge of its three-dimensional structure at atomic resolution. At present, there are two methods available for determining such structures. The first method, which has been established for many years, is x-ray diffraction of protein single crystals. The second method has blossomed only in the last 5 years and is based on the application of nuclear magnetic resonance (NMR) spectroscopy to proteins in solution. This review paper describes three- and four-dimensional NMR methods applied to protein structure determination and was adapted from Clore and Gronenborn. The review focuses on the underlying principals and practice of multidimensional NMR and the structural information obtained.

  18. Comparison of NMR and crystal structures for the proteins TM1112 and TM1367

    International Nuclear Information System (INIS)

    Mohanty, Biswaranjan; Serrano, Pedro; Pedrini, Bill; Jaudzems, Kristaps; Geralt, Michael; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

    2010-01-01

    NMR structures of the proteins TM1112 and TM1367 solved by the JCSG in solution at 298 K could be superimposed with the corresponding crystal structures at 100 K with r.m.s.d. values of <1.0 Å for the backbone heavy atoms. For both proteins the structural differences between multiple molecules in the asymmetric unit of the crystals correlated with structural variations within the bundles of conformers used to represent the NMR solution structures. A recently introduced JCSG NMR structure-determination protocol, which makes use of the software package UNIO for extensive automation, was further evaluated by comparison of the TM1112 structure obtained using these automated methods with another NMR structure that was independently solved in another PSI center, where a largely interactive approach was applied. The NMR structures of the TM1112 and TM1367 proteins from Thermotoga maritima in solution at 298 K were determined following a new protocol which uses the software package UNIO for extensive automation. The results obtained with this novel procedure were evaluated by comparison with the crystal structures solved by the JCSG at 100 K to 1.83 and 1.90 Å resolution, respectively. In addition, the TM1112 solution structure was compared with an NMR structure solved by the NESG using a conventional largely interactive methodology. For both proteins, the newly determined NMR structure could be superimposed with the crystal structure with r.m.s.d. values of <1.0 Å for the backbone heavy atoms, which provided a starting platform to investigate local structure variations, which may arise from either the methods used or from the different chemical environments in solution and in the crystal. Thereby, these comparative studies were further explored with the use of reference NMR and crystal structures, which were computed using the NMR software with input of upper-limit distance constraints derived from the molecular models that represent the results of structure

  19. Protein-observed (19)F-NMR for fragment screening, affinity quantification and druggability assessment.

    Science.gov (United States)

    Gee, Clifford T; Arntson, Keith E; Urick, Andrew K; Mishra, Neeraj K; Hawk, Laura M L; Wisniewski, Andrea J; Pomerantz, William C K

    2016-08-01

    NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed 'fragments'; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed (19)F-NMR (PrOF NMR) using (19)F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as Kd determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.

  20. The second round of Critical Assessment of Automated Structure Determination of Proteins by NMR: CASD-NMR-2013

    International Nuclear Information System (INIS)

    Rosato, Antonio; Vranken, Wim; Fogh, Rasmus H.; Ragan, Timothy J.; Tejero, Roberto; Pederson, Kari; Lee, Hsiau-Wei; Prestegard, James H.; Yee, Adelinda; Wu, Bin; Lemak, Alexander; Houliston, Scott; Arrowsmith, Cheryl H.; Kennedy, Michael; Acton, Thomas B.; Xiao, Rong; Liu, Gaohua; Montelione, Gaetano T.; Vuister, Geerten W.

    2015-01-01

    The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100 % of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90 % of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged

  1. The second round of Critical Assessment of Automated Structure Determination of Proteins by NMR: CASD-NMR-2013

    Energy Technology Data Exchange (ETDEWEB)

    Rosato, Antonio [University of Florence, Department of Chemistry and Magnetic Resonance Center (Italy); Vranken, Wim [Vrije Universiteit Brussel, Structural Biology Brussels (Belgium); Fogh, Rasmus H.; Ragan, Timothy J. [University of Leicester, Department of Biochemistry, School of Biological Sciences (United Kingdom); Tejero, Roberto [Universidad de Valencia, Departamento de Química Física (Spain); Pederson, Kari; Lee, Hsiau-Wei; Prestegard, James H. [University of Georgia, Complex Carbohydrate Research Center and Northeast Structural Genomics Consortium (United States); Yee, Adelinda; Wu, Bin; Lemak, Alexander; Houliston, Scott; Arrowsmith, Cheryl H. [University of Toronto, Department of Medical Biophysics, Cancer Genomics and Proteomics, Ontario Cancer Institute, Northeast Structural Genomics Consortium (Canada); Kennedy, Michael [Miami University, Department of Chemistry and Biochemistry, Northeast Structural Genomics Consortium (United States); Acton, Thomas B.; Xiao, Rong; Liu, Gaohua; Montelione, Gaetano T., E-mail: guy@cabm.rutgers.edu [The State University of New Jersey, Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Northeast Structural Genomics Consortium, Rutgers (United States); Vuister, Geerten W., E-mail: gv29@le.ac.uk [University of Leicester, Department of Biochemistry, School of Biological Sciences (United Kingdom)

    2015-08-15

    The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100 % of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90 % of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged.

  2. Algal autolysate medium to label proteins for NMR in mammalian cells.

    Science.gov (United States)

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  3. Algal autolysate medium to label proteins for NMR in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia [University of Florence, Magnetic Resonance Center (CERM) (Italy); Neri, Sara [Giotto Biotech S.R.L. (Italy); Fragai, Marco, E-mail: fragai@cerm.unifi.it [University of Florence, Magnetic Resonance Center (CERM) (Italy)

    2016-04-15

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in {sup 15}N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  4. Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures

    Science.gov (United States)

    2014-01-01

    We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5–22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10–25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function. PMID:24392845

  5. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    Science.gov (United States)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  6. CASD-NMR 2: robust and accurate unsupervised analysis of raw NOESY spectra and protein structure determination with UNIO

    International Nuclear Information System (INIS)

    Guerry, Paul; Duong, Viet Dung; Herrmann, Torsten

    2015-01-01

    UNIO is a comprehensive software suite for protein NMR structure determination that enables full automation of all NMR data analysis steps involved—including signal identification in NMR spectra, sequence-specific backbone and side-chain resonance assignment, NOE assignment and structure calculation. Within the framework of the second round of the community-wide stringent blind NMR structure determination challenge (CASD-NMR 2), we participated in two categories of CASD-NMR 2, namely using either raw NMR spectra or unrefined NOE peak lists as input. A total of 15 resulting NMR structure bundles were submitted for 9 out of 10 blind protein targets. All submitted UNIO structures accurately coincided with the corresponding blind targets as documented by an average backbone root mean-square deviation to the reference proteins of only 1.2 Å. Also, the precision of the UNIO structure bundles was virtually identical to the ensemble of reference structures. By assessing the quality of all UNIO structures submitted to the two categories, we find throughout that only the UNIO–ATNOS/CANDID approach using raw NMR spectra consistently yielded structure bundles of high quality for direct deposition in the Protein Data Bank. In conclusion, the results obtained in CASD-NMR 2 are another vital proof for robust, accurate and unsupervised NMR data analysis by UNIO for real-world applications

  7. Facilitated assignment of large protein NMR signals with covariance sequential spectra using spectral derivatives.

    Science.gov (United States)

    Harden, Bradley J; Nichols, Scott R; Frueh, Dominique P

    2014-09-24

    Nuclear magnetic resonance (NMR) studies of larger proteins are hampered by difficulties in assigning NMR resonances. Human intervention is typically required to identify NMR signals in 3D spectra, and subsequent procedures depend on the accuracy of this so-called peak picking. We present a method that provides sequential connectivities through correlation maps constructed with covariance NMR, bypassing the need for preliminary peak picking. We introduce two novel techniques to minimize false correlations and merge the information from all original 3D spectra. First, we take spectral derivatives prior to performing covariance to emphasize coincident peak maxima. Second, we multiply covariance maps calculated with different 3D spectra to destroy erroneous sequential correlations. The maps are easy to use and can readily be generated from conventional triple-resonance experiments. Advantages of the method are demonstrated on a 37 kDa nonribosomal peptide synthetase domain subject to spectral overlap.

  8. Identifying secondary structures in proteins using NMR chemical shift 3D correlation maps

    Science.gov (United States)

    Kumari, Amrita; Dorai, Kavita

    2013-06-01

    NMR chemical shifts are accurate indicators of molecular environment and have been extensively used as aids in protein structure determination. This work focuses on creating empirical 3D correlation maps of backbone chemical shift nuclei for use as identifiers of secondary structure elements in proteins. A correlated database of backbone nuclei chemical shifts was constructed from experimental structural data gathered from entries in the Protein Data Bank (PDB) as well as isotropic chemical shift values from the RefDB database. Rigorous statistical analysis of the maps led to the conclusion that specific correlations between triplets of backbone chemical shifts are best able to differentiate between different secondary structures such as α-helices, β-strands and turns. The method is compared with similar techniques that use NMR chemical shift information as aids in biomolecular structure determination and performs well in tests done on experimental data determined for different types of proteins, including large multi-domain proteins and membrane proteins.

  9. An approach for high-throughput structure determination of proteins by NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Medek, Ales; Olejniczak, Edward T.; Meadows, Robert P.; Fesik, Stephen W. [Abbott Laboratories, Pharmaceutical Discovery Division (United States)

    2000-11-15

    An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing {sup 13}C-methyl labeled Val, Leu, and Ile ({delta}1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the hydrophobic core and overall fold of the protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acquisition. The approach is demonstrated by determining the overall fold of the antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refinement of the Bcl-xL structure to a backbone rmsd of 0.95 A was accomplished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data suggests that our method will allow the global folds of a wide variety of proteins to be determined.

  10. Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites

    International Nuclear Information System (INIS)

    Serrano, Pedro; Pedrini, Bill; Geralt, Michael; Jaudzems, Kristaps; Mohanty, Biswaranjan; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

    2010-01-01

    Tools for systematic comparisons of NMR and crystal structures developed by the JCSG were applied to two proteins with known functions: the T. maritima anti-σ factor antagonist TM1081 and the mouse γ-glutamylamine cyclotransferase A2LD1 (gi:13879369). In an attempt to exploit the complementarity of crystal and NMR data, the combined use of the two structure-determination techniques was explored for the initial steps in the challenge of searching proteins of unknown functions for putative active sites. The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Å resolution and at 100 K and 1.9 Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the

  11. Ring current shifts in {sup 19}F-NMR of membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Dongsheng, E-mail: liudsh@shanghaitech.edu.cn; Wüthrich, Kurt, E-mail: kwuthrich@shanghaitech.edu.cn [ShanghaiTech University, iHuman Institute (China)

    2016-05-15

    Fluorine-19 NMR markers are attractive reporter groups for use in studies of complex biomacromolecular systems, in particular also for studies of function-related conformational equilibria and rate processes in membrane proteins. Advantages of {sup 19}F-NMR probes include high sensitivity of the {sup 19}F chemical shifts to variations in the non-covalent environment. Nonetheless, in studies of G protein-coupled receptors (GPCR) we encountered situations where {sup 19}F chemical shifts were not responsive to conformational changes that had been implicated by other methods. This prompted us to examine possible effects of aromatic ring current fields on the chemical shifts of {sup 19}F-NMR probes used in GPCRs. Analysis of previously reported {sup 19}F-NMR data on the β{sub 2}-adrenergic receptor and mammalian rhodopsin showed that all {sup 19}F-labeling sites which manifested conformational changes are located near aromatic residues. Although ring current effects are small when compared to other known non-covalent effects on {sup 19}F chemical shifts, there is thus an indication that their contributions are significant when studying activation processes in GPCRs, since the observed activation-related {sup 19}F-NMR chemical shifts are comparable in size to the calculated ring current shifts. Considering the impact of ring current shifts may thus be helpful in identifying promising indigenous or engineered labeling sites for future {sup 19}F-NMR studies of GPCR activation, and novel information may be obtained on the nature of conformational rearrangements near the {sup 19}F-labels. It will then also be interesting to see if the presently indicated role of ring current shifts in membrane protein studies with {sup 19}F-NMR markers can be substantiated by a more extensive data base resulting from future studies.

  12. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra

    KAUST Repository

    Cannistraci, Carlo Vittorio

    2015-01-26

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet\\'s performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis.

  13. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra

    KAUST Repository

    Cannistraci, Carlo Vittorio; Abbas, Ahmed; Gao, Xin

    2015-01-01

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet's performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis.

  14. Practical aspects of NMR signal assignment in larger and challenging proteins

    Science.gov (United States)

    Frueh, Dominique P.

    2014-01-01

    NMR has matured into a technique routinely employed for studying proteins in near physiological conditions. However, applications to larger proteins are impeded by the complexity of the various correlation maps necessary to assign NMR signals. This article reviews the data analysis techniques traditionally employed for resonance assignment and describes alternative protocols necessary for overcoming challenges in large protein spectra. In particular, simultaneous analysis of multiple spectra may help overcome ambiguities or may reveal correlations in an indirect manner. Similarly, visualization of orthogonal planes in a multidimensional spectrum can provide alternative assignment procedures. We describe examples of such strategies for assignment of backbone, methyl, and nOe resonances. We describe experimental aspects of data acquisition for the related experiments and provide guidelines for preliminary studies. Focus is placed on large folded monomeric proteins and examples are provided for 37, 48, 53, and 81 kDa proteins. PMID:24534088

  15. REDOR NMR of stable-isotope-labeled protein binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Schaefer, J. [Washington Univ., St. Louis, MO (United States)

    1994-12-01

    Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.

  16. Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra

    International Nuclear Information System (INIS)

    Emami, Sanaz; Fan Ying; Munro, Rachel; Ladizhansky, Vladimir; Brown, Leonid S.

    2013-01-01

    One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a homogeneous natively folded sample giving high spectral resolution sufficient for structural studies. Eukaryotic membrane proteins are especially difficult and expensive targets in this respect. Methylotrophic yeast Pichia pastoris is a reliable producer of eukaryotic membrane proteins for crystallography and a promising economical source of isotopically labeled proteins for NMR. We show that eukaryotic membrane protein human aquaporin 1 can be doubly ( 13 C/ 15 N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.

  17. Blind testing of routine, fully automated determination of protein structures from NMR data.

    NARCIS (Netherlands)

    Rosato, A.; Aramini, J.M.; Arrowsmith, C.; Bagaria, A.; Baker, D.; Cavalli, A.; Doreleijers, J.; Eletsky, A.; Giachetti, A.; Guerry, P.; Gutmanas, A.; Guntert, P.; He, Y.; Herrmann, T.; Huang, Y.J.; Jaravine, V.; Jonker, H.R.; Kennedy, M.A.; Lange, O.F.; Liu, G.; Malliavin, T.E.; Mani, R.; Mao, B.; Montelione, G.T.; Nilges, M.; Rossi, P.; Schot, G. van der; Schwalbe, H.; Szyperski, T.A.; Vendruscolo, M.; Vernon, R.; Vranken, W.F.; Vries, S.D. de; Vuister, G.W.; Wu, B.; Yang, Y.; Bonvin, A.M.

    2012-01-01

    The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by

  18. Blind Testing of Routine, Fully Automated Determination of Protein Structures from NMR Data

    NARCIS (Netherlands)

    Rosato, A.; Aramini, J.M.; van der Schot, G.; de Vries, S.J.|info:eu-repo/dai/nl/304837717; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238

    2012-01-01

    The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by

  19. A novel strategy for NMR resonance assignment and protein structure determination

    International Nuclear Information System (INIS)

    Lemak, Alexander; Gutmanas, Aleksandras; Chitayat, Seth; Karra, Murthy; Farès, Christophe; Sunnerhagen, Maria; Arrowsmith, Cheryl H.

    2011-01-01

    The quality of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy is contingent on the number and quality of experimentally-derived resonance assignments, distance and angular restraints. Two key features of protein NMR data have posed challenges for the routine and automated structure determination of small to medium sized proteins; (1) spectral resolution – especially of crowded nuclear Overhauser effect spectroscopy (NOESY) spectra, and (2) the reliance on a continuous network of weak scalar couplings as part of most common assignment protocols. In order to facilitate NMR structure determination, we developed a semi-automated strategy that utilizes non-uniform sampling (NUS) and multidimensional decomposition (MDD) for optimal data collection and processing of selected, high resolution multidimensional NMR experiments, combined it with an ABACUS protocol for sequential and side chain resonance assignments, and streamlined this procedure to execute structure and refinement calculations in CYANA and CNS, respectively. Two graphical user interfaces (GUIs) were developed to facilitate efficient analysis and compilation of the data and to guide automated structure determination. This integrated method was implemented and refined on over 30 high quality structures of proteins ranging from 5.5 to 16.5 kDa in size.

  20. A combined rheology and time domain NMR approach for determining water distributions in protein blends

    NARCIS (Netherlands)

    Dekkers, Birgit L.; Kort, de Daan W.; Grabowska, Katarzyna J.; Tian, Bei; As, Van Henk; Goot, van der Atze Jan

    2016-01-01

    We present a combined time domain NMR and rheology approach to quantify the water distribution in a phase separated protein blend. The approach forms the basis for a new tool to assess the microstructural properties of phase separated biopolymer blends, making it highly relevant for many food and

  1. NMR assignments of juvenile hormone binding protein in complex with JH III.

    Science.gov (United States)

    Suzuki, Rintaro; Tase, Akira; Fujimoto, Zui; Shiotsuki, Takahiro; Yamazaki, Toshimasa

    2009-06-01

    A hemolymph juvenile hormone binding protein (JHBP) shuttles hydrophobic JH, a key hormone in regulation of the insect life cycle, from the site of the JH biosynthesis to the cells of target organs. We report complete NMR chemical shift assignments of Bombyx mori JHBP in the JH III-bound state.

  2. Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

    NARCIS (Netherlands)

    Peters, Fabian; Maestre-Martinez, M.; Leonov, A.; Kovacic, L.; Becker, S.; Boelens, R.; Griesinger, C.

    2011-01-01

    Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large

  3. A robust algorithm for optimizing protein structures with NMR chemical shifts

    Energy Technology Data Exchange (ETDEWEB)

    Berjanskii, Mark; Arndt, David; Liang, Yongjie; Wishart, David S., E-mail: david.wishart@ualberta.ca [University of Alberta, Department of Computing Science (Canada)

    2015-11-15

    Over the past decade, a number of methods have been developed to determine the approximate structure of proteins using minimal NMR experimental information such as chemical shifts alone, sparse NOEs alone or a combination of comparative modeling data and chemical shifts. However, there have been relatively few methods that allow these approximate models to be substantively refined or improved using the available NMR chemical shift data. Here, we present a novel method, called Chemical Shift driven Genetic Algorithm for biased Molecular Dynamics (CS-GAMDy), for the robust optimization of protein structures using experimental NMR chemical shifts. The method incorporates knowledge-based scoring functions and structural information derived from NMR chemical shifts via a unique combination of multi-objective MD biasing, a genetic algorithm, and the widely used XPLOR molecular modelling language. Using this approach, we demonstrate that CS-GAMDy is able to refine and/or fold models that are as much as 10 Å (RMSD) away from the correct structure using only NMR chemical shift data. CS-GAMDy is also able to refine of a wide range of approximate or mildly erroneous protein structures to more closely match the known/correct structure and the known/correct chemical shifts. We believe CS-GAMDy will allow protein models generated by sparse restraint or chemical-shift-only methods to achieve sufficiently high quality to be considered fully refined and “PDB worthy”. The CS-GAMDy algorithm is explained in detail and its performance is compared over a range of refinement scenarios with several commonly used protein structure refinement protocols. The program has been designed to be easily installed and easily used and is available at http://www.gamdy.ca http://www.gamdy.ca.

  4. Describing intrinsically disordered proteins at atomic resolution by NMR

    International Nuclear Information System (INIS)

    Ringkjobing Jensen, Malene; Blackledge, Martin; Ruigrok, Rob WH

    2013-01-01

    There is growing interest in the development of physical methods to study the conformational behaviour and biological activity of intrinsically disordered proteins (IDPs). In this review recent advances in the elucidation of quantitative descriptions of disordered proteins from nuclear magnetic resonance spectroscopy are presented. Ensemble approaches are particularly well adapted to map the conformational energy landscape sampled by the protein at atomic resolution. Significant advances in development of calibrated approaches to the statistical representation of the conformational behaviour of IDPs are presented, as well as applications to some biologically important systems where disorder plays a crucial role. (authors)

  5. NMRNet: A deep learning approach to automated peak picking of protein NMR spectra.

    Science.gov (United States)

    Klukowski, Piotr; Augoff, Michal; Zieba, Maciej; Drwal, Maciej; Gonczarek, Adam; Walczak, Michal J

    2018-03-14

    Automated selection of signals in protein NMR spectra, known as peak picking, has been studied for over 20 years, nevertheless existing peak picking methods are still largely deficient. Accurate and precise automated peak picking would accelerate the structure calculation, and analysis of dynamics and interactions of macromolecules. Recent advancement in handling big data, together with an outburst of machine learning techniques, offer an opportunity to tackle the peak picking problem substantially faster than manual picking and on par with human accuracy. In particular, deep learning has proven to systematically achieve human-level performance in various recognition tasks, and thus emerges as an ideal tool to address automated identification of NMR signals. We have applied a convolutional neural network for visual analysis of multidimensional NMR spectra. A comprehensive test on 31 manually-annotated spectra has demonstrated top-tier average precision (AP) of 0.9596, 0.9058 and 0.8271 for backbone, side-chain and NOESY spectra, respectively. Furthermore, a combination of extracted peak lists with automated assignment routine, FLYA, outperformed other methods, including the manual one, and led to correct resonance assignment at the levels of 90.40%, 89.90% and 90.20% for three benchmark proteins. The proposed model is a part of a Dumpling software (platform for protein NMR data analysis), and is available at https://dumpling.bio/. michaljerzywalczak@gmail.compiotr.klukowski@pwr.edu.pl. Supplementary data are available at Bioinformatics online.

  6. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    International Nuclear Information System (INIS)

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M.

    2015-01-01

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales

  7. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M., E-mail: fmarassi@sbmri.org [Sanford-Burnham Medical Research Institute (United States)

    2015-04-15

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales.

  8. Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

    Energy Technology Data Exchange (ETDEWEB)

    Antanasijevic, Aleksandar; Kingsley, Carolyn [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Basu, Arnab; Bowlin, Terry L. [Microbiotix Inc. (United States); Rong, Lijun [University of Illinois at Chicago, Department of Microbiology and Immunology (United States); Caffrey, Michael, E-mail: caffrey@uic.edu [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)

    2016-03-15

    The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.

  9. NMR structure of the protein NP-247299.1: comparison with the crystal structure

    International Nuclear Information System (INIS)

    Jaudzems, Kristaps; Geralt, Michael; Serrano, Pedro; Mohanty, Biswaranjan; Horst, Reto; Pedrini, Bill; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

    2010-01-01

    Comparison of the NMR and crystal structures of a protein determined using largely automated methods has enabled the interpretation of local differences in the highly similar structures. These differences are found in segments of higher B values in the crystal and correlate with dynamic processes on the NMR chemical shift timescale observed in solution. The NMR structure of the protein NP-247299.1 in solution at 313 K has been determined and is compared with the X-ray crystal structure, which was also solved in the Joint Center for Structural Genomics (JCSG) at 100 K and at 1.7 Å resolution. Both structures were obtained using the current largely automated crystallographic and solution NMR methods used by the JCSG. This paper assesses the accuracy and precision of the results from these recently established automated approaches, aiming for quantitative statements about the location of structure variations that may arise from either one of the methods used or from the different environments in solution and in the crystal. To evaluate the possible impact of the different software used for the crystallographic and the NMR structure determinations and analysis, the concept is introduced of reference structures, which are computed using the NMR software with input of upper-limit distance constraints derived from the molecular models representing the results of the two structure determinations. The use of this new approach is explored to quantify global differences that arise from the different methods of structure determination and analysis versus those that represent interesting local variations or dynamics. The near-identity of the protein core in the NMR and crystal structures thus provided a basis for the identification of complementary information from the two different methods. It was thus observed that locally increased crystallographic B values correlate with dynamic structural polymorphisms in solution, including that the solution state of the protein involves

  10. Site-specific labeling of proteins with NMR-active unnatural amino acids

    International Nuclear Information System (INIS)

    Jones, David H.; Cellitti, Susan E.; Hao Xueshi; Zhang Qiong; Jahnz, Michael; Summerer, Daniel; Schultz, Peter G.; Uno, Tetsuo; Geierstanger, Bernhard H.

    2010-01-01

    A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

  11. APSY-NMR for protein backbone assignment in high-throughput structural biology

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, Samit Kumar; Serrano, Pedro; Proudfoot, Andrew; Geralt, Michael [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States); Pedrini, Bill [Paul Scherrer Institute (PSI), SwissFEL Project (Switzerland); Herrmann, Torsten [Université de Lyon, Institut des Sciences Analytiques, Centre de RMN à Très Hauts Champs, UMR 5280 CNRS, ENS Lyon, UCB Lyon 1 (France); Wüthrich, Kurt, E-mail: wuthrich@scripps.edu [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States)

    2015-01-15

    A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [{sup 1}H,{sup 1}H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.

  12. Synthesis of Structures Related to Antifreeze Glycoproteins

    OpenAIRE

    Fyrner, Timmy

    2005-01-01

    In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzo...

  13. EPR and NMR spectroscopy on spin-labeled proteins

    NARCIS (Netherlands)

    Finiguerra, Michelina Giuseppina

    2011-01-01

    Spin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in frozen solution were studied using 275 GHz EPR (J-band), with the advantage compared to 9 GHz (X-band) and

  14. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, Brian Finley [Univ. of California, Berkeley, CA (United States)

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  15. On the Analytical Superiority of 1D NMR for Fingerprinting the Higher Order Structure of Protein Therapeutics Compared to Multidimensional NMR Methods.

    Science.gov (United States)

    Poppe, Leszek; Jordan, John B; Rogers, Gary; Schnier, Paul D

    2015-06-02

    An important aspect in the analytical characterization of protein therapeutics is the comprehensive characterization of higher order structure (HOS). Nuclear magnetic resonance (NMR) is arguably the most sensitive method for fingerprinting HOS of a protein in solution. Traditionally, (1)H-(15)N or (1)H-(13)C correlation spectra are used as a "structural fingerprint" of HOS. Here, we demonstrate that protein fingerprint by line shape enhancement (PROFILE), a 1D (1)H NMR spectroscopy fingerprinting approach, is superior to traditional two-dimensional methods using monoclonal antibody samples and a heavily glycosylated protein therapeutic (Epoetin Alfa). PROFILE generates a high resolution structural fingerprint of a therapeutic protein in a fraction of the time required for a 2D NMR experiment. The cross-correlation analysis of PROFILE spectra allows one to distinguish contributions from HOS vs protein heterogeneity, which is difficult to accomplish by 2D NMR. We demonstrate that the major analytical limitation of two-dimensional methods is poor selectivity, which renders these approaches problematic for the purpose of fingerprinting large biological macromolecules.

  16. Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology.

    Science.gov (United States)

    Horst, Reto; Wüthrich, Kurt

    2015-07-20

    Reconstitution of integral membrane proteins (IMP) in aqueous solutions of detergent micelles has been extensively used in structural biology, using either X-ray crystallography or NMR in solution. Further progress could be achieved by establishing a rational basis for the selection of detergent and buffer conditions, since the stringent bottleneck that slows down the structural biology of IMPs is the preparation of diffracting crystals or concentrated solutions of stable isotope labeled IMPs. Here, we describe procedures to monitor the quality of aqueous solutions of [ 2 H, 15 N]-labeled IMPs reconstituted in detergent micelles. This approach has been developed for studies of β-barrel IMPs, where it was successfully applied for numerous NMR structure determinations, and it has also been adapted for use with α-helical IMPs, in particular GPCRs, in guiding crystallization trials and optimizing samples for NMR studies (Horst et al ., 2013). 2D [ 15 N, 1 H]-correlation maps are used as "fingerprints" to assess the foldedness of the IMP in solution. For promising samples, these "inexpensive" data are then supplemented with measurements of the translational and rotational diffusion coefficients, which give information on the shape and size of the IMP/detergent mixed micelles. Using microcoil equipment for these NMR experiments enables data collection with only micrograms of protein and detergent. This makes serial screens of variable solution conditions viable, enabling the optimization of parameters such as the detergent concentration, sample temperature, pH and the composition of the buffer.

  17. Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

    International Nuclear Information System (INIS)

    Peters, Fabian; Maestre-Martinez, Mitcheell; Leonov, Andrei; Kovačič, Lidija; Becker, Stefan; Boelens, Rolf; Griesinger, Christian

    2011-01-01

    Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.

  18. Microscopic insights into the NMR relaxation based protein conformational entropy meter

    Science.gov (United States)

    Kasinath, Vignesh; Sharp, Kim A.; Wand, A. Joshua

    2013-01-01

    Conformational entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of conformational entropy are necessary for an understanding of its role but have been difficult to obtain. An empirical method that utilizes changes in conformational dynamics as a proxy for changes in conformational entropy has recently been introduced. Here we probe the microscopic origins of the link between conformational dynamics and conformational entropy using molecular dynamics simulations. Simulation of seven pro! teins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side-chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain conformational entropy. These results tend to validate the use of experimentally accessible measures of methyl motion - the NMR-derived generalized order parameters - as a proxy from which to derive changes in protein conformational entropy. PMID:24007504

  19. Automated solid-state NMR resonance assignment of protein microcrystals and amyloids

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, Elena [Goethe University Frankfurt am Main, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry (Germany); Gath, Julia [ETH Zurich, Physical Chemistry (Switzerland); Habenstein, Birgit [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Ravotti, Francesco; Szekely, Kathrin; Huber, Matthias [ETH Zurich, Physical Chemistry (Switzerland); Buchner, Lena [Goethe University Frankfurt am Main, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry (Germany); Boeckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Guentert, Peter, E-mail: guentert@em.uni-frankfurt.de [Goethe University Frankfurt am Main, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry (Germany)

    2013-07-15

    Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218-289) and {alpha}-synuclein yielded 88-97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77-90 % correctness if also assignments classified as tentative by the algorithm are included.

  20. Automated solid-state NMR resonance assignment of protein microcrystals and amyloids

    International Nuclear Information System (INIS)

    Schmidt, Elena; Gath, Julia; Habenstein, Birgit; Ravotti, Francesco; Székely, Kathrin; Huber, Matthias; Buchner, Lena; Böckmann, Anja; Meier, Beat H.; Güntert, Peter

    2013-01-01

    Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218–289) and α-synuclein yielded 88–97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77–90 % correctness if also assignments classified as tentative by the algorithm are included

  1. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

    International Nuclear Information System (INIS)

    Kobayashi, Hiroshi; Swapna, G. V. T.; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T.; Inouye, Masayori

    2012-01-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS 2 ) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS 2 -tag is replaced with non-isotope labeled PrS 2 -tag, silencing the NMR signals from PrS 2 -tag in isotope-filtered 1 H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS 2 -tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS 2 (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS 2 -tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS 2 -tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone 1 H, 15 N and 13 C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear 1 H– 15 N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

  2. PARAssign-paramagnetic NMR assignments of protein nuclei on the basis of pseudocontact shifts

    Energy Technology Data Exchange (ETDEWEB)

    Skinner, Simon P., E-mail: skinnersp@chem.leidenuniv.nl [Leiden University, Gorlaeus Laboratories, Leiden Institute of Chemistry (Netherlands); Moshev, Mois, E-mail: mois@monomon.me [Leiden University, Leiden Institute of Advanced Computer Science (Netherlands); Hass, Mathias A. S., E-mail: hassmas@chem.leidenuniv.nl; Ubbink, Marcellus, E-mail: m.ubbink@chem.leidenuniv.nl [Leiden University, Gorlaeus Laboratories, Leiden Institute of Chemistry (Netherlands)

    2013-04-15

    The use of paramagnetic NMR data for the refinement of structures of proteins and protein complexes is widespread. However, the power of paramagnetism for protein assignment has not yet been fully exploited. PARAssign is software that uses pseudocontact shift data derived from several paramagnetic centers attached to the protein to obtain amide and methyl assignments. The ability of PARAssign to perform assignment when the positions of the paramagnetic centers are known and unknown is demonstrated. PARAssign has been tested using synthetic data for methyl assignment of a 47 kDa protein, and using both synthetic and experimental data for amide assignment of a 14 kDa protein. The complex fitting space involved in such an assignment procedure necessitates that good starting conditions are found, both regarding placement and strength of paramagnetic centers. These starting conditions are obtained through automated tensor placement and user-defined tensor parameters. The results presented herein demonstrate that PARAssign is able to successfully perform resonance assignment in large systems with a high degree of reliability. This software provides a method for obtaining the assignments of large systems, which may previously have been unassignable, by using 2D NMR spectral data and a known protein structure.

  3. High resolution NMR spectroscopy of nanocrystalline proteins at ultra-high magnetic field

    International Nuclear Information System (INIS)

    Sperling, Lindsay J.; Nieuwkoop, Andrew J.; Lipton, Andrew S.; Berthold, Deborah A.; Rienstra, Chad M.

    2010-01-01

    Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly- 13 C, 15 N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7, 17.6 and 21.1 Tesla ( 1 H frequencies of 500, 750, and 900 MHz). For two protein systems-GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline protein-line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function of field, and for DsbA, resolution in the C-C region increases by 42%, according to the number of peaks that can be uniquely picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely observed in the highest field spectra for at least two isoleucine Cδ1 sites in DsbA. These results further illustrate the benefits of high-field MAS SSNMR spectroscopy for protein structural studies.

  4. Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

    International Nuclear Information System (INIS)

    Ducat, Thierry; Declerck, Nathalie; Gostan, Thierry; Kochoyan, Michel; Demene, Helene

    2006-01-01

    Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters

  5. Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

    KAUST Repository

    Li, Jianping; Pilla, Kala Bharath; Li, Qingfeng; Zhang, Zhengfeng; Su, Xuncheng; Huber, Thomas; Yang, Jun

    2013-01-01

    Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromolecules which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is shown that high-resolution structure of proteins in solid phase can be determined without the use of traditional dipolar-dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomethyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunoglobulin-binding domain of protein G (GB1). The long-range structural restraints with metal-nucleus distance of up to ∼20 Å are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances back-calculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic susceptibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 Å root-mean-square deviation relative to X-ray structure. © 2013 American Chemical Society.

  6. Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

    KAUST Repository

    Li, Jianping

    2013-06-05

    Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromolecules which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is shown that high-resolution structure of proteins in solid phase can be determined without the use of traditional dipolar-dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomethyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunoglobulin-binding domain of protein G (GB1). The long-range structural restraints with metal-nucleus distance of up to ∼20 Å are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances back-calculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic susceptibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 Å root-mean-square deviation relative to X-ray structure. © 2013 American Chemical Society.

  7. Automated sequence-specific protein NMR assignment using the memetic algorithm MATCH

    International Nuclear Information System (INIS)

    Volk, Jochen; Herrmann, Torsten; Wuethrich, Kurt

    2008-01-01

    MATCH (Memetic Algorithm and Combinatorial Optimization Heuristics) is a new memetic algorithm for automated sequence-specific polypeptide backbone NMR assignment of proteins. MATCH employs local optimization for tracing partial sequence-specific assignments within a global, population-based search environment, where the simultaneous application of local and global optimization heuristics guarantees high efficiency and robustness. MATCH thus makes combined use of the two predominant concepts in use for automated NMR assignment of proteins. Dynamic transition and inherent mutation are new techniques that enable automatic adaptation to variable quality of the experimental input data. The concept of dynamic transition is incorporated in all major building blocks of the algorithm, where it enables switching between local and global optimization heuristics at any time during the assignment process. Inherent mutation restricts the intrinsically required randomness of the evolutionary algorithm to those regions of the conformation space that are compatible with the experimental input data. Using intact and artificially deteriorated APSY-NMR input data of proteins, MATCH performed sequence-specific resonance assignment with high efficiency and robustness

  8. NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

    Directory of Open Access Journals (Sweden)

    Choe Senyon

    2007-11-01

    Full Text Available Abstract Background Structural studies of integral membrane proteins (IMPs are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs. The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. Conclusion The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.

  9. Principal components analysis of protein structure ensembles calculated using NMR data

    International Nuclear Information System (INIS)

    Howe, Peter W.A.

    2001-01-01

    One important problem when calculating structures of biomolecules from NMR data is distinguishing converged structures from outlier structures. This paper describes how Principal Components Analysis (PCA) has the potential to classify calculated structures automatically, according to correlated structural variation across the population. PCA analysis has the additional advantage that it highlights regions of proteins which are varying across the population. To apply PCA, protein structures have to be reduced in complexity and this paper describes two different representations of protein structures which achieve this. The calculated structures of a 28 amino acid peptide are used to demonstrate the methods. The two different representations of protein structure are shown to give equivalent results, and correct results are obtained even though the ensemble of structures used as an example contains two different protein conformations. The PCA analysis also correctly identifies the structural differences between the two conformations

  10. Study of the hydration of globular proteins by broad NMR lines method

    Energy Technology Data Exchange (ETDEWEB)

    Blicharska, B [Uniwersytet Jagiellonski, Krakow (Poland). Instytut Fizyki

    1973-01-01

    Spectra of proteins and polypeptides obtained by means of a NMR broad line spectrometer consist of broad and thin lines. These broad and thin lines are attributed to proteins and to water absorbed on the surfaces of proteins respectively. The behaviour of the thin line in the spectra of lyophilizated albumin of the egg white has been studied in the temperature range from -42 to 20/sup 0/C. The amount of water has been found by the simple method of weighing and has been equal about 7% of the total weight. It has been found that the water absorbed on the surface of the lyophilizated proteins gives a thinner line in comparison to the water absorbed on molecules of proteins in water solutions and that the correlation time is about 10/sup 3/ times greater.

  11. CASA: An Efficient Automated Assignment of Protein Mainchain NMR Data Using an Ordered Tree Search Algorithm

    International Nuclear Information System (INIS)

    Wang Jianyong; Wang Tianzhi; Zuiderweg, Erik R. P.; Crippen, Gordon M.

    2005-01-01

    Rapid analysis of protein structure, interaction, and dynamics requires fast and automated assignments of 3D protein backbone triple-resonance NMR spectra. We introduce a new depth-first ordered tree search method of automated assignment, CASA, which uses hand-edited peak-pick lists of a flexible number of triple resonance experiments. The computer program was tested on 13 artificially simulated peak lists for proteins up to 723 residues, as well as on the experimental data for four proteins. Under reasonable tolerances, it generated assignments that correspond to the ones reported in the literature within a few minutes of CPU time. The program was also tested on the proteins analyzed by other methods, with both simulated and experimental peaklists, and it could generate good assignments in all relevant cases. The robustness was further tested under various situations

  12. Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

    International Nuclear Information System (INIS)

    Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Onuchic, José N.; Schug, Alexander

    2015-01-01

    The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein’s functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism

  13. Accurate protein structure modeling using sparse NMR data and homologous structure information.

    Science.gov (United States)

    Thompson, James M; Sgourakis, Nikolaos G; Liu, Gaohua; Rossi, Paolo; Tang, Yuefeng; Mills, Jeffrey L; Szyperski, Thomas; Montelione, Gaetano T; Baker, David

    2012-06-19

    While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining (1)H(N), (13)C, and (15)N backbone and (13)Cβ chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2-1.9 Å relative to the conventional determined NMR ensembles and of 0.9-1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.

  14. NMR structure of the N-terminal domain of the replication initiator protein DnaA

    Energy Technology Data Exchange (ETDEWEB)

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  15. Assignment strategies in homonuclear three-dimensional 1H NMR spectra of proteins

    International Nuclear Information System (INIS)

    Vuister, G.W.; Boelens, R.; Padilla, A.; Kleywegt, G.J.; Kaptein, R.

    1990-01-01

    The increase in dimensionality of three-dimensional (3D) NMR greatly enhances the spectral resolution in comparison to 2D NMR. It alleviates the problem of resonance overlap and may extend the range of molecules amenable to structure determination by high-resolution NMR spectroscopy. Here, the authors present strategies for the assignment of protein resonances from homonuclear nonselective 3D NOE-HOHAHA spectra. A notation for connectivities between protons, corresponding to cross peaks in 3D spectra, is introduced. They show how spin systems can be identified by tracing cross-peak patterns in cross sections perpendicular to the three frequency axes. The observable 3D sequential connectivities in proteins are tabulated, and estimates for the relative intensities of the corresponding cross peaks are given for α-helical and β-sheet conformations. Intensities of the cross peaks in the 3D spectrum of pike III paravalbumin follow the predictions. The sequential-assignment procedure is illustrated for loop regions, extended and α-helical conformations for the residues Ala 54-Leu 63 of paravalbumin. NOEs that were not previously identified in 2D spectra of paravalbumin due to overlap are found

  16. On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

    Science.gov (United States)

    Sharp, Kim A; O'Brien, Evan; Kasinath, Vignesh; Wand, A Joshua

    2015-05-01

    Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH  entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. © 2015 Wiley Periodicals, Inc.

  17. Optimizing nanodiscs and bicelles for solution NMR studies of two β-barrel membrane proteins

    International Nuclear Information System (INIS)

    Kucharska, Iga; Edrington, Thomas C.; Liang, Binyong; Tamm, Lukas K.

    2015-01-01

    Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1ΔH5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1ΔH5 nanodiscs

  18. Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

    International Nuclear Information System (INIS)

    Haselhorst, Thomas; Muenster-Kuehnel, Anja K.; Oschlies, Melanie; Tiralongo, Joe; Gerardy-Schahn, Rita; Itzstein, Mark von

    2007-01-01

    We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand (α/β-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) no specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available

  19. NMR in a crystallography-based high-throughput protein structure-determination environment

    International Nuclear Information System (INIS)

    Wüthrich, Kurt

    2010-01-01

    As an introduction to three papers on comparisons of corresponding crystal and NMR solution structures determined by the Joint Center for Structural Genomics (JCSG), an outline is provided of the JCSG strategy for combined use of the two techniques. A special commentary addresses the potentialities of the concept of ‘reference crystal structures’, which is introduced in the following three papers. An introduction is provided to three papers which compare corresponding protein crystal and NMR solution structures determined by the Joint Center for Structural Genomics (JCSG). Special mention is made of the JCSG strategy for combined use of the two techniques, and of potential applications of the concept of ‘reference crystal structures’, which is introduced in the following three papers

  20. Measuring 13Cβ chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

    International Nuclear Information System (INIS)

    Lundstroem, Patrik; Lin Hong; Kay, Lewis E.

    2009-01-01

    A labeling scheme is introduced that facilitates the measurement of accurate 13 C β chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of 13 C enrichment (30-40%) at C β side-chain carbon positions for 15 of the amino acids with little 13 C label at positions one bond removed (∼5%). A pair of samples are produced using [1- 13 C]-glucose/NaH 12 CO 3 or [2- 13 C]-glucose as carbon sources with isolated and enriched (>30%) 13 C β positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of 13 C β chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples

  1. Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay.

    Science.gov (United States)

    Hodgson, Derek J; Aubin, Yves

    2017-05-10

    A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  2. Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

    International Nuclear Information System (INIS)

    Nucci, Nathaniel V.; Marques, Bryan S.; Bédard, Sabrina; Dogan, Jakob; Gledhill, John M.; Moorman, Veronica R.; Peterson, Ronald W.; Valentine, Kathleen G.; Wand, Alison L.; Wand, A. Joshua

    2011-01-01

    Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30 kDa require complex experimental methods, such as TROSY in conjunction with isotopic labeling schemes that are often expensive and generally reduce the potential information available. We have developed the reverse micelle encapsulation strategy as an alternative approach. Encapsulation of proteins within the protective nano-scale water pool of a reverse micelle dissolved in ultra-low viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins. Here, we characterize the contributions from the various components of the protein-containing reverse micelle system to the rotational correlation time of the encapsulated protein. Importantly, we demonstrate that the protein encapsulated in the reverse micelle maintains a hydration shell comparable in size to that seen in bulk solution. Using moderate pressures, encapsulation in ultra-low viscosity propane or ethane can be used to magnify this advantage. We show that encapsulation in liquid ethane can be used to reduce the tumbling time of the 43 kDa maltose binding protein from ∼23 to ∼10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43 kDa encapsulated maltose binding protein dissolved in liquid ethane, which is typically impossible for proteins of such size without use of extensive deuteration or the TROSY effect.

  3. NMR of proteins (4Fe-4S): structural properties and intramolecular electron transfer

    International Nuclear Information System (INIS)

    Huber, J.G.

    1996-01-01

    NMR started to be applied to Fe-S proteins in the seventies. Its use has recently been enlarged as the problems arising from the paramagnetic polymetallic clusters ware overcome. Applications to [4Fe-4S] are presented herein. The information derived thereof deepens the understanding of the redox properties of these proteins which play a central role in the metabolism of bacterial cells. The secondary structure elements and the overall folding of Chromatium vinosum ferredoxin (Cv Fd) in solution have been established by NMR. The unique features of this sequence have been shown to fold as an α helix at the C-terminus and as a loop between two cysteines ligand of one cluster: these two parts localize in close proximity from one another. The interaction between nuclear and electronic spins is a source of additional structural information for (4Fe-AS] proteins. The conformation of the cysteine-ligands, as revealed by the Fe-(S γ -C β -H β )Cys dihedral angles, is related to the chemical shifts of the signals associated with the protons of these residues. The longitudinal relaxation times of the protons depend on their distance to the cluster. A quantitative relationship has been established and used to show that the solution structure of the high-potential ferredoxin from Cv differs significantly from the crystal structure around Phe-48. Both parameters (chemical shifts and longitudinal relaxation times) give also insight into the electronic and magnetic properties of the [4Fe-4S] clusters. The rate of intramolecular electron transfer between the two [4FE-4S] clusters of ferredoxins has been measured by NMR. It is far slower in the case of Cv Fd than for shorter ferredoxins. The difference may be associated with changes in the magnetic and/or electronic properties of one cluster. The strong paramagnetism of the [4Fe-4S] clusters, which originally limited the applicability of NMR to proteins containing these cofactors, has been proven instrumental in affording new

  4. PINE-SPARKY.2 for automated NMR-based protein structure research.

    Science.gov (United States)

    Lee, Woonghee; Markley, John L

    2018-05-01

    Nuclear magnetic resonance (NMR) spectroscopy, along with X-ray crystallography and cryoelectron microscopy, is one of the three major tools that enable the determination of atomic-level structural models of biological macromolecules. Of these, NMR has the unique ability to follow important processes in solution, including conformational changes, internal dynamics and protein-ligand interactions. As a means for facilitating the handling and analysis of spectra involved in these types of NMR studies, we have developed PINE-SPARKY.2, a software package that integrates and automates discrete tasks that previously required interaction with separate software packages. The graphical user interface of PINE-SPARKY.2 simplifies chemical shift assignment and verification, automated detection of secondary structural elements, predictions of flexibility and hydrophobic cores, and calculation of three-dimensional structural models. PINE-SPARKY.2 is available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers to the SBGrid (https://sbgrid.org). For a detailed description of the program, see http://www.nmrfam.wisc.edu/pine-sparky2.htm. whlee@nmrfam.wisc.edu or markley@nmrfam.wisc.edu. Supplementary data are available at Bioinformatics online.

  5. Efficient DNP NMR of Membrane Proteins: Sample Preparation Protocols, Sensitivity, and Radical Location

    Science.gov (United States)

    Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V.; Hong, Mei

    2016-01-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

  6. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo [Massachusetts Institute of Technology, Department of Chemistry (United States); Sergeyev, Ivan V. [Bruker Biospin (United States); Hong, Mei, E-mail: meihong@mit.edu [Massachusetts Institute of Technology, Department of Chemistry (United States)

    2016-03-15

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

  7. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts

    Energy Technology Data Exchange (ETDEWEB)

    Shen Yang; Delaglio, Frank [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Cornilescu, Gabriel [National Magnetic Resonance Facility (United States); Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)], E-mail: bax@nih.gov

    2009-08-15

    NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between {sup 13}C, {sup 15}N and {sup 1}H chemical shifts and backbone torsion angles {phi} and {psi} (Cornilescu et al. J Biomol NMR 13 289-302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted {phi} and {psi} angles, equals {+-}13{sup o}. Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.

  8. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

    International Nuclear Information System (INIS)

    Eddy, Matthew T.; Su, Yongchao; Silvers, Robert; Andreas, Loren; Clark, Lindsay; Wagner, Gerhard; Pintacuda, Guido; Emsley, Lyndon; Griffin, Robert G.

    2015-01-01

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13 C line widths and <0.5 ppm 15 N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported

  9. Insights into the interaction between nucleoid-associated proteins H ha and H-NS by NMR and fluorescence anisotropy

    Energy Technology Data Exchange (ETDEWEB)

    Cordeiro, T.N.; Garcia, J. [Institut de Recerca Biomedica-Parc Cientific de (Spain). Lab. of Biomolecular NMR; Pons, M. [Universitat de Barcelona (Spain). Dept. de Quimica Organica]. E-mail: mpons@ub.edu

    2005-07-01

    NMR and fluorescence anisotropy are both valuable tools for studying bio molecular interactions. NMR can provide structural insights at atomic resolution. Still, it can be wisely complemented by lower-resolution biophysical techniques, such as fluorescence anisotropy. In this article we report the combination of NMR and fluorescence anisotropy in establishing novel structure-function insights into the interaction between two bacterial nucleoid-associated proteins, H ha and H-NS. H ha (H-NS) complexes are known to play an important role in modulating the expression of some environmentally regulated genes that confer survival advantage in a particular growth condition. (author)

  10. Insights into the interaction between nucleoid-associated proteins H ha and H-NS by NMR and fluorescence anisotropy

    International Nuclear Information System (INIS)

    Cordeiro, T.N.; Garcia, J.; Pons, M.

    2005-01-01

    NMR and fluorescence anisotropy are both valuable tools for studying bio molecular interactions. NMR can provide structural insights at atomic resolution. Still, it can be wisely complemented by lower-resolution biophysical techniques, such as fluorescence anisotropy. In this article we report the combination of NMR and fluorescence anisotropy in establishing novel structure-function insights into the interaction between two bacterial nucleoid-associated proteins, H ha and H-NS. H ha (H-NS) complexes are known to play an important role in modulating the expression of some environmentally regulated genes that confer survival advantage in a particular growth condition. (author)

  11. High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.

    Science.gov (United States)

    Zhang, Yi; Kitazawa, Soichiro; Peran, Ivan; Stenzoski, Natalie; McCallum, Scott A; Raleigh, Daniel P; Royer, Catherine A

    2016-11-23

    Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.

  12. Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

    Science.gov (United States)

    Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

    2010-05-01

    High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

  13. Exploring abiotic stress on asynchronous protein metabolism in single kernels of wheat studied by NMR spectroscopy and chemometrics

    DEFF Research Database (Denmark)

    Winning, H.; Viereck, N.; Wollenweber, B.

    2009-01-01

    at the vegetative growth stage had little effect on the parameters investigated. For the first time, H-1 HR-MAS NMR spectra of grains taken during grain-filling were analysed by an advanced multiway model. In addition to the results from the chemical protein analysis and the H-1 HR-MAS NMR spectra of single kernels...... was to examine the implications of different drought treatments on the protein fractions in grains of winter wheat using H-1 nuclear magnetic resonance spectroscopy followed by chemometric analysis. Triticum aestivum L. cv. Vinjett was studied in a semi-field experiment and subjected to drought episodes either...... at terminal spikelet, during grain-filling or at both stages. Principal component trajectories of the total protein content and the protein fractions of flour as well as the H-1 NMR spectra of single wheat kernels, wheat flour, and wheat methanol extracts were analysed to elucidate the metabolic development...

  14. RESCUE: An artificial neural network tool for the NMR spectral assignment of proteins

    International Nuclear Information System (INIS)

    Pons, J.L.; Delsuc, M.A.

    1999-01-01

    The assignment of the 1 H spectrum of a protein or a polypeptide is the prerequisite for advanced NMR studies. We present here an assignment tool based on the artificial neural network technology, which determines the type of the amino acid from the chemical shift values observed in the 1 H spectrum. Two artificial neural networks have been trained and extensively tested against a non-redundant subset of the BMRB chemical shift data bank [Seavey, B.R. et al. (1991) J. Biomol. NMR, 1, 217-236]. The most promising of the two accomplishes the analysis in two steps, grouping related amino acids together. It presents a mean rate of success above 80% on the test set. The second network tested separates down to the single amino acid; it presents a mean rate of success of 63%. This tool has been used to assist the manual assignment of peptides and proteins and can also be used as a block in an automated approach to assignment. The program has been called RESCUE and is made publicly available at the following URL: http://www.infobiosud.univ-montp1.fr/rescue

  15. PASA - A Program for Automated Protein NMR Backbone Signal Assignment by Pattern-Filtering Approach

    International Nuclear Information System (INIS)

    Xu Yizhuang; Wang Xiaoxia; Yang Jun; Vaynberg, Julia; Qin Jun

    2006-01-01

    We present a new program, PASA (Program for Automated Sequential Assignment), for assigning protein backbone resonances based on multidimensional heteronuclear NMR data. Distinct from existing programs, PASA emphasizes a per-residue-based pattern-filtering approach during the initial stage of the automated 13 C α and/or 13 C β chemical shift matching. The pattern filter employs one or multiple constraints such as 13 C α /C β chemical shift ranges for different amino acid types and side-chain spin systems, which helps to rule out, in a stepwise fashion, improbable assignments as resulted from resonance degeneracy or missing signals. Such stepwise filtering approach substantially minimizes early false linkage problems that often propagate, amplify, and ultimately cause complication or combinatorial explosion of the automation process. Our program (http://www.lerner.ccf.org/moleccard/qin/) was tested on four representative small-large sized proteins with various degrees of resonance degeneracy and missing signals, and we show that PASA achieved the assignments efficiently and rapidly that are fully consistent with those obtained by laborious manual protocols. The results demonstrate that PASA may be a valuable tool for NMR-based structural analyses, genomics, and proteomics

  16. Relationship between recombinant protein expression and host metabolome as determined by two-dimensional NMR spectroscopy.

    Directory of Open Access Journals (Sweden)

    Young Kee Chae

    Full Text Available Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming and labor-intensive trial-and-error. We hypothesized that different metabolomic fingerprints might be associated with different protein production outcomes. If so, then it might be possible to change the expression pattern by manipulating the metabolite environment. As a first step in testing this hypothesis, we probed a subset of the intracellular metabolites by partially labeling it with 13C-glucose. We tested 71 genes and identified 17 metabolites by employing the two-dimensional NMR spectroscopy. The statistical analysis showed that there existed the metabolite compositions favoring protein production. We hope that this work would help devise a systematic and predictive approach to the recombinant protein production.

  17. The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data

    International Nuclear Information System (INIS)

    Lee, Woonghee; Petit, Chad M.; Cornilescu, Gabriel; Stark, Jaime L.; Markley, John L.

    2016-01-01

    We introduce AUDANA (Automated Database-Assisted NOE Assignment), an algorithm for determining three-dimensional structures of proteins from NMR data that automates the assignment of 3D-NOE spectra, generates distance constraints, and conducts iterative high temperature molecular dynamics and simulated annealing. The protein sequence, chemical shift assignments, and NOE spectra are the only required inputs. Distance constraints generated automatically from ambiguously assigned NOE peaks are validated during the structure calculation against information from an enlarged version of the freely available PACSY database that incorporates information on protein structures deposited in the Protein Data Bank (PDB). This approach yields robust sets of distance constraints and 3D structures. We evaluated the performance of AUDANA with input data for 14 proteins ranging in size from 6 to 25 kDa that had 27–98 % sequence identity to proteins in the database. In all cases, the automatically calculated 3D structures passed stringent validation tests. Structures were determined with and without database support. In 9/14 cases, database support improved the agreement with manually determined structures in the PDB and in 11/14 cases, database support lowered the r.m.s.d. of the family of 20 structural models.

  18. The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Woonghee, E-mail: whlee@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison and Biochemistry Department (United States); Petit, Chad M. [University of Alabama at Birmingham, Department of Biochemistry and Molecular Genetics (United States); Cornilescu, Gabriel; Stark, Jaime L.; Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison and Biochemistry Department (United States)

    2016-06-15

    We introduce AUDANA (Automated Database-Assisted NOE Assignment), an algorithm for determining three-dimensional structures of proteins from NMR data that automates the assignment of 3D-NOE spectra, generates distance constraints, and conducts iterative high temperature molecular dynamics and simulated annealing. The protein sequence, chemical shift assignments, and NOE spectra are the only required inputs. Distance constraints generated automatically from ambiguously assigned NOE peaks are validated during the structure calculation against information from an enlarged version of the freely available PACSY database that incorporates information on protein structures deposited in the Protein Data Bank (PDB). This approach yields robust sets of distance constraints and 3D structures. We evaluated the performance of AUDANA with input data for 14 proteins ranging in size from 6 to 25 kDa that had 27–98 % sequence identity to proteins in the database. In all cases, the automatically calculated 3D structures passed stringent validation tests. Structures were determined with and without database support. In 9/14 cases, database support improved the agreement with manually determined structures in the PDB and in 11/14 cases, database support lowered the r.m.s.d. of the family of 20 structural models.

  19. Equilibrium simulations of proteins using molecular fragment replacement and NMR chemical shifts.

    Science.gov (United States)

    Boomsma, Wouter; Tian, Pengfei; Frellsen, Jes; Ferkinghoff-Borg, Jesper; Hamelryck, Thomas; Lindorff-Larsen, Kresten; Vendruscolo, Michele

    2014-09-23

    Methods of protein structure determination based on NMR chemical shifts are becoming increasingly common. The most widely used approaches adopt the molecular fragment replacement strategy, in which structural fragments are repeatedly reassembled into different complete conformations in molecular simulations. Although these approaches are effective in generating individual structures consistent with the chemical shift data, they do not enable the sampling of the conformational space of proteins with correct statistical weights. Here, we present a method of molecular fragment replacement that makes it possible to perform equilibrium simulations of proteins, and hence to determine their free energy landscapes. This strategy is based on the encoding of the chemical shift information in a probabilistic model in Markov chain Monte Carlo simulations. First, we demonstrate that with this approach it is possible to fold proteins to their native states starting from extended structures. Second, we show that the method satisfies the detailed balance condition and hence it can be used to carry out an equilibrium sampling from the Boltzmann distribution corresponding to the force field used in the simulations. Third, by comparing the results of simulations carried out with and without chemical shift restraints we describe quantitatively the effects that these restraints have on the free energy landscapes of proteins. Taken together, these results demonstrate that the molecular fragment replacement strategy can be used in combination with chemical shift information to characterize not only the native structures of proteins but also their conformational fluctuations.

  20. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks

    Energy Technology Data Exchange (ETDEWEB)

    Shen Yang; Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2013-07-15

    A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, {>=}90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed ({phi}, {psi}) torsion angles of ca 12 Masculine-Ordinal-Indicator . TALOS-N also reports sidechain {chi}{sup 1} rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.

  1. Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets

    International Nuclear Information System (INIS)

    Bellstedt, Peter; Seiboth, Thomas; Häfner, Sabine; Kutscha, Henriette; Ramachandran, Ramadurai; Görlach, Matthias

    2013-01-01

    NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ('scrambling”) of NH and CO groups in a standard Escherichia coli expression host is provided

  2. Protein analysis by 31p NMR spectroscopy in ionic liquid: quantitative determination of enzymatically created cross-links.

    Science.gov (United States)

    Monogioudi, Evanthia; Permi, Perttu; Filpponen, Ilari; Lienemann, Michael; Li, Bin; Argyropoulos, Dimitris; Buchert, Johanna; Mattinen, Maija-Liisa

    2011-02-23

    Cross-linking of β-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by (31)P nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to (31)P NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative (31)P NMR in IL was shown to be a simple and efficient method for the study of protein modification.

  3. Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain.

    Directory of Open Access Journals (Sweden)

    Christos Tzitzilonis

    Full Text Available Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [(15N,(1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [(15N,(1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.

  4. A natural and readily available crowding agent: NMR studies of proteins in hen egg white.

    Science.gov (United States)

    Martorell, Gabriel; Adrover, Miquel; Kelly, Geoff; Temussi, Piero Andrea; Pastore, Annalisa

    2011-05-01

    In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media. Copyright © 2010 Wiley-Liss, Inc.

  5. A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

    Science.gov (United States)

    Hikone, Yuya; Hirai, Go; Mishima, Masaki; Inomata, Kohsuke; Ikeya, Teppei; Arai, Souichiro; Shirakawa, Masahiro; Sodeoka, Mikiko; Ito, Yutaka

    2016-10-01

    Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

  6. A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Hikone, Yuya [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Hirai, Go [RIKEN, Synthetic Organic Chemistry Laboratory (Japan); Mishima, Masaki [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Inomata, Kohsuke [RIKEN, Quantitative Biology Center (Japan); Ikeya, Teppei; Arai, Souichiro [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Shirakawa, Masahiro [Japan Agency for Medical Research and Development, AMED-CREST (Japan); Sodeoka, Mikiko [RIKEN, Synthetic Organic Chemistry Laboratory (Japan); Ito, Yutaka, E-mail: ito-yutaka@tmu.ac.jp [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan)

    2016-10-15

    Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N–H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

  7. A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells

    International Nuclear Information System (INIS)

    Hikone, Yuya; Hirai, Go; Mishima, Masaki; Inomata, Kohsuke; Ikeya, Teppei; Arai, Souichiro; Shirakawa, Masahiro; Sodeoka, Mikiko; Ito, Yutaka

    2016-01-01

    Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N–H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

  8. Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR

    Science.gov (United States)

    Hong, Mei; Su, Yongchao

    2011-01-01

    Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. PMID:21344534

  9. Quantifying protein dynamics in the ps–ns time regime by NMR relaxation

    Energy Technology Data Exchange (ETDEWEB)

    Hernández, Griselda; LeMaster, David M., E-mail: david.lemaster@health.ny.gov [University at Albany - SUNY, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, School of Public Health (United States)

    2016-11-15

    Both {sup 15}N chemical shift anisotropy (CSA) and sufficiently rapid exchange linebroadening transitions exhibit relaxation contributions that are proportional to the square of the magnetic field. Deconvoluting these contributions is further complicated by residue-dependent variations in protein amide {sup 15}N CSA values which have proven difficult to accurately measure. Exploiting recently reported improvements for the implementation of T{sub 1} and T{sub 1ρ} experiments, field strength-dependent studies have been carried out on the B3 domain of protein G (GB3) as well as on the immunophilin FKBP12 and a H87V variant of that protein in which the major conformational exchange linebroadening transition is suppressed. By applying a zero frequency spectral density rescaling analysis to the relaxation data collected at magnetic fields from 500 to 900 MHz {sup 1}H, differential residue-specific {sup 15}N CSA values have been obtained for GB3 which correlate with those derived from solid state and liquid crystalline NMR measurements to a level similar to the correlation among those previously reported studies. Application of this analysis protocol to FKBP12 demonstrated an efficient quantitation of both weak exchange linebroadening contributions and differential residue-specific {sup 15}N CSA values. Experimental access to such differential residue-specific {sup 15}N CSA values should significantly facilitate more accurate comparisons with molecular dynamics simulations of protein motion that occurs within the timeframe of global molecular tumbling.

  10. Protein-Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy.

    Science.gov (United States)

    Diniz, Ana; Dias, Jorge S; Jiménez-Barbero, Jesús; Marcelo, Filipa; Cabrita, Eurico J

    2017-09-21

    Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1 H 15 N-HSQC signals. The intensity of the Gal-3 1 H 15 N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1 H 15 N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Zero in on Key Open Problems in Automated NMR Protein Structure Determination

    KAUST Repository

    Abbas, Ahmed

    2015-11-12

    Nuclear magnetic resonance (NMR) is one of the main approaches for protein struc- ture determination. The biggest advantage of this approach is that it can determine the three-dimensional structure of the protein in the solution phase. Thus, the natural dynamics of the protein can be studied. However, NMR protein structure determina- tion is an expertise intensive and time-consuming process. If the structure determi- nation process can be accelerated or even automated by computational methods, that will significantly advance the structural biology field. Our goal in this dissertation is to propose highly efficient and error tolerant methods that can work well on real and noisy data sets of NMR. Our first contribution in this dissertation is the development of a novel peak pick- ing method (WaVPeak). First, WaVPeak denoises the NMR spectra using wavelet smoothing. A brute force method is then used to identify all the candidate peaks. Af- ter that, the volume of each candidate peak is estimated. Finally, the peaks are sorted according to their volumes. WaVPeak is tested on the same benchmark data set that was used to test the state-of-the-art method, PICKY. WaVPeak shows significantly better performance than PICKY in terms of recall and precision. Our second contribution is to propose an automatic method to select peaks pro- duced by peak picking methods. This automatic method is used to overcome the limitations of fixed number-based methods. Our method is based on the Benjamini- Hochberg (B-H) algorithm. The method is used with both WaVPeak and PICKY to automatically select the number of peaks to return from out of hundreds of candidate peaks. The volume (in WaVPeak) and the intensity (in PICKY) are converted into p-values. Peaks that have p-values below some certain threshold are selected. Ex- perimental results show that the new method is better than the fixed number-based method in terms of recall. To improve precision, we tried to eliminate false peaks using

  12. Resolution-by-proxy: a simple measure for assessing and comparing the overall quality of NMR protein structures

    International Nuclear Information System (INIS)

    Berjanskii, Mark; Zhou Jianjun; Liang Yongjie; Lin Guohui; Wishart, David S.

    2012-01-01

    In protein X-ray crystallography, resolution is often used as a good indicator of structural quality. Diffraction resolution of protein crystals correlates well with the number of X-ray observables that are used in structure generation and, therefore, with protein coordinate errors. In protein NMR, there is no parameter identical to X-ray resolution. Instead, resolution is often used as a synonym of NMR model quality. Resolution of NMR structures is often deduced from ensemble precision, torsion angle normality and number of distance restraints per residue. The lack of common techniques to assess the resolution of X-ray and NMR structures complicates the comparison of structures solved by these two methods. This problem is sometimes approached by calculating “equivalent resolution” from structure quality metrics. However, existing protocols do not offer a comprehensive assessment of protein structure as they calculate equivalent resolution from a relatively small number (<5) of protein parameters. Here, we report a development of a protocol that calculates equivalent resolution from 25 measurable protein features. This new method offers better performance (correlation coefficient of 0.92, mean absolute error of 0.28 Å) than existing predictors of equivalent resolution. Because the method uses coordinate data as a proxy for X-ray diffraction data, we call this measure “Resolution-by-Proxy” or ResProx. We demonstrate that ResProx can be used to identify under-restrained, poorly refined or inaccurate NMR structures, and can discover structural defects that the other equivalent resolution methods cannot detect. The ResProx web server is available at http://www.resprox.cahttp://www.resprox.ca.

  13. Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Trempe, Jean-Francois; Morin, Frederick G.; Xia Zhicheng; Marchessault, Robert H.; Gehring, Kalle [McGill University, Department of Biochemistry and Department of Chemistry (Canada)], E-mail: kalle@bri.nrc.ca

    2002-01-15

    A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the {sup 2}H NMR water signal and by the measurement of {sup 1}H-{sup 15}N residual dipolar couplings (RDC) in the archeal translation elongation factor 1{beta}. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.

  14. Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

    International Nuclear Information System (INIS)

    Trempe, Jean-Francois; Morin, Frederick G.; Xia Zhicheng; Marchessault, Robert H.; Gehring, Kalle

    2002-01-01

    A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the 2 H NMR water signal and by the measurement of 1 H- 15 N residual dipolar couplings (RDC) in the archeal translation elongation factor 1β. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample

  15. Smartnotebook: A semi-automated approach to protein sequential NMR resonance assignments

    International Nuclear Information System (INIS)

    Slupsky, Carolyn M.; Boyko, Robert F.; Booth, Valerie K.; Sykes, Brian D.

    2003-01-01

    Complete and accurate NMR spectral assignment is a prerequisite for high-throughput automated structure determination of biological macromolecules. However, completely automated assignment procedures generally encounter difficulties for all but the most ideal data sets. Sources of these problems include difficulty in resolving correlations in crowded spectral regions, as well as complications arising from dynamics, such as weak or missing peaks, or atoms exhibiting more than one peak due to exchange phenomena. Smartnotebook is a semi-automated assignment software package designed to combine the best features of the automated and manual approaches. The software finds and displays potential connections between residues, while the spectroscopist makes decisions on which connection is correct, allowing rapid and robust assignment. In addition, smartnotebook helps the user fit chains of connected residues to the primary sequence of the protein by comparing the experimentally determined chemical shifts with expected shifts derived from a chemical shift database, while providing bookkeeping throughout the assignment procedure

  16. Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

    International Nuclear Information System (INIS)

    Roder, H.; Wuethrich, K.

    1986-01-01

    A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin, which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1 H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1 H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes

  17. Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR

    International Nuclear Information System (INIS)

    Gruschus, James M.; Ferretti, James A.

    2001-01-01

    Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant

  18. Probing membrane protein structure using water polarization transfer solid-state NMR.

    Science.gov (United States)

    Williams, Jonathan K; Hong, Mei

    2014-10-01

    Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected (1)H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins. Copyright © 2014 Elsevier Inc. All

  19. Protein samples for NMR: expression and analysis without purification, and stabilization by covalent cyclization

    International Nuclear Information System (INIS)

    Otting, G.; Ozawa, K.; Prosselkov, P.; Williams, N.K.; Dixon, N.E.; Liepinsh, E.

    2002-01-01

    Full text: A modified cell-free in vitro expression system was established for the expression of milligram quantities of protein per mL reaction medium. Expression levels of the E coli cytoplasmic peptidyl-prolyl cis-trans isomerase, PpiB, in 0 6 mL reaction medium were sufficient for the direct recording of clean 15N-HSQC spectra without chromatographic purification or sample concentration steps, using a 600 MHz NMR spectrometer with cryoprobe. Besides providing a route to high-throughput sample preparation, in vitro expression systems are known to be highly economic in their utilization of selectively labelled ammo acids. Using dual-selective labelling with 15N- and 13C-labelled amino acids, the 15N-HSQC cross peaks of strategically selected ammo acids can readily be identified and monitored for their response to the presence of ligand molecules, again without sample purification. 2) The N-terminal domain of E coli DnaB is a protein of ca 110 residues with a structured core composed of 6 helices. Additional segments of 10 residues each at the N- and C-termini are highly mobile. Both ends are close in space and can be linked together in a covalent peptide bond using intern technology. The core structures of linear (lin-DnaB-N) and cyclized (cz-DnaB-N) protein are conserved, as evidenced by superimposable NOESY spectra and chemical shifts. The linker segment in cz-DnaB-N is mobile as shown by 1H-15N NOEs. Yet, the cyclic protein melts about 10 degrees higher than the linear version. A stabilization free energy of ca 2 kcal/mol is in agreement with predictions based on the reduced entropy in the unfolded state. Amide proton exchange rates are much slower in the cyclic protein and reveal cooperative exchange through total, global unfolding at a rate of once every 100 minutes in the linear protein

  20. General framework for studying the dynamics of folded and nonfolded proteins by NMR relaxation spectroscopy and MD simulation

    NARCIS (Netherlands)

    Prompers, J.J.; Brüschweiler, R.

    2002-01-01

    A general framework is presented for the interpretation of NMR relaxation data of proteins. The method, termed isotropic reorientational eigenmode dynamics (iRED), relies on a principal component analysis of the isotropically averaged covariance matrix of the lattice functions of the spin

  1. Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins

    International Nuclear Information System (INIS)

    Englander, S.W.; Wand, A.J.

    1987-01-01

    A strategy for assigning the resonances in two-dimensional (2D) NMR spectra of proteins is described. The method emphasizes the analysis of through-space relationships between protons by use of the two-dimensional nuclear Overhauser effect (NOE) experiment. NOE patterns used in the algorithm were derived from a statistical analysis of the combinations of short proton-proton distances observed in the high-resolution crystal structures of 21 proteins. One starts with a search for authentic main-chain NH-C/sub α/H-C/sub β/H J-coupled units, which can be found with high reliability. The many main-chain units of a protein are then placed in their proper juxtaposition by recognition of predefined NOE connectivity patterns. To discover these connectivities, the 2D NOE spectrum is examined, in a prescribed order, for the distinct NOE patterns characteristic of helices, sheets, turns, and extended chain. Finally, the recognition of a few amino acid side-chain types places the discovered secondary structure elements within the polypeptide sequences. Unlike the sequential assignment approach, the main-chain-directed strategy does not rely on the difficult task of recognizing many side-chain spin systems in J-correlated spectra, the assignment process is not in general sequential with the polypeptide chain, and the prescribed connectivity patterns are cyclic rather than linear. The latter characteristic avoids ambiguous branch points in the analysis and imposed an internally confirmatory property on each forward step

  2. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    International Nuclear Information System (INIS)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

    1991-01-01

    Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

  3. REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment

    Directory of Open Access Journals (Sweden)

    Hao Yang

    2018-01-01

    Full Text Available Bryostatin 1 (henceforth bryostatin is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

  4. Recommendations for the presentation of NMR structures of proteins and nucleic acids - IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy

    International Nuclear Information System (INIS)

    Markley, John L.; Bax, Ad; Arata, Yoji; Hilbers, C. W.; Kaptein, Robert; Sykes, Brian D.; Wright, Peter E.; Wuethrich, Kurt

    1998-01-01

    The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms

  5. Efficient Stereospecific Hβ2/3 NMR Assignment Strategy for Mid-Size Proteins

    Directory of Open Access Journals (Sweden)

    Alexandra Born

    2018-06-01

    Full Text Available We present a strategy for stereospecific NMR assignment of Hβ2 and Hβ3 protons in mid-size proteins (~150 residues. For such proteins, resonance overlap in standard experiments is severe, thereby preventing unambiguous assignment of a large fraction of β-methylenes. To alleviate this limitation, assignment experiments may be run in high static fields, where higher decoupling power is required. Three-bond Hα–Hβ J-couplings (3JHα–Hβ are critical for stereospecific assignments of β-methylene protons, and for determining rotameric χ1 states. Therefore, we modified a pulse sequence designed to measure accurate 3JHα–Hβ couplings such that probe heating was reduced, while the decoupling performance was improved. To further increase the resolution, we applied non-uniform sampling (NUS schemes in the indirect 1H and 13C dimensions. The approach was applied to two medium-sized proteins, odorant binding protein 22 (OBP22; 14.4 kDa and Pin1 (18.2 kDa, at 900 MHz polarizing fields. The coupling values obtained from NUS and linear sampling were extremely well correlated. However, NUS decreased the overlap of Hβ2/3 protons, thus supplying a higher yield of extracted 3JHα-Hβ coupling values when compared with linear sampling. A similar effect could be achieved with linear prediction applied to the linearly sampled data prior to the Fourier transformation. Finally, we used 3JHα–Hβ couplings from Pin1 in combination with either conventional or exact nuclear Overhauser enhancement (eNOE restraints to determine the stereospecific assignments of β-methylene protons. The use of eNOEs further increased the fraction of unambiguously assigned resonances when compared with procedures using conventional NOEs.

  6. Probing Early Misfolding Events in Prion Protein Mutants by NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Gregor Ilc

    2013-08-01

    Full Text Available The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify “hot spots” in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

  7. Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations

    International Nuclear Information System (INIS)

    Hong, M.; Jakes, K.

    1999-01-01

    The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated

  8. Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

    Science.gov (United States)

    Chen, Jia-Liang; Zhao, Yu; Gong, Yan-Jun; Pan, Bin-Bin; Wang, Xiao; Su, Xun-Cheng

    2018-02-01

    Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

  9. High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

    International Nuclear Information System (INIS)

    Yao, Yong; Dutta, Samit Kumar; Park, Sang Ho; Rai, Ratan; Fujimoto, L. Miya; Bobkov, Andrey A.; Opella, Stanley J.; Marassi, Francesca M.

    2017-01-01

    The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13 C or 1 H detection, have very narrow line widths (0.40–0.60 ppm for 13 C, 0.11–0.15 ppm for 1 H, and 0.46–0.64 ppm for 15 N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1 H-detected solid-state NMR 1 H/ 15 N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1 H/ 15 N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

  10. High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Yong; Dutta, Samit Kumar [Sanford Burnham Prebys Medical Discovery Institute (United States); Park, Sang Ho; Rai, Ratan [University of California San Diego, Department of Chemistry and Biochemistry (United States); Fujimoto, L. Miya; Bobkov, Andrey A. [Sanford Burnham Prebys Medical Discovery Institute (United States); Opella, Stanley J. [University of California San Diego, Department of Chemistry and Biochemistry (United States); Marassi, Francesca M., E-mail: fmarassi@sbp.edu [Sanford Burnham Prebys Medical Discovery Institute (United States)

    2017-03-15

    The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with {sup 13}C or {sup 1}H detection, have very narrow line widths (0.40–0.60 ppm for {sup 13}C, 0.11–0.15 ppm for {sup 1}H, and 0.46–0.64 ppm for {sup 15}N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The {sup 1}H-detected solid-state NMR {sup 1}H/{sup 15}N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR {sup 1}H/{sup 15}N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

  11. NMR approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes.

    Science.gov (United States)

    Dias, David M; Ciulli, Alessio

    2014-01-01

    Nuclear magnetic resonance (NMR) spectroscopy is a pivotal method for structure-based and fragment-based lead discovery because it is one of the most robust techniques to provide information on protein structure, dynamics and interaction at an atomic level in solution. Nowadays, in most ligand screening cascades, NMR-based methods are applied to identify and structurally validate small molecule binding. These can be high-throughput and are often used synergistically with other biophysical assays. Here, we describe current state-of-the-art in the portfolio of available NMR-based experiments that are used to aid early-stage lead discovery. We then focus on multi-protein complexes as targets and how NMR spectroscopy allows studying of interactions within the high molecular weight assemblies that make up a vast fraction of the yet untargeted proteome. Finally, we give our perspective on how currently available methods could build an improved strategy for drug discovery against such challenging targets. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR

    International Nuclear Information System (INIS)

    Takeuchi, Koh; Arthanari, Haribabu; Shimada, Ichio; Wagner, Gerhard

    2015-01-01

    Detection of 15 N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached 15 N nuclei (TROSY 15 N H ) yields high quality spectra in high field magnets (>600 MHz) by taking advantage of the slow 15 N transverse relaxation and compensating for the inherently low 15 N sensitivity. The 15 N TROSY transverse relaxation rates increase modestly with molecular weight but the TROSY gain in peak heights depends strongly on the magnetic field strength. Theoretical simulations predict that the narrowest line width for the TROSY 15 N H component can be obtained at 900 MHz, but sensitivity reaches its maximum around 1.2 GHz. Based on these considerations, a 15 N-detected 2D 1 H– 15 N TROSY-HSQC ( 15 N-detected TROSY-HSQC) experiment was developed and high-quality 2D spectra were recorded at 800 MHz in 2 h for 1 mM maltose-binding protein at 278 K (τ c  ∼ 40 ns). Unlike for 1 H detected TROSY, deuteration is not mandatory to benefit 15 N detected TROSY due to reduced dipolar broadening, which facilitates studies of proteins that cannot be deuterated, especially in cases where production requires eukaryotic expression systems. The option of recording 15 N TROSY of proteins expressed in H 2 O media also alleviates the problem of incomplete amide proton back exchange, which often hampers the detection of amide groups in the core of large molecular weight proteins that are expressed in D 2 O culture media and cannot be refolded for amide back exchange. These results illustrate the potential of 15 N H -detected TROSY experiments as a means to exploit the high resolution offered by high field magnets near and above 1 GHz

  13. Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR

    Energy Technology Data Exchange (ETDEWEB)

    Takeuchi, Koh [National Institute for Advanced Industrial Science and Technology, Molecular Profiling Research Center for Drug Discovery (Japan); Arthanari, Haribabu [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States); Shimada, Ichio, E-mail: shimada@iw-nmr.f.u-tokyo.ac.jp [National Institute for Advanced Industrial Science and Technology, Molecular Profiling Research Center for Drug Discovery (Japan); Wagner, Gerhard, E-mail: gerhard-wagner@hms.harvard.edu [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States)

    2015-12-15

    Detection of {sup 15}N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached {sup 15}N nuclei (TROSY {sup 15}N{sub H}) yields high quality spectra in high field magnets (>600 MHz) by taking advantage of the slow {sup 15}N transverse relaxation and compensating for the inherently low {sup 15}N sensitivity. The {sup 15}N TROSY transverse relaxation rates increase modestly with molecular weight but the TROSY gain in peak heights depends strongly on the magnetic field strength. Theoretical simulations predict that the narrowest line width for the TROSY {sup 15}N{sub H} component can be obtained at 900 MHz, but sensitivity reaches its maximum around 1.2 GHz. Based on these considerations, a {sup 15}N-detected 2D {sup 1}H–{sup 15}N TROSY-HSQC ({sup 15}N-detected TROSY-HSQC) experiment was developed and high-quality 2D spectra were recorded at 800 MHz in 2 h for 1 mM maltose-binding protein at 278 K (τ{sub c} ∼ 40 ns). Unlike for {sup 1}H detected TROSY, deuteration is not mandatory to benefit {sup 15}N detected TROSY due to reduced dipolar broadening, which facilitates studies of proteins that cannot be deuterated, especially in cases where production requires eukaryotic expression systems. The option of recording {sup 15}N TROSY of proteins expressed in H{sub 2}O media also alleviates the problem of incomplete amide proton back exchange, which often hampers the detection of amide groups in the core of large molecular weight proteins that are expressed in D{sub 2}O culture media and cannot be refolded for amide back exchange. These results illustrate the potential of {sup 15}N{sub H}-detected TROSY experiments as a means to exploit the high resolution offered by high field magnets near and above 1 GHz.

  14. Probing the Structure and Dynamics of Proteins by Combining Molecular Dynamics Simulations and Experimental NMR Data.

    Science.gov (United States)

    Allison, Jane R; Hertig, Samuel; Missimer, John H; Smith, Lorna J; Steinmetz, Michel O; Dolenc, Jožica

    2012-10-09

    NMR experiments provide detailed structural information about biological macromolecules in solution. However, the amount of information obtained is usually much less than the number of degrees of freedom of the macromolecule. Moreover, the relationships between experimental observables and structural information, such as interatomic distances or dihedral angle values, may be multiple-valued and may rely on empirical parameters and approximations. The extraction of structural information from experimental data is further complicated by the time- and ensemble-averaged nature of NMR observables. Combining NMR data with molecular dynamics simulations can elucidate and alleviate some of these problems, as well as allow inconsistencies in the NMR data to be identified. Here, we use a number of examples from our work to highlight the power of molecular dynamics simulations in providing a structural interpretation of solution NMR data.

  15. Gravity-driven pH adjustment for site-specific protein pKa measurement by solution-state NMR

    Science.gov (United States)

    Li, Wei

    2017-12-01

    To automate pH adjustment in site-specific protein pKa measurement by solution-state NMR, I present a funnel with two caps for the standard 5 mm NMR tube. The novelty of this simple-to-build and inexpensive apparatus is that it allows automatic gravity-driven pH adjustment within the magnet, and consequently results in a fully automated NMR-monitored pH titration without any hardware modification on the NMR spectrometer.

  16. Myristoylation as a general method for immobilization and alignment of soluble proteins for solid-state NMR structural studies

    International Nuclear Information System (INIS)

    Mesleh, M.F.; Valentine, K.G.; Opella, S.J.; Louis, J.M.; Gronenborn, A.M.

    2003-01-01

    N-terminal myristoylation of the immunoglobulin-binding domain of protein G (GB1) from group G Streptococcus provides the means to bind the protein to aligned phospholipid bilayers for solid-state NMR structural studies. The myristoylated protein is immobilized by its interactions with bilayers, and the sample alignment enables orientationally dependent 15 N chemical shifts and 1 H- 15 N-dipolar couplings to be measured. Spectra calculated for the average solution NMR structure of the protein at various orientations with respect to the magnetic field direction were compared to the experimental spectrum. The best fit identified the orientation of the myristoylated protein on the lipid bilayers, and demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms, and that the structure is not grossly distorted by its interaction with the phosholipid bilayer surface or by its location in the restricted aqueous space between bilayer leaflets. The protein is oriented such that its charged sides face the phosphatidylcholine headgroups of the lipids with the single amphiphilic helix running parallel to the bilayer surface

  17. Potential Antifreeze Compounds in Present-Day Martian Seepage Groundwater

    Directory of Open Access Journals (Sweden)

    Jiin-Shuh Jean

    2008-01-01

    Full Text Available Is the recently found seepage groundwater on Mars pure H2O, or mixed with salts and other antifreeze compounds? Given the surface conditions of Mars, it is unlikely that pure water could either exist in its liquid state or have shaped Mars¡¦ fluid erosional landforms (gullies, channels, and valley networks. More likely is that Mars¡¦ seepage groundwater contains antifreeze and salt compounds that resist freezing and suppress evaporation. This model better accounts for Mars¡¦ enigmatic surface erosion. This paper suggests 17 antifreeze compounds potentially present in Martian seepage groundwater. Given their liquid state and physical properties, triethylene glycol, diethylene glycol, ethylene glycol, and 1,3-propylene glycol are advanced as the most likely candidate compounds. This paper also explores how a mixing of glycol or glycerol with salts in the Martian seepage groundwater may have lowered water¡¦s freezing point and raised its boiling point, with consequences that created fluid gully and channel erosion. Ethylene glycol and related hydrocarbon compounds have been identified in Martian and other interstellar meteorites. We suggest that these compounds and their proportions to water be included for detection in future explorations.

  18. Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study

    Energy Technology Data Exchange (ETDEWEB)

    Rajagopal, P.; Klevit, R.E. [Univ. of Washington, Seattle, WA (United States)

    1994-12-01

    HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residue at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.

  19. Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles

    International Nuclear Information System (INIS)

    Stanczak, Pawel; Zhang Qinghai; Horst, Reto; Serrano, Pedro; Wüthrich, Kurt

    2012-01-01

    Optimization of aqueous solutions of the integral membrane protein (IMP) OmpW for NMR structure determination has been monitored with micro-coil NMR, which enables the acquisition of NMR spectra using only micrograms of protein and detergent. The detergent 30-Fos (2-undecylphosphocholine) was found to yield the best 2D [ 15 N, 1 H]-TROSY correlation NMR spectra of [ 2 H, 15 N]-labeled OmpW. For the OmpW structure determination we then optimized the 30-Fos concentration, the sample temperature and long-time stability, and the deuteration level of the protein. Some emerging guidelines for reconstitution of β-barrel integral membrane proteins in structural biology are discussed.

  20. Site-specific tagging proteins with a rigid, small and stable transition metal chelator, 8-hydroxyquinoline, for paramagnetic NMR analysis

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yin; Huang, Feng [Nankai University, State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) (China); Huber, Thomas [Australian National University, Research School of Chemistry (Australia); Su, Xun-Cheng, E-mail: xunchengsu@nankai.edu.cn [Nankai University, State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) (China)

    2016-02-15

    Design of a paramagnetic metal binding motif in a protein is a valuable way for understanding the function, dynamics and interactions of a protein by paramagnetic NMR spectroscopy. Several strategies have been proposed to site-specifically tag proteins with paramagnetic lanthanide ions. Here we report a simple approach of engineering a transition metal binding motif via site-specific labelling of a protein with 2-vinyl-8-hydroxyquinoline (2V-8HQ). The protein-2V-8HQ adduct forms a stable complex with transition metal ions, Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). The paramagnetic effects generated by these transition metal ions were evaluated by NMR spectroscopy. We show that 2V-8HQ is a rigid and stable transition metal binding tag. The coordination of the metal ion can be assisted by protein sidechains. More importantly, tunable paramagnetic tensors are simply obtained in an α-helix that possesses solvent exposed residues in positions i and i + 3, where i is the residue to be mutated to cysteine, i + 3 is Gln or Glu or i − 4 is His. The coordination of a sidechain carboxylate/amide or imidazole to cobalt(II) results in different structural geometries, leading to different paramagnetic tensors as shown by experimental data.

  1. Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

    Science.gov (United States)

    Panthu, Baptiste; Ohlmann, Théophile; Perrier, Johan; Schlattner, Uwe; Jalinot, Pierre; Elena-Herrmann, Bénédicte; Rautureau, Gilles J P

    2018-01-19

    A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

  2. NMR spin relaxation in proteins: The patterns of motion that dissipate power to the bath

    Energy Technology Data Exchange (ETDEWEB)

    Shapiro, Yury E., E-mail: eva.meirovitch@biu.ac.il, E-mail: yuryeshapiro@gmail.com; Meirovitch, Eva, E-mail: eva.meirovitch@biu.ac.il, E-mail: yuryeshapiro@gmail.com [The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900-02 (Israel)

    2014-04-21

    We developed in recent years the two-body coupled-rotator slowly relaxing local structure (SRLS) approach for the analysis of NMR relaxation in proteins. The two bodies/rotators are the protein (diffusion tensor D{sub 1}) and the spin-bearing probe, e.g., the {sup 15}N−{sup 1}H bond (diffusion tensor, D{sub 2}), coupled by a local potential (u). A Smoluchowski equation is solved to yield the generic time correlation functions (TCFs), which are sums of weighted exponentials (eigenmodes). By Fourier transformation one obtains the generic spectral density functions (SDFs) which underlie the experimental relaxation parameters. The typical paradigm is to characterize structural dynamics in terms of the best-fit values of D{sub 1}, D{sub 2}, and u. Additional approaches we pursued employ the SRLS TCFs, SDFs, or eigenmodes as descriptors. In this study we develop yet another perspective. We consider the SDF as function of the angular velocity associated with the fluctuating fields underlying NMR relaxation. A parameter called j-fraction, which represents the relative contribution of eigenmode, i, to a given value of the SDF function at a specific frequency, ω, is defined. j-fraction profiles of the dominant eigenmodes are derived for 0 ≤ ω ≤ 10{sup 12} rad/s. They reveal which patterns of motion actuate power dissipation at given ω-values, what are their rates, and what is their relative contribution. Simulations are carried out to determine the effect of timescale separation, D{sub 1}/D{sub 2}, axial potential strength, and local diffusion axiality. For D{sub 1}/D{sub 2} ≤ 0.01 and strong local potential of 15 k{sub B}T, power is dissipated by global diffusion, renormalized (by the strong potential) local diffusion, and probe diffusion on the surface of a cone (to be called cone diffusion). For D{sub 1}/D{sub 2} = 0.1, power is dissipated by mixed eigenmodes largely of a global-diffusion-type or cone-diffusion-type, and a nearly bare renormalized

  3. Bacterial Ice Crystal Controlling Proteins

    Science.gov (United States)

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  4. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    International Nuclear Information System (INIS)

    Zanzoni, Serena; D’Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

    2012-01-01

    Highlights: ► Bile acid binding proteins from different constructs retain structural integrity. ► NMR 15 N-T 1 relaxation data of BABPs show differences if LVPR extension is present. ► Deviations from a 15 N-T 1 /molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  5. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zanzoni, Serena; D' Onofrio, Mariapina; Molinari, Henriette [Department of Biotechnology, University of Verona, 37134 Verona (Italy); Assfalg, Michael, E-mail: michael.assfalg@univr.it [Department of Biotechnology, University of Verona, 37134 Verona (Italy)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  6. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    Energy Technology Data Exchange (ETDEWEB)

    Smet-Nocca, Caroline, E-mail: caroline.smet@univ-lille1.fr; Launay, Helene; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle, E-mail: isabelle.landrieu@univ-lille1.fr [Universite de Lille-Nord de France, Institut Federatif de Recherches 147, CNRS UMR 8576 (France)

    2013-04-15

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the {sup 1}H,{sup 15}N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  7. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    International Nuclear Information System (INIS)

    Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle

    2013-01-01

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1 H, 15 N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  8. Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

    Energy Technology Data Exchange (ETDEWEB)

    Monneau, Yoan R. [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Ishida, Yojiro [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Rossi, Paolo; Saio, Tomohide; Tzeng, Shiou-Ru [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Inouye, Masayori, E-mail: inouye@cabm.rutgers.edu [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Kalodimos, Charalampos G., E-mail: ckalodim@umn.edu [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States)

    2016-06-15

    A simple and cost effective method to independently and stereo-specifically incorporate [{sup 1}H,{sup 13}C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [{sup 1}H,{sup 13}C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [{sup 1}H,{sup 13}C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-{sup 13}C,2,3-{sup 2}H{sub 2},{sup 15}N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[{sup 13}CH{sub 3}]{sup γ2} labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for {sup 2}H-glycine to prevent scrambling.

  9. Flow-through lipid nanotube arrays for structure-function studies of membrane proteins by solid-state NMR spectroscopy.

    Science.gov (United States)

    Chekmenev, Eduard Y; Gor'kov, Peter L; Cross, Timothy A; Alaouie, Ali M; Smirnov, Alex I

    2006-10-15

    A novel method for studying membrane proteins in a native lipid bilayer environment by solid-state NMR spectroscopy is described and tested. Anodic aluminum oxide (AAO) substrates with flow-through 175 nm wide and 60-mum-long nanopores were employed to form macroscopically aligned peptide-containing lipid bilayers that are fluid and highly hydrated. We demonstrate that the surfaces of both leaflets of such bilayers are fully accessible to aqueous solutes. Thus, high hydration levels as well as pH and desirable ion and/or drug concentrations could be easily maintained and modified as desired in a series of experiments with the same sample. The method allows for membrane protein NMR experiments in a broad pH range that could be extended to as low as 1 and as high as 12 units for a period of up to a few hours and temperatures as high as 70 degrees C without losing the lipid alignment or bilayers from the nanopores. We demonstrate the utility of this method by a solid-state 19.6 T (17)O NMR study of reversible binding effects of mono- and divalent ions on the chemical shift properties of the Leu(10) carbonyl oxygen of transmembrane pore-forming peptide gramicidin A (gA). We further compare the (17)O shifts induced by binding metal ions to the binding of protons in the pH range from 1 to 12 and find a significant difference. This unexpected result points to a difference in mechanisms for ion and proton conduction by the gA pore. We believe that a large number of solid-state NMR-based studies, including structure-function, drug screening, proton exchange, pH, and other titration experiments, will benefit significantly from the method described here.

  10. Towards fully automated structure-based NMR resonance assignment of 15N-labeled proteins from automatically picked peaks

    KAUST Repository

    Jang, Richard; Gao, Xin; Li, Ming

    2011-01-01

    In NMR resonance assignment, an indispensable step in NMR protein studies, manually processed peaks from both N-labeled and C-labeled spectra are typically used as inputs. However, the use of homologous structures can allow one to use only N-labeled NMR data and avoid the added expense of using C-labeled data. We propose a novel integer programming framework for structure-based backbone resonance assignment using N-labeled data. The core consists of a pair of integer programming models: one for spin system forming and amino acid typing, and the other for backbone resonance assignment. The goal is to perform the assignment directly from spectra without any manual intervention via automatically picked peaks, which are much noisier than manually picked peaks, so methods must be error-tolerant. In the case of semi-automated/manually processed peak data, we compare our system with the Xiong-Pandurangan-Bailey- Kellogg's contact replacement (CR) method, which is the most error-tolerant method for structure-based resonance assignment. Our system, on average, reduces the error rate of the CR method by five folds on their data set. In addition, by using an iterative algorithm, our system has the added capability of using the NOESY data to correct assignment errors due to errors in predicting the amino acid and secondary structure type of each spin system. On a publicly available data set for human ubiquitin, where the typing accuracy is 83%, we achieve 91% accuracy, compared to the 59% accuracy obtained without correcting for such errors. In the case of automatically picked peaks, using assignment information from yeast ubiquitin, we achieve a fully automatic assignment with 97% accuracy. To our knowledge, this is the first system that can achieve fully automatic structure-based assignment directly from spectra. This has implications in NMR protein mutant studies, where the assignment step is repeated for each mutant. © Copyright 2011, Mary Ann Liebert, Inc.

  11. Towards fully automated structure-based NMR resonance assignment of 15N-labeled proteins from automatically picked peaks

    KAUST Repository

    Jang, Richard

    2011-03-01

    In NMR resonance assignment, an indispensable step in NMR protein studies, manually processed peaks from both N-labeled and C-labeled spectra are typically used as inputs. However, the use of homologous structures can allow one to use only N-labeled NMR data and avoid the added expense of using C-labeled data. We propose a novel integer programming framework for structure-based backbone resonance assignment using N-labeled data. The core consists of a pair of integer programming models: one for spin system forming and amino acid typing, and the other for backbone resonance assignment. The goal is to perform the assignment directly from spectra without any manual intervention via automatically picked peaks, which are much noisier than manually picked peaks, so methods must be error-tolerant. In the case of semi-automated/manually processed peak data, we compare our system with the Xiong-Pandurangan-Bailey- Kellogg\\'s contact replacement (CR) method, which is the most error-tolerant method for structure-based resonance assignment. Our system, on average, reduces the error rate of the CR method by five folds on their data set. In addition, by using an iterative algorithm, our system has the added capability of using the NOESY data to correct assignment errors due to errors in predicting the amino acid and secondary structure type of each spin system. On a publicly available data set for human ubiquitin, where the typing accuracy is 83%, we achieve 91% accuracy, compared to the 59% accuracy obtained without correcting for such errors. In the case of automatically picked peaks, using assignment information from yeast ubiquitin, we achieve a fully automatic assignment with 97% accuracy. To our knowledge, this is the first system that can achieve fully automatic structure-based assignment directly from spectra. This has implications in NMR protein mutant studies, where the assignment step is repeated for each mutant. © Copyright 2011, Mary Ann Liebert, Inc.

  12. Measurement of conformational constraints in an elastin-mimetic protein by residue-pair selected solid-state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Mei [Iowa State University, Department of Chemistry (United States)], E-mail: mhong@iastate.edu; McMillan, R. Andrew; Conticello, Vincent P. [Emory University, Department of Chemistry (United States)

    2002-02-15

    We introduce a solid-state NMR technique for selective detection of a residue pair in multiply labeled proteins to obtain site-specific structural constraints. The method exploits the frequency-offset dependence of cross polarization to achieve {sup 13}CO{sub i} {sup {yields}} {sup 15}N{sub i} {sup {yields}} {sup 13}C{alpha}{sub i} transfer between two residues. A {sup 13}C, {sup 15}N-labeled elastin mimetic protein (VPGVG){sub n} is used to demonstrate the method. The technique selected the Gly3 C{alpha} signal while suppressing the Gly5 C{alpha} signal, and allowed the measurement of the Gly3 C{alpha} chemical shift anisotropy to derive information on the protein conformation. This residue-pair selection technique should simplify the study of protein structure at specific residues.

  13. Measurement of conformational constraints in an elastin-mimetic protein by residue-pair selected solid-state NMR

    International Nuclear Information System (INIS)

    Hong, Mei; McMillan, R. Andrew; Conticello, Vincent P.

    2002-01-01

    We introduce a solid-state NMR technique for selective detection of a residue pair in multiply labeled proteins to obtain site-specific structural constraints. The method exploits the frequency-offset dependence of cross polarization to achieve 13 CO i → 15 N i → 13 Cα i transfer between two residues. A 13 C, 15 N-labeled elastin mimetic protein (VPGVG) n is used to demonstrate the method. The technique selected the Gly3 Cα signal while suppressing the Gly5 Cα signal, and allowed the measurement of the Gly3 Cα chemical shift anisotropy to derive information on the protein conformation. This residue-pair selection technique should simplify the study of protein structure at specific residues

  14. MERA: a webserver for evaluating backbone torsion angle distributions in dynamic and disordered proteins from NMR data

    Energy Technology Data Exchange (ETDEWEB)

    Mantsyzov, Alexey B. [M.V. Lomonosov Moscow State University, Faculty of Fundamental Medicine (Russian Federation); Shen, Yang; Lee, Jung Ho [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Hummer, Gerhard [Max Planck Institute of Biophysics (Germany); Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2015-09-15

    MERA (Maximum Entropy Ramachandran map Analysis from NMR data) is a new webserver that generates residue-by-residue Ramachandran map distributions for disordered proteins or disordered regions in proteins on the basis of experimental NMR parameters. As input data, the program currently utilizes up to 12 different parameters. These include three different types of short-range NOEs, three types of backbone chemical shifts ({sup 15}N, {sup 13}C{sup α}, and {sup 13}C′), six types of J couplings ({sup 3}J{sub HNHα}, {sup 3}J{sub C′C′}, {sup 3}J{sub C′Hα}, {sup 1}J{sub HαCα}, {sup 2}J{sub CαN} and {sup 1}J{sub CαN}), as well as the {sup 15}N-relaxation derived J(0) spectral density. The Ramachandran map distributions are reported in terms of populations of their 15° × 15° voxels, and an adjustable maximum entropy weight factor is available to ensure that the obtained distributions will not deviate more from a newly derived coil library distribution than required to account for the experimental data. MERA output includes the agreement between each input parameter and its distribution-derived value. As an application, we demonstrate performance of the program for several residues in the intrinsically disordered protein α-synuclein, as well as for several static and dynamic residues in the folded protein GB3.

  15. Application of NMR Screening Methods with 19F Detection to Fluorinated Compounds Bound to Proteins

    Directory of Open Access Journals (Sweden)

    Kazuo Furihata

    2017-12-01

    Full Text Available The combinational use of one-dimensional (1D NMR-based screening techniques with 1H and 19F detections were applied to a human serum albumin–diflunisal complex. Since most NMR screening methods observe 1H spectra, the overlapped 1H signals were unavailable in the binding epitope mapping. However, the NMR experiments with 19F detection can be used as an effective complementary method. For the purpose of identifying the 1H and 19F binding epitopes of diflunisal, this paper carries out a combinatorial analysis using 1H{1H} and 19F{1H} saturation transfer difference experiments. The differences of the 1H-inversion recovery rates with and without target irradiation are also analyzed for a comprehensive interpretation of binding epitope mapping.

  16. 1H NMR of High-Potential Iron-Sulfur Protein from the Purple Non-Sulfur Bacterium Rhodoferax fermentans

    DEFF Research Database (Denmark)

    Ciurli, Stefano; Cremonini, Mauro Andrea; Kofod, Pauli

    1996-01-01

    residues bound to the [4Fe-4S]3+/2+ cluster have been performed using one-dimensional NOE and exchange spectroscopy experiments. 1H-NMR hyperfine shifts and relaxation rates of cluster-bound Cys β-CH2 protons indicate that in the [4Fe-4S]3+ cluster one iron ion can be formally described as Fe(III), while......Oxidized and reduced forms of high-potential iron-sulfur protein (HiPIP) from the purple non-sulfur photosynthetic bacterium Rhodoferux fermentans have been characterized using 1H-NMR spectroscopy. Pairwise and sequence-specific assignments of hyperfine-shifted 1H-NMR signals to protons of cysteine...... longitudinal relaxation rates of Cys β-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-Cβ-Cα dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligeable contribution from a ligand-centered dipolar mechanism which involves...

  17. Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies

    Science.gov (United States)

    2018-01-01

    Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents. PMID:29488756

  18. Anti-freezing of air-cooled heat exchanger by switching off sectors

    International Nuclear Information System (INIS)

    Wang, Weijia; Kong, Yanqiang; Huang, Xianwei; Yang, Lijun; Du, Xiaoze; Yang, Yongping

    2017-01-01

    Highlights: • The anti-freezing of air-cooled heat exchanger by switching off sectors is studied. • The water side heat loads of various sectors are compared for different cases. • Anti-freezing turbine back pressure is proposed and obtained for various cases. • As wind speed increases, the energy efficiency can be clearly improved by sector off. • By switching frontal sector off, anti-freezing operation is most energy efficient. - Abstract: With the air side huge heat transfer surface, the air-cooled heat exchanger will take a serious freezing risk in cold winter. Therefore, it is of benefit to the safe operation of natural draft dry cooling system to propose the anti-freezing measures. In this work, the flow and heat transfer models of the cooling air coupling with the circulating water, are developed and numerically simulated for the anti-freezing by switching various sectors off. The local thermo-flow fields of cooling air are presented, and the water side heat loads of various sectors are compared for various cases. The anti-freezing turbine back pressure is proposed and obtained for the energy efficiency analysis. The results show that the sector switching off approach can effectively prevent the air-cooled heat exchanger from freezing and improve the energy efficiency of the cooling system, especially at high wind speeds. Moreover, with the frontal sector switching off, the most energy efficient anti-freezing operation of natural draft dry cooling system can be achieved.

  19. Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements

    Energy Technology Data Exchange (ETDEWEB)

    Eghbalnia, Hamid R.; Wang Liya; Bahrami, Arash [National Magnetic Resonance Facility at Madison, Biochemistry Department (United States); Assadi, Amir [University of Wisconsin-Madison, Mathematics Department (United States); Markley, John L. [National Magnetic Resonance Facility at Madison, Biochemistry Department (United States)], E-mail: eghbalni@nmrfam.wisc.edu

    2005-05-15

    We present an energy model that combines information from the amino acid sequence of a protein and available NMR chemical shifts for the purposes of identifying low energy conformations and determining elements of secondary structure. The model ('PECAN', Protein Energetic Conformational Analysis from NMR chemical shifts) optimizes a combination of sequence information and residue-specific statistical energy function to yield energetic descriptions most favorable to predicting secondary structure. Compared to prior methods for secondary structure determination, PECAN provides increased accuracy and range, particularly in regions of extended structure. Moreover, PECAN uses the energetics to identify residues located at the boundaries between regions of predicted secondary structure that may not fit the stringent secondary structure class definitions. The energy model offers insights into the local energetic patterns that underlie conformational preferences. For example, it shows that the information content for defining secondary structure is localized about a residue and reaches a maximum when two residues on either side are considered. The current release of the PECAN software determines the well-defined regions of secondary structure in novel proteins with assigned chemical shifts with an overall accuracy of 90%, which is close to the practical limit of achievable accuracy in classifying the states.

  20. Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements

    International Nuclear Information System (INIS)

    Eghbalnia, Hamid R.; Wang Liya; Bahrami, Arash; Assadi, Amir; Markley, John L.

    2005-01-01

    We present an energy model that combines information from the amino acid sequence of a protein and available NMR chemical shifts for the purposes of identifying low energy conformations and determining elements of secondary structure. The model ('PECAN', Protein Energetic Conformational Analysis from NMR chemical shifts) optimizes a combination of sequence information and residue-specific statistical energy function to yield energetic descriptions most favorable to predicting secondary structure. Compared to prior methods for secondary structure determination, PECAN provides increased accuracy and range, particularly in regions of extended structure. Moreover, PECAN uses the energetics to identify residues located at the boundaries between regions of predicted secondary structure that may not fit the stringent secondary structure class definitions. The energy model offers insights into the local energetic patterns that underlie conformational preferences. For example, it shows that the information content for defining secondary structure is localized about a residue and reaches a maximum when two residues on either side are considered. The current release of the PECAN software determines the well-defined regions of secondary structure in novel proteins with assigned chemical shifts with an overall accuracy of 90%, which is close to the practical limit of achievable accuracy in classifying the states

  1. Nano-mole scale sequential signal assignment by 1 H-detected protein solid-state NMR

    KAUST Repository

    Wang, Songlin; Parthasarathy, Sudhakar; Xiao, Yiling; Nishiyama, Yusuke; Long, Fei; Matsuda, Isamu; Endo, Yuki; Nemoto, Takahiro; Yamauchi, Kazuo; Asakura, Tetsuo; Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune; Ishii, Yoshitaka

    2015-01-01

    We present a 3D 1H-detected solid-state NMR (SSNMR) approach for main-chain signal assignments of 10-100 nmol of fully protonated proteins using ultra-fast magic-angle spinning (MAS) at ∼80 kHz by a novel spectral-editing method, which permits drastic spectral simplification. The approach offers ∼110 fold time saving over a traditional 3D 13C-detected SSNMR approach. This journal is © The Royal Society of Chemistry 2015.

  2. NMR structure of the N-terminal domain of capsid protein from the Mason-Pfizer monkey virus

    Czech Academy of Sciences Publication Activity Database

    Macek, Pavel; Chmelík, Josef; Křížová, Ivana; Kadeřávek, P.; Padrta, P.; Žídek, L.; Wildová, Marcela; Hadravová, Romana; Chaloupková, R.; Pichová, Iva; Ruml, T.; Rumlová, Michaela; Sklenář, V.

    2009-01-01

    Roč. 392, č. 1 (2009), s. 100-114 ISSN 0022-2836 R&D Projects: GA MŠk LC545; GA MŠk 1M0508; GA ČR GA204/09/1388; GA ČR GESCO/06/E001 Grant - others:GA MŠk(CZ) 1M0520; MŠk(CZ) LC06030 Program:1M; LC Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50200510 Keywords : M-PMV * betaretroviruses * capsid protein * NMR structure * internal dynamics Subject RIV: CE - Biochemistry Impact factor: 3.871, year: 2009

  3. High-field EPR on membrane proteins - crossing the gap to NMR.

    Science.gov (United States)

    Möbius, Klaus; Lubitz, Wolfgang; Savitsky, Anton

    2013-11-01

    In this review on advanced EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR concerning the measurement of molecular interactions in large biomolecules. From these interactions, detailed information can be revealed on structure and dynamics of macromolecules embedded in solution- or solid-state environments. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed to new horizons the limits of EPR spectroscopy and its multifrequency extensions concerning the sensitivity of detection, the selectivity with respect to interactions, and the resolution in frequency and time domains. One of the most important advances has been the extension of EPR to high magnetic fields and microwave frequencies, very much in analogy to what happens in NMR. This is exemplified by referring to ongoing efforts for signal enhancement in both NMR and EPR double-resonance techniques by exploiting dynamic nuclear or electron spin polarization via unpaired electron spins and their electron-nuclear or electron-electron interactions. Signal and resolution enhancements are particularly spectacular for double-resonance techniques such as ENDOR and PELDOR at high magnetic fields. They provide greatly improved orientational selection for disordered samples that approaches single-crystal resolution at canonical g-tensor orientations - even for molecules with small g-anisotropies. Exchange of experience between the EPR and NMR communities allows for handling polarization and resolution improvement strategies in an optimal manner. Consequently, a dramatic improvement of EPR detection sensitivity could be achieved, even for short-lived paramagnetic reaction intermediates. Unique structural and dynamic information is thus revealed that can hardly be obtained by any other analytical techniques. Micromolar

  4. Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mote, Kaustubh R. [University of Minnesota, Department of Chemistry (United States); Gopinath, T. [University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics (United States); Veglia, Gianluigi, E-mail: vegli001@umn.edu [University of Minnesota, Department of Chemistry (United States)

    2013-10-15

    The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD {approx}0.44 A, a tilt angle of 24 Degree-Sign {+-} 1 Degree-Sign , and an azimuthal angle of 55 Degree-Sign {+-} 6 Degree-Sign . This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.

  5. Crystallization and preliminary X-ray crystallographic analysis of the NmrA-like DDB-G0286605 protein from the social amoeba Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Kim, Min-Kyu; Yim, Hyung-Soon; Kang, Sa-Ouk

    2010-01-01

    In order to investigate its structure and function, the NmrA-like domain-containing DDB-G0286605 protein from D. discoideum was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 1.64 Å. The DDB-G0286605 gene product from Dictyostelium discoideum, an NmrA-like protein that belongs to the short-chain dehydrogenase/reductase family, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.64 Å resolution data set was collected using synchrotron radiation. The DDB-G0286605 protein crystals belonged to space group P2 1 , with unit-cell parameters a = 67.598, b = 54.935, c = 84.219 Å, β = 109.620°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 43.25% with 99% probability. Molecular-replacement trials were attempted with three NmrA-like proteins, NmrA, HSCARG and QOR2, as search models, but failed. This may be a consequence of the low sequence identity between the DDB-G0286605 protein and the search models (DDB-G0286605 has a primary-sequence identity of 28, 32 and 19% to NmrA, HCARG and QOR2, respectively)

  6. An improved ultrafast 2D NMR experiment: Towards atom-resolved real-time studies of protein kinetics at multi-Hz rates

    International Nuclear Information System (INIS)

    Gal, Maayan; Kern, Thomas; Schanda, Paul; Frydman, Lucio; Brutscher, Bernhard

    2009-01-01

    Multidimensional NMR spectroscopy is a well-established technique for the characterization of structure and fast-time-scale dynamics of highly populated ground states of biological macromolecules. The investigation of short-lived excited states that are important for molecular folding, misfolding and function, however, remains a challenge for modern biomolecular NMR techniques. Off-equilibrium real-time kinetic NMR methods allow direct observation of conformational or chemical changes by following peak positions and intensities in a series of spectra recorded during a kinetic event. Because standard multidimensional NMR methods required to yield sufficient atom-resolution are intrinsically time-consuming, many interesting phenomena are excluded from real-time NMR analysis. Recently, spatially encoded ultrafast 2D NMR techniques have been proposed that allow one to acquire a 2D NMR experiment within a single transient. In addition, when combined with the SOFAST technique, such ultrafast experiments can be repeated at high rates. One of the problems detected for such ultrafast protein NMR experiments is related to the heteronuclear decoupling during detection with interferences between the pulses and the oscillatory magnetic field gradients arising in this scheme. Here we present a method for improved ultrafast data acquisition yielding higher signal to noise and sharper lines in single-scan 2D NMR spectra. In combination with a fast-mixing device, the recording of 1 H- 15 N correlation spectra with repetition rates of up to a few Hertz becomes feasible, enabling real-time studies of protein kinetics occurring on time scales down to a few seconds

  7. Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex

    International Nuclear Information System (INIS)

    Hennig, Janosch; Wang, Iren; Sonntag, Miriam; Gabel, Frank; Sattler, Michael

    2013-01-01

    Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.

  8. Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex

    Energy Technology Data Exchange (ETDEWEB)

    Hennig, Janosch; Wang, Iren; Sonntag, Miriam [Institute of Structural Biology, Helmholtz Zentrum Muenchen (Germany); Gabel, Frank [Extremophiles and Large Molecular Assemblies Group (ELMA), Institut de Biologie Structurale (IBS) CEA-CNRS-UJF (France); Sattler, Michael, E-mail: sattler@helmholtz-muenchen.de [Institute of Structural Biology, Helmholtz Zentrum Muenchen (Germany)

    2013-05-15

    Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.

  9. Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations

    Science.gov (United States)

    Baxa, Michael C.; Haddadian, Esmael J.; Jumper, John M.; Freed, Karl F.; Sosnick, Tobin R.

    2014-01-01

    The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔSTotal = 1.4 kcal⋅mol−1 per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (ΩU/ΩN). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔShelix−sheet = 0.5 kcal⋅mol−1), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR. PMID:25313044

  10. Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations

    International Nuclear Information System (INIS)

    Bernado, Pau; Fernandes, Miguel X.; Jacobs, Doris M.; Fiebig, Klaus; Garcia de la Torre, Jose; Pons, Miquel

    2004-01-01

    Many important proteins contain multiple domains connected by flexible linkers. Inter-domain motion is suggested to play a key role in many processes involving molecular recognition. Heteronuclear NMR relaxation is sensitive to motions in the relevant time scales and could provide valuable information on the dynamics of multi-domain proteins. However, the standard analysis based on the separation of global tumbling and fast local motions is no longer valid for multi-domain proteins undergoing internal motions involving complete domains and that take place on the same time scale than the overall motion.The complexity of the motions experienced even for the simplest two-domain proteins are difficult to capture with simple extensions of the classical Lipari-Szabo approach. Hydrodynamic effects are expected to dominate the motion of the individual globular domains, as well as that of the complete protein. Using Pin1 as a test case, we have simulated its motion at the microsecond time scale, at a reasonable computational expense, using Brownian Dynamic simulations on simplified models. The resulting trajectories provide insight on the interplay between global and inter-domain motion and can be analyzed using the recently published method of isotropic Reorientational Mode Dynamics which offer a way of calculating their contribution to heteronuclear relaxation rates. The analysis of trajectories computed with Pin1 models of different flexibility provides a general framework to understand the dynamics of multi-domain proteins and explains some of the observed features in the relaxation rate profile of free Pin1

  11. Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations

    Energy Technology Data Exchange (ETDEWEB)

    Bernado, Pau [Institut de Biologie Structurale, Jean Pierre Ebel (France); Fernandes, Miguel X. [Universidad de Murcia, Departamento de Quimica Fisica, Facultad de Quimica (Spain); Jacobs, Doris M. [Johann Wolfgang Goethe-Universitaet Frankfurt, Institut fuer Organische Chemie und Chemische Biologie (Germany); Fiebig, Klaus [Affinium Pharmaceuticals (Canada); Garcia de la Torre, Jose [Universidad de Murcia, Departamento de Quimica Fisica, Facultad de Quimica (Spain); Pons, Miquel [Laboratori de RMN de Biomolecules, Parc Cientific de Barcelona (Spain)], E-mail: mpons@ub.edu

    2004-05-15

    Many important proteins contain multiple domains connected by flexible linkers. Inter-domain motion is suggested to play a key role in many processes involving molecular recognition. Heteronuclear NMR relaxation is sensitive to motions in the relevant time scales and could provide valuable information on the dynamics of multi-domain proteins. However, the standard analysis based on the separation of global tumbling and fast local motions is no longer valid for multi-domain proteins undergoing internal motions involving complete domains and that take place on the same time scale than the overall motion.The complexity of the motions experienced even for the simplest two-domain proteins are difficult to capture with simple extensions of the classical Lipari-Szabo approach. Hydrodynamic effects are expected to dominate the motion of the individual globular domains, as well as that of the complete protein. Using Pin1 as a test case, we have simulated its motion at the microsecond time scale, at a reasonable computational expense, using Brownian Dynamic simulations on simplified models. The resulting trajectories provide insight on the interplay between global and inter-domain motion and can be analyzed using the recently published method of isotropic Reorientational Mode Dynamics which offer a way of calculating their contribution to heteronuclear relaxation rates. The analysis of trajectories computed with Pin1 models of different flexibility provides a general framework to understand the dynamics of multi-domain proteins and explains some of the observed features in the relaxation rate profile of free Pin1.

  12. NMR of proteins (4Fe-4S): structural properties and intramolecular electron transfer; RMN de proteines (4Fe-4S): proprietes structurales et transfert electronique intramoleculaire

    Energy Technology Data Exchange (ETDEWEB)

    Huber, J G

    1996-10-17

    NMR started to be applied to Fe-S proteins in the seventies. Its use has recently been enlarged as the problems arising from the paramagnetic polymetallic clusters ware overcome. Applications to [4Fe-4S] are presented herein. The information derived thereof deepens the understanding of the redox properties of these proteins which play a central role in the metabolism of bacterial cells. The secondary structure elements and the overall folding of Chromatium vinosum ferredoxin (Cv Fd) in solution have been established by NMR. The unique features of this sequence have been shown to fold as an {alpha} helix at the C-terminus and as a loop between two cysteines ligand of one cluster: these two parts localize in close proximity from one another. The interaction between nuclear and electronic spins is a source of additional structural information for (4Fe-AS] proteins. The conformation of the cysteine-ligands, as revealed by the Fe-(S{sub {gamma}}-C{sub {beta}}-H{sub {beta}})Cys dihedral angles, is related to the chemical shifts of the signals associated with the protons of these residues. The longitudinal relaxation times of the protons depend on their distance to the cluster. A quantitative relationship has been established and used to show that the solution structure of the high-potential ferredoxin from Cv differs significantly from the crystal structure around Phe-48. Both parameters (chemical shifts and longitudinal relaxation times) give also insight into the electronic and magnetic properties of the [4Fe-4S] clusters. The rate of intramolecular electron transfer between the two [4FE-4S] clusters of ferredoxins has been measured by NMR. It is far slower in the case of Cv Fd than for shorter ferredoxins. The difference may be associated with changes in the magnetic and/or electronic properties of one cluster. The strong paramagnetism of the [4Fe-4S] clusters, which originally limited the applicability of NMR to proteins containing these cofactors, has been proven

  13. A suite of Mathematica notebooks for the analysis of protein main chain 15N NMR relaxation data

    International Nuclear Information System (INIS)

    Spyracopoulos, Leo

    2006-01-01

    A suite of Mathematica notebooks has been designed to ease the analysis of protein main chain 15 N NMR relaxation data collected at a single magnetic field strength. Individual notebooks were developed to perform the following tasks: nonlinear fitting of 15 N-T 1 and -T 2 relaxation decays to a two parameter exponential decay, calculation of the principal components of the inertia tensor from protein structural coordinates, nonlinear optimization of the principal components and orientation of the axially symmetric rotational diffusion tensor, model-free analysis of 15 N-T 1 , -T 2 , and { 1 H}- 15 N NOE data, and reduced spectral density analysis of the relaxation data. The principle features of the notebooks include use of a minimal number of input files, integrated notebook data management, ease of use, cross-platform compatibility, automatic visualization of results and generation of high-quality graphics, and output of analyses in text format

  14. Selective {sup 2}H and {sup 13}C labeling in NMR analysis of solution protein structure and dynamics

    Energy Technology Data Exchange (ETDEWEB)

    LeMaster, D.M. [Northwestern Univ., Evanston, IL (United States)

    1994-12-01

    Preparation of samples bearing combined isotope enrichment patterns has played a central role in the recent advances in NMR analysis of proteins in solution. In particular, uniform {sup 13}C, {sup 15}N enrichment has made it possible to apply heteronuclear multidimensional correlation experiments for the mainchain assignments of proteins larger than 30 KDa. In contrast, selective labeling approaches can offer advantages in terms of the directedness of the information provided, such as chirality and residue type assignments, as well as through enhancements in resolution and sensitivity that result from editing the spectral complexity, the relaxation pathways and the scalar coupling networks. In addition, the combination of selective {sup 13}C and {sup 2}H enrichment can greatly facilitate the determination of heteronuclear relaxation behavior.

  15. Determination of muscle protein synthesis rates in fish using (2)H2O and (2)H NMR analysis of alanine.

    Science.gov (United States)

    Marques, Cátia; Viegas, Filipa; Rito, João; Jones, John; Viegas, Ivan

    2016-09-15

    Following administration of deuterated water ((2)H2O), the fractional synthetic rate (FSR) of a given endogenous protein can be estimated by (2)H-enrichment quantification of its alanine residues. Currently, this is measured by mass spectrometry following a derivatization procedure. Muscle FSR was measured by (1)H/(2)H NMR analysis of alanine from seabass kept for 6 days in 5% (2)H-enriched saltwater, following acid hydrolysis and amino acid isolation by cation-exchange chromatography of muscle tissue. The analysis is simple and robust, and provides precise measurements of excess alanine (2)H-enrichment in the 0.1-0.4% range from 50 mmol of alanine recovered from muscle protein. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Structure/Function Studies of Insect Antifreeze Proteins

    National Research Council Canada - National Science Library

    Duman, John

    1997-01-01

    ...-x5-x6-Cys-X8-X9-Ala-X11-Th-X13 where X3 and X1 tend toward charged residues, X5 toward threonine or serine, X9 toward asparagine or aspartate, X6 toward asparagine or lysine, and X13 toward alanine...

  17. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    1. Acaenamagellanica. Prickly burr. Doucet et al. 2000. 2. Acer saccharoides. Maple. Doucet et al. 2000. 3. Agrostistenuis. Creeping bentgrass. Doucet et al. 2000. 4. Alliarapetiolata. Garlic mustard. Urrutia et al. 1992. 5. Ammopiptanthusmongolicus. Evergreen legume. Wang et al. 2003. 6. Aster cordifolius. Wood aster.

  18. Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment

    International Nuclear Information System (INIS)

    Fan Ying; Shi Lichi; Ladizhansky, Vladimir; Brown, Leonid S.

    2011-01-01

    Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly 13 C, 15 N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution 13 C and 15 N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

  19. NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide

    Science.gov (United States)

    Wang, Tuo; Cady, Sarah D.; Hong, Mei

    2012-01-01

    The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use 31P and 13C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. 31P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct 31P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. 31P- and 13C-detected 1H T2 relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. 31P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides. PMID:22385849

  20. Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

    Energy Technology Data Exchange (ETDEWEB)

    Mas, Guillaume; Crublet, Elodie [Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) (France); Hamelin, Olivier [CNRS (France); Gans, Pierre; Boisbouvier, Jérôme, E-mail: jerome.boisbouvier@ibs.fr [Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) (France)

    2013-09-28

    The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D{sub 2}O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d{sub 10}. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.

  1. A comparison of chemical shift sensitivity of trifluoromethyl tags: optimizing resolution in {sup 19}F NMR studies of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Libin; Larda, Sacha Thierry; Frank Li, Yi Feng [University of Toronto, UTM, Department of Chemistry (Canada); Manglik, Aashish [Stanford University School of Medicine, Department of Molecular and Cellular Physiology (United States); Prosser, R. Scott, E-mail: scott.prosser@utoronto.ca [University of Toronto, UTM, Department of Chemistry (Canada)

    2015-05-15

    The elucidation of distinct protein conformers or states by fluorine ({sup 19}F) NMR requires fluorinated moieties whose chemical shifts are most sensitive to subtle changes in the local dielectric and magnetic shielding environment. In this study we evaluate the effective chemical shift dispersion of a number of thiol-reactive trifluoromethyl probes [i.e. 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA), N-(4-bromo-3-(trifluoromethyl)phenyl)acetamide (3-BTFMA), 3-bromo-1,1,1-trifluoropropan-2-ol (BTFP), 1-bromo-3,3,4,4,4-pentafluorobutan-2-one (BPFB), 3-bromo-1,1,1-trifluoropropan-2-one (BTFA), and 2,2,2-trifluoroethyl-1-thiol (TFET)] under conditions of varying polarity. In considering the sensitivity of the {sup 19}F NMR chemical shift to the local environment, a series of methanol/water mixtures were prepared, ranging from relatively non-polar (MeOH:H{sub 2}O = 4) to polar (MeOH:H{sub 2}O = 0.25). {sup 19}F NMR spectra of the tripeptide, glutathione ((2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl] -2-sulfanylethyl]carbamoyl}butanoic acid), conjugated to each of the above trifluoromethyl probes, revealed that the BTFMA tag exhibited a significantly greater range of chemical shift as a function of solvent polarity than did either BTFA or TFET. DFT calculations using the B3LYP hybrid functional and the 6-31G(d,p) basis set, confirmed the observed trend in chemical shift dispersion with solvent polarity.

  2. Sparse "1"3C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins

    International Nuclear Information System (INIS)

    Liu, Jing; Liu, Chang; Fan, Ying; Munro, Rachel A.; Ladizhansky, Vladimir; Brown, Leonid S.; Wang, Shenlin

    2016-01-01

    We demonstrate a novel sparse "1"3C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically "1"3C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

  3. Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR

    Science.gov (United States)

    Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

    2017-10-01

    In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

  4. Solution NMR structure and functional analysis of the integral membrane protein YgaP from Escherichia coli.

    Science.gov (United States)

    Eichmann, Cédric; Tzitzilonis, Christos; Bordignon, Enrica; Maslennikov, Innokentiy; Choe, Senyon; Riek, Roland

    2014-08-22

    The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

    Science.gov (United States)

    Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Clore, G Marius

    2016-06-03

    Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Controlling residual dipolar couplings in high-resolution NMR of proteins by strain induced alignment in a gel

    International Nuclear Information System (INIS)

    Ishii, Yoshitaka; Markus, Michelle A.; Tycko, Robert

    2001-01-01

    Water-soluble biological macromolecules can be weakly aligned by dissolution in a strained, hydrated gel such as cross-linked polyacrylamide, an effect termed 'strain-induced alignment in a gel' (SAG). SAG induces nonzero nuclear magnetic dipole-dipole couplings that can be measured in high-resolution NMR spectra and used as structural constraints. The dependence of experimental 15 N- 1 H dipolar couplings extracted from two-dimensional heteronuclear single quantum coherence (HSQC) spectra on several properties of compressed polyacrylamide, including the extent of compression, the polyacrylamide concentration, and the cross-link density, is reported for the B1 immunoglobulin binding domain of streptococcal protein G (protein G/B1, 57 residues). It is shown that the magnitude of macromolecular alignment can be widely varied by adjusting these properties, although the orientation and asymmetry of the alignment tensor are not affected significantly. The dependence of the 15 N relaxation times T 1 and T 2 of protein G/B1 on polyacrylamide concentration are also reported. In addition, the results of 15 N relaxation and HSQC experiments on the RNA binding domain of prokaryotic protein S4 from Bacillus stearothermophilus (S4 Δ41, residues 43-200) in a compressed polyacrylamide gel are presented. These results demonstrate the applicability of SAG to proteins of higher molecular weight and greater complexity. A modified in-phase/anti-phase (IPAP) HSQC technique is described that suppresses natural-abundance 15 N background signals from amide groups in polyacrylamide, resulting in cleaner HSQC spectra in SAG experiments. The mechanism of protein alignment in strained polyacrylamide gels is contrasted with that in liquid crystalline media

  7. Mimicking the properties of antifreeze glycoproteins: synthesis and characterization of a model system for ice nucleation and antifreeze studies.

    Science.gov (United States)

    Hederos, Markus; Konradsson, Peter; Borgh, Annika; Liedberg, Bo

    2005-08-25

    Synthesis of beta-D-Gal-(1 --> 3)-beta-D-GalNAc coupled to HOC2H4NHCOC15H30SH is described. This compound was coadsorbed at various proportions with C2H5OC2H4NHCOC15H30SH to form statistically mixed self-assembled monolayers (SAMs) on gold in an attempt to mimic the properties of the active domain in antifreeze glycoproteins (AFGPs). The monolayers were characterized by null ellipsometry, contact angle goniometry, X-ray photoelectron spectroscopy, and infrared reflection-absorption spectroscopy. The disaccharide compound adsorbed preferentially, and SAMs prepared at a solution molar ratio >0.3 displayed total wetting. The mixed SAMs showed well-organized alkyl chains up to a disaccharide surface fraction of 0.8. The amount of gauche conformers in the alkyls increased rapidly above this point, and the monolayers became disordered and less densely packed. Furthermore, the generated mixed SAMs were subjected to water vapor at constant relative humidity and the subsequent ice crystallization on a cooled substrate was monitored via an optical microscope. Interestingly, rapid crystallization occurred within a narrow range of temperatures on mixed SAMs with a high disaccharide content, surface fraction >0.3. The reported crystallization temperatures and the ice layer topography were compared with results obtained for a much simpler reference system composed of -OH/-CH3 terminated n-alkanethiols in order to account for changes in topography of the water/ice layer with surface energy. Although preliminary, the obtained results can be useful in the search for the molecular mechanism behind the antifreeze activity of AFGPs.

  8. NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

    OpenAIRE

    Choe Senyon; Riek Roland; Johnson Casey; Kefala Georgia; Maslennikov Innokentiy; Kwiatkowski Witek

    2007-01-01

    Abstract Background Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characteriza...

  9. BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions

    International Nuclear Information System (INIS)

    Markley, John L.; Ulrich, Eldon L.; Berman, Helen M.; Henrick, Kim; Nakamura, Haruki; Akutsu, Hideo

    2008-01-01

    We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems

  10. An Introduction to Drug Discovery by Probing Protein-Substrate Interactions Using Saturation Transfer Difference-Nuclear Magnetic Resonance (STD-NMR)

    Science.gov (United States)

    Guegan, Jean-Paul; Daniellou, Richard

    2012-01-01

    NMR spectroscopy is a powerful tool for characterizing and identifying molecules and nowadays is even used to characterize complex systems in biology. In the experiment presented here, students learned how to apply this modern technique to probe interactions between small molecules and proteins. With the use of simple organic synthesis, students…

  11. Solid state NMR sequential resonance assignments and conformational analysis of the 2x10.4 kDa dimeric form of the Bacillus subtilis protein Crh

    Energy Technology Data Exchange (ETDEWEB)

    Boeckmann, Anja [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France)], E-mail: a.bockmann@ibcp.fr; Lange, Adam [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Galinier, Anne [Institut de Biologie Structurale et Microbiologie, C.N.R.S UPR 9043 (France); Luca, Sorin [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Giraud, Nicolas; Juy, Michel [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France); Heise, Henrike [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Montserret, Roland; Penin, Francois [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France); Baldus, Marc [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany)], E-mail: maba@mpibpc.mpg.de

    2003-12-15

    Solid state NMR sample preparation and resonance assignments of the U-[{sup 13}C,{sup 15}N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all {sup 15}N, {sup 13}C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

  12. Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments

    Energy Technology Data Exchange (ETDEWEB)

    Motackova, Veronika; Novacek, Jiri [Masaryk University, Faculty of Science, National Centre for Biomolecular Research (Czech Republic); Zawadzka-Kazimierczuk, Anna; Kazimierczuk, Krzysztof [University of Warsaw, Faculty of Chemistry (Poland); Zidek, Lukas, E-mail: lzidek@chemi.muni.c [Masaryk University, Faculty of Science, National Centre for Biomolecular Research (Czech Republic); Sanderova, Hana; Krasny, Libor [Academy of Sciences of the Czech Republic, Laboratory of Molecular Genetics of Bacteria and Department of Bacteriology, Institute of Microbiology (Czech Republic); Kozminski, Wiktor [University of Warsaw, Faculty of Chemistry (Poland); Sklenar, Vladimir [Masaryk University, Faculty of Science, National Centre for Biomolecular Research (Czech Republic)

    2010-11-15

    A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, {delta} subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.

  13. NMR and molecular modeling of wine tannins binding to saliva proteins: revisiting astringency from molecular and colloidal prospects.

    Science.gov (United States)

    Cala, Olivier; Pinaud, Noël; Simon, Cécile; Fouquet, Eric; Laguerre, Michel; Dufourc, Erick J; Pianet, Isabelle

    2010-11-01

    In organoleptic science, the association of tannins to saliva proteins leads to the poorly understood phenomenon of astringency. To decipher this interaction at molecular and colloidal levels, the binding of 4 procyanidin dimers (B1-4) and 1 trimer (C2) to a human saliva proline-rich peptide, IB7(14), was studied. Interactions have been characterized by measuring dissociation constants, sizes of complexes, number, and nature of binding sites using NMR (chemical shift variations, diffusion-ordered spectroscopy, and saturation transfer diffusion). The binding sites were identified using molecular mechanics, and the hydrophilic/hydrophobic nature of the interactions was resolved by calculating the molecular lipophilicity potential within the complexes. The following comprehensive scheme can be proposed: 1) below the tannin critical micelle concentration (CMC), interaction is specific, and the procyanidin anchorage always occurs on the same three IB7(14) sites. The tannin 3-dimensional structure plays a key role in the binding force and in the tannin's ability to act as a bidentate ligand: tannins adopting an extended conformation exhibit higher affinity toward protein and initiate the formation of a network. 2) Above the CMC, after the first specific hydrophilic interaction has taken place, a random hydrophobic stacking occurs between tannins and proteins. The whole process is discussed in the general frame of wine tannins eliciting astringency.

  14. RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins

    International Nuclear Information System (INIS)

    Hoffman, D.W.; Query, C.C.; Golden, B.L.; White, S.W.; Keene, J.D.

    1991-01-01

    An RNA recognition motif (RRM) of ∼80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel β-strands and two α-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent β-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface to the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins

  15. Equilibrium simulations of proteins using molecular fragment replacement and NMR chemical shifts

    DEFF Research Database (Denmark)

    Boomsma, Wouter; Tian, Pengfei; Frellsen, J.

    2014-01-01

    recently been shown that using such information directly as input in molecular simulations based on the molecular fragment replacement strategy can help the process of protein structure determination. Here, we show how to implement this strategy to determine not only the structures of proteins but also...

  16. A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP

    Energy Technology Data Exchange (ETDEWEB)

    Rozentur-Shkop, Eva; Goobes, Gil; Chill, Jordan H., E-mail: Jordan.Chill@biu.ac.il [Bar Ilan University, Department of Chemistry (Israel)

    2016-12-15

    Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J({sup 15}N,{sup 13}Cα) couplings. This concept was realized using a unidirectional (H)NCO {sup 13}C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J({sup 15}N,{sup 13}Cα) couplings were acquired for WIP residues 2–65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16–23 and 45–60, and a helical tendency at residues 28–42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP{sup 2–65} conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein–protein interactions that stabilize the unfolded

  17. Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Fogeron, Marie-Laure [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Jirasko, Vlastimil; Penzel, Susanne [ETH Zurich, Physical Chemistry (Switzerland); Paul, David [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Montserret, Roland; Danis, Clément; Lacabanne, Denis; Badillo, Aurélie [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Gouttenoire, Jérôme; Moradpour, Darius [University of Lausanne, Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois (Switzerland); Bartenschlager, Ralf [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Penin, François [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); and others

    2016-06-15

    We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [{sup 2}H,{sup 13}C,{sup 15}N]-labeled protein are shown to yield narrow {sup 13}C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

  18. Water-Protein Hydrogen Exchange in the Micro-Crystalline Protein Crh as Observed by Solid State NMR Spectroscopy

    International Nuclear Information System (INIS)

    Boeckmann, Anja; Juy, Michel; Bettler, Emmanuel; Emsley, Lyndon; Galinier, Anne; Penin, Francois; Lesage, Anne

    2005-01-01

    We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T 2 ' -selective 1 H- 13 C- 13 C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T 2 ' selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on 13 C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates

  19. Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR & X-ray Diffraction

    Science.gov (United States)

    Jenkins, Janelle E.; Sampath, Sujatha; Butler, Emily; Kim, Jihyun; Henning, Robert W.; Holland, Gregory P.; Yarger, Jeffery L.

    2013-01-01

    This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked β-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) 13C-13C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and hence to the amino acids that make up the motifs. Specifically, alanine is incorporated in β-sheet (poly(Alan) and poly(Gly-Ala)), 31-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in β-sheet (poly(Gly-Ala)) and 31-helical (poly(Gly-Gly-Xaa)) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 31-helix (poly(Gly-Gly-Ser)), and β-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk. PMID:24024617

  20. Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

    International Nuclear Information System (INIS)

    Korzhnev, Dmitry M.; Neculai, Dante; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.; Bezsonova, Irina

    2016-01-01

    HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

  1. Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

    Energy Technology Data Exchange (ETDEWEB)

    Korzhnev, Dmitry M. [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States); Neculai, Dante [Zhejiang University, School of Medicine (China); Dhe-Paganon, Sirano [Dana-Farber Cancer Institute, Department of Cancer Biology (United States); Arrowsmith, Cheryl H. [University of Toronto, Structural Genomics Consortium (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States)

    2016-11-15

    HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

  2. Resonance assignment of the NMR spectra of disordered proteins using a multi-objective non-dominated sorting genetic algorithm

    International Nuclear Information System (INIS)

    Yang, Yu; Fritzsching, Keith J.; Hong, Mei

    2013-01-01

    A multi-objective genetic algorithm is introduced to predict the assignment of protein solid-state NMR (SSNMR) spectra with partial resonance overlap and missing peaks due to broad linewidths, molecular motion, and low sensitivity. This non-dominated sorting genetic algorithm II (NSGA-II) aims to identify all possible assignments that are consistent with the spectra and to compare the relative merit of these assignments. Our approach is modeled after the recently introduced Monte-Carlo simulated-annealing (MC/SA) protocol, with the key difference that NSGA-II simultaneously optimizes multiple assignment objectives instead of searching for possible assignments based on a single composite score. The multiple objectives include maximizing the number of consistently assigned peaks between multiple spectra (“good connections”), maximizing the number of used peaks, minimizing the number of inconsistently assigned peaks between spectra (“bad connections”), and minimizing the number of assigned peaks that have no matching peaks in the other spectra (“edges”). Using six SSNMR protein chemical shift datasets with varying levels of imperfection that was introduced by peak deletion, random chemical shift changes, and manual peak picking of spectra with moderately broad linewidths, we show that the NSGA-II algorithm produces a large number of valid and good assignments rapidly. For high-quality chemical shift peak lists, NSGA-II and MC/SA perform similarly well. However, when the peak lists contain many missing peaks that are uncorrelated between different spectra and have chemical shift deviations between spectra, the modified NSGA-II produces a larger number of valid solutions than MC/SA, and is more effective at distinguishing good from mediocre assignments by avoiding the hazard of suboptimal weighting factors for the various objectives. These two advantages, namely diversity and better evaluation, lead to a higher probability of predicting the correct

  3. Resonance assignment of the NMR spectra of disordered proteins using a multi-objective non-dominated sorting genetic algorithm.

    Science.gov (United States)

    Yang, Yu; Fritzsching, Keith J; Hong, Mei

    2013-11-01

    A multi-objective genetic algorithm is introduced to predict the assignment of protein solid-state NMR (SSNMR) spectra with partial resonance overlap and missing peaks due to broad linewidths, molecular motion, and low sensitivity. This non-dominated sorting genetic algorithm II (NSGA-II) aims to identify all possible assignments that are consistent with the spectra and to compare the relative merit of these assignments. Our approach is modeled after the recently introduced Monte-Carlo simulated-annealing (MC/SA) protocol, with the key difference that NSGA-II simultaneously optimizes multiple assignment objectives instead of searching for possible assignments based on a single composite score. The multiple objectives include maximizing the number of consistently assigned peaks between multiple spectra ("good connections"), maximizing the number of used peaks, minimizing the number of inconsistently assigned peaks between spectra ("bad connections"), and minimizing the number of assigned peaks that have no matching peaks in the other spectra ("edges"). Using six SSNMR protein chemical shift datasets with varying levels of imperfection that was introduced by peak deletion, random chemical shift changes, and manual peak picking of spectra with moderately broad linewidths, we show that the NSGA-II algorithm produces a large number of valid and good assignments rapidly. For high-quality chemical shift peak lists, NSGA-II and MC/SA perform similarly well. However, when the peak lists contain many missing peaks that are uncorrelated between different spectra and have chemical shift deviations between spectra, the modified NSGA-II produces a larger number of valid solutions than MC/SA, and is more effective at distinguishing good from mediocre assignments by avoiding the hazard of suboptimal weighting factors for the various objectives. These two advantages, namely diversity and better evaluation, lead to a higher probability of predicting the correct assignment for a

  4. Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: Application to microcrystalline and membrane protein preparations

    Science.gov (United States)

    Gopinath, T.; Veglia, Gianluigi

    2015-04-01

    Solid-state NMR spectroscopy of proteins is a notoriously low-throughput technique. Relatively low-sensitivity and poor resolution of protein samples require long acquisition times for multidimensional NMR experiments. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments, all while using conventional probes with spectrometers equipped with one receiver. POE allow the concatenation of multiple 2D or 3D pulse sequences into a single experiment, thus potentially combining all of the aforementioned advances, boosting the capability of ssNMR spectrometers at least two-fold without the addition of any hardware. In this perspective, we describe the first generation of POE, such as dual acquisition MAS (or DUMAS) methods, and then illustrate the evolution of these experiments into MEIOSIS, a method that enables the simultaneous acquisition of multiple 2D and 3D spectra. Using these new pulse schemes for the solid-state NMR investigation of biopolymers makes it possible to obtain sequential resonance assignments, as well as distance restraints, in about half the experimental time. While designed for acquisition of heteronuclei, these new experiments can be easily implemented for proton detection and coupled with other recent advancements, such as dynamic nuclear polarization (DNP), to improve signal to noise. Finally, we illustrate the application of these methods to microcrystalline protein preparations as well as single and multi-span membrane proteins reconstituted in lipid membranes.

  5. CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts.

    Science.gov (United States)

    Hafsa, Noor E; Arndt, David; Wishart, David S

    2015-07-01

    The Chemical Shift Index or CSI 3.0 (http://csi3.wishartlab.com) is a web server designed to accurately identify the location of secondary and super-secondary structures in protein chains using only nuclear magnetic resonance (NMR) backbone chemical shifts and their corresponding protein sequence data. Unlike earlier versions of CSI, which only identified three types of secondary structure (helix, β-strand and coil), CSI 3.0 now identifies total of 11 types of secondary and super-secondary structures, including helices, β-strands, coil regions, five common β-turns (type I, II, I', II' and VIII), β hairpins as well as interior and edge β-strands. CSI 3.0 accepts experimental NMR chemical shift data in multiple formats (NMR Star 2.1, NMR Star 3.1 and SHIFTY) and generates colorful CSI plots (bar graphs) and secondary/super-secondary structure assignments. The output can be readily used as constraints for structure determination and refinement or the images may be used for presentations and publications. CSI 3.0 uses a pipeline of several well-tested, previously published programs to identify the secondary and super-secondary structures in protein chains. Comparisons with secondary and super-secondary structure assignments made via standard coordinate analysis programs such as DSSP, STRIDE and VADAR on high-resolution protein structures solved by X-ray and NMR show >90% agreement between those made with CSI 3.0. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Characterization of pH titration shifts for all the nonlabile proton resonances in a protein by two-dimensional NMR: The case of mouse epidermal growth factor

    International Nuclear Information System (INIS)

    Kohda, Daisuke; Sawada, Toshie; Inagaki, Fuyuhiko

    1991-01-01

    The pH titration shifts for all the nonlabile proton resonances in a 53-residue protein (mouse epidermal growth factor) were measured in the p 2 H range 1.5-9 with two-dimensional (2D) 1 H NMR. The 2D NMR pH titration experiment made it possible to determine the pK values for all the ionizable group which were titrated in the pH range 1.5-9 in the protein. The pK values of the nine ionizable groups (α-amino group, four Asp, two Glu, one His, and α-carboxyl group) were found to be near their normal values. The 2D titration experiment also provided a detailed description of the pH-dependent behavior of the proton chemical shifts and enabled us to characterize the pH-dependent changes of protein conformation. Analysis of the pH-dependent shifts of ca. 200 proton resonances offered evidence of conformational changes in slightly basic pH solution: The deprotonation of the N-terminal α-amino group induced a widespread conformational change over the β-sheet structure in the protein, while the effects of deprotonation of the His22 imidazole group were relatively localized. The authors found that the 2D NMR pH titration experiment is a powerful tool for investigating the structural and dynamic properties of proteins

  7. Mapping protein–protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Changmiao; Hou, Guangjin, E-mail: hou@udel.edu; Lu, Xingyu; Polenova, Tatyana, E-mail: tpolenov@udel.edu [University of Delaware, Department of Chemistry and Biochemistry (United States)

    2017-02-15

    REDOR-based experiments with simultaneous {sup 1}H–{sup 13}C and {sup 1}H−{sup 15}N dipolar dephasing are explored for investigating intermolecular protein–protein interfaces in complexes formed by a U–{sup 13}C,{sup 15}N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U–{sup 13}C,{sup 15}N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by {sup 1}H–{sup 13}C or {sup 1}H–{sup 15}N cross polarization, permitting to establish the residues of the U–{sup 13}C,{sup 15}N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into {sup 13}C–{sup 13}C correlation experiments. We established the validity of this approach on U–{sup 13}C,{sup 15}N-histidine and on a structurally characterized complex of dynactin’s U–{sup 13}C,{sup 15}N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.

  8. Carbohydrate-protein interaction studies by laser photo CIDNP NMR methods

    NARCIS (Netherlands)

    Siebert, HC; Kaptein, R; Beintema, JJ; Soedjanaatmadja, UM; Wright, CS; Rice, A; Kleineidam, RG; Kruse, S; Schauer, R; Pouwels, PJW; Kamerling, JP; Gabius, HJ; Vliegenthart, JFG

    The side chains of tyrosine, tryptophan and histidine are able to produce CIDNP (Chemically Induced Dynamic Nuclear Polarization) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies surface accessibility of the

  9. Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

    Science.gov (United States)

    Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

    2010-05-05

    Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  10. Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

    Directory of Open Access Journals (Sweden)

    Anne N Shemon

    2010-05-01

    Full Text Available Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  11. Antifreeze polymeric additives for fuels; Aditivos polimericos anticongelantes para combustiveis

    Energy Technology Data Exchange (ETDEWEB)

    Muniz, Aline S.; Carvalho, Agne Roani de; Sakae, George Hideki; Oliveira, Angelo R.S.; Cesar-Oliveira, Maria Aparecida F. [Universidade Federal do Parana - UFPR - Departamento de Quimica - LABPOL-Laboratorio de Polimeros Sinteticos, Centro Politecnico, Curitiba, PR (Brazil)], e-mails: mafco@ufpr.br, alinemuniz@ufpr.br

    2011-07-01

    Owing to the current interest in the reduction of environmental pollution, several researchers are seeking renewable sources of energy which can at least partially replace combustibles derived from petroleum. Diesel oil is the combustible that most seriously pollutes the environment and is thus the biodiesel that is being considered as a fuel which can be replaced by a renewable combustible; this can possibly be used in diesel engines without any modifications. However, certain problems have to be overcome with regard to the temperature at which the biodiesel should be stored and used, since there is a tendency for biodiesel to solidify at low temperatures. This suggests that there is a need for the use of anti-freeze additives. This work behind the main focus additives with only 25 ppm, were able to reduce the pour point of fuel, achieving significant results, for example, the additive M14A18 lowered the pour point (PP) of B20 to -20 degree C, showing that the use of increasing amounts of biodiesel to diesel can aggregate. The main focus of work behind the development of additives that with only 25 ppm, were able to reduce the pour point of fuel, producing significant results such as those obtained with the use of additive M14A18 which lowered the pour point of the B20 to -20 degree C, showing the possibility of using increasing amounts of biodiesel added to diesel. (author)

  12. Synthesis of Cyclic Antifreeze Glycopeptide and Glycopeptoids and Their Ice Recrystallization Inhibition Activity

    International Nuclear Information System (INIS)

    Ahn, Mija; Murugan, Ravichandran N.; Bang, Jeong Kyu; Kim, Hak Jun; Shin, Song Yub; Kim, Eunjung; Lee, Jun Hyuck

    2012-01-01

    Until now, few groups reported the antifreeze activity of cyclic glycopeptides; however, the tedious synthetic procedure is not amenable to study the intensive structure activity relationship. A series of N-linked cyclic glycopeptoids and glycopeptide have been prepared to evaluate antifreeze activity as a function of peptide backbone cyclization and methyl stereochemical effect on the rigid Thr position. This study has combined the cyclization protocol with solid phase peptide synthesis and obtained significant quantities of homogeneous cyclic glycopeptide and glycopeptoids. Analysis of antifreeze activity revealed that our cyclic peptide demonstrated RI activity while cyclic glycopeptoids showed no RI activity. These results suggest that the subtle changes in conformation and Thr orientation dramatically influence RI activity of N-linked glycopeptoids

  13. J-UNIO protocol used for NMR structure determination of the 206-residue protein NP-346487.1 from Streptococcus pneumoniae TIGR4

    Energy Technology Data Exchange (ETDEWEB)

    Jaudzems, Kristaps [Latvian Institute of Organic Synthesis (Latvia); Pedrini, Bill [Paul Scherrer Institute (PSI), SwissFEL Project (Switzerland); Geralt, Michael; Serrano, Pedro; Wüthrich, Kurt, E-mail: wuthrich@scripps.edu [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States)

    2015-01-15

    The NMR structure of the 206-residue protein NP-346487.1 was determined with the J-UNIO protocol, which includes extensive automation of the structure determination. With input from three APSY-NMR experiments, UNIO-MATCH automatically yielded 77 % of the backbone assignments, which were interactively validated and extended to 97 %. With an input of the near-complete backbone assignments and three 3D heteronuclear-resolved [{sup 1}H,{sup 1}H]-NOESY spectra, automated side chain assignment with UNIO-ATNOS/ASCAN resulted in 77 % of the expected assignments, which was extended interactively to about 90 %. Automated NOE assignment and structure calculation with UNIO-ATNOS/CANDID in combination with CYANA was used for the structure determination of this two-domain protein. The individual domains in the NMR structure coincide closely with the crystal structure, and the NMR studies further imply that the two domains undergo restricted hinge motions relative to each other in solution. NP-346487.1 is so far the largest polypeptide chain to which the J-UNIO structure determination protocol has successfully been applied.

  14. BioMagResBank databases DOCR and FRED containing converted and filtered sets of experimental NMR restraints and coordinates from over 500 protein PDB structures

    Energy Technology Data Exchange (ETDEWEB)

    Doreleijers, Jurgen F. [University of Wisconsin-Madison, BioMagResBank, Department of Biochemistry (United States); Nederveen, Aart J. [Utrecht University, Bijvoet Center for Biomolecular Research (Netherlands); Vranken, Wim [European Bioinformatics Institute, Macromolecular Structure Database group (United Kingdom); Lin Jundong [University of Wisconsin-Madison, BioMagResBank, Department of Biochemistry (United States); Bonvin, Alexandre M.J.J.; Kaptein, Robert [Utrecht University, Bijvoet Center for Biomolecular Research (Netherlands); Markley, John L.; Ulrich, Eldon L. [University of Wisconsin-Madison, BioMagResBank, Department of Biochemistry (United States)], E-mail: elu@bmrb.wisc.edu

    2005-05-15

    We present two new databases of NMR-derived distance and dihedral angle restraints: the Database Of Converted Restraints (DOCR) and the Filtered Restraints Database (FRED). These databases currently correspond to 545 proteins with NMR structures deposited in the Protein Databank (PDB). The criteria for inclusion were that these should be unique, monomeric proteins with author-provided experimental NMR data and coordinates available from the PDB capable of being parsed and prepared in a consistent manner. The Wattos program was used to parse the files, and the CcpNmr FormatConverter program was used to prepare them semi-automatically. New modules, including a new implementation of Aqua in the BioMagResBank (BMRB) software Wattos were used to analyze the sets of distance restraints (DRs) for inconsistencies, redundancies, NOE completeness, classification and violations with respect to the original coordinates. Restraints that could not be associated with a known nomenclature were flagged. The coordinates of hydrogen atoms were recalculated from the positions of heavy atoms to allow for a full restraint analysis. The DOCR database contains restraint and coordinate data that is made consistent with each other and with IUPAC conventions. The FRED database is based on the DOCR data but is filtered for use by test calculation protocols and longitudinal analyses and validations. These two databases are available from websites of the BMRB and the Macromolecular Structure Database (MSD) in various formats: NMR-STAR, CCPN XML, and in formats suitable for direct use in the software packages CNS and CYANA.

  15. BioMagResBank databases DOCR and FRED containing converted and filtered sets of experimental NMR restraints and coordinates from over 500 protein PDB structures

    International Nuclear Information System (INIS)

    Doreleijers, Jurgen F.; Nederveen, Aart J.; Vranken, Wim; Lin Jundong; Bonvin, Alexandre M.J.J.; Kaptein, Robert; Markley, John L.; Ulrich, Eldon L.

    2005-01-01

    We present two new databases of NMR-derived distance and dihedral angle restraints: the Database Of Converted Restraints (DOCR) and the Filtered Restraints Database (FRED). These databases currently correspond to 545 proteins with NMR structures deposited in the Protein Databank (PDB). The criteria for inclusion were that these should be unique, monomeric proteins with author-provided experimental NMR data and coordinates available from the PDB capable of being parsed and prepared in a consistent manner. The Wattos program was used to parse the files, and the CcpNmr FormatConverter program was used to prepare them semi-automatically. New modules, including a new implementation of Aqua in the BioMagResBank (BMRB) software Wattos were used to analyze the sets of distance restraints (DRs) for inconsistencies, redundancies, NOE completeness, classification and violations with respect to the original coordinates. Restraints that could not be associated with a known nomenclature were flagged. The coordinates of hydrogen atoms were recalculated from the positions of heavy atoms to allow for a full restraint analysis. The DOCR database contains restraint and coordinate data that is made consistent with each other and with IUPAC conventions. The FRED database is based on the DOCR data but is filtered for use by test calculation protocols and longitudinal analyses and validations. These two databases are available from websites of the BMRB and the Macromolecular Structure Database (MSD) in various formats: NMR-STAR, CCPN XML, and in formats suitable for direct use in the software packages CNS and CYANA

  16. Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

    Science.gov (United States)

    Delaforge, Elise; Kragelj, Jaka; Tengo, Laura; Palencia, Andrés; Milles, Sigrid; Bouvignies, Guillaume; Salvi, Nicola; Blackledge, Martin; Jensen, Malene Ringkjøbing

    2018-01-24

    Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

  17. NMR studies on the mechanism of structural destabilization of the globular proteins and DNA by aliphatic alcohols

    International Nuclear Information System (INIS)

    Lubas, B.; Witman, B.; Wieniewska, T.; Soltysik, M.

    1977-01-01

    The concept that the mechanism of structural destabilization of the biologically active macromolecules by typical denaturing agents should find a reflection in the NMR spectra of the denaturants themselves has been followed by proton NMR for some aliphatic alcohols in the system containing the serum albumin of DNA. (author)

  18. Visualizing the principal component of 1H,15N-HSQC NMR spectral changes that reflect protein structural or functional properties: application to troponin C

    International Nuclear Information System (INIS)

    Robertson, Ian M.; Boyko, Robert F.; Sykes, Brian D.

    2011-01-01

    Laboratories often repeatedly determine the structure of a given protein under a variety of conditions, mutations, modifications, or in a number of states. This approach can be cumbersome and tedious. Given then a database of structures, identifiers, and corresponding 1 H, 15 N-HSQC NMR spectra for homologous proteins, we investigated whether structural information could be ascertained for a new homolog solely from its 1 H, 15 N-HSQC NMR spectrum. We addressed this question with two different approaches. First, we used a semi-automated approach with the program, ORBplus. ORBplus looks for patterns in the chemical shifts and correlates these commonalities to the explicit property of interest. ORBplus ranks resonances based on consistency of the magnitude and direction of the chemical shifts within the database, and the chemical shift correlation of the unknown protein with the database. ORBplus visualizes the results by a histogram and a vector diagram, and provides residue specific predictions on structural similarities with the database. The second method we used was partial least squares (PLS), which is a multivariate statistical technique used to correlate response and predictor variables. We investigated the ability of these methods to predict the tertiary structure of the contractile regulatory protein troponin C. Troponin C undergoes a closed-to-open conformational change, which is coupled to its function in muscle. We found that both ORBplus and PLS were able to identify patterns in the 1 H, 15 N-HSQC NMR data from different states of troponin C that correlated to its conformation.

  19. 1H-detected MAS solid-state NMR experiments enable the simultaneous mapping of rigid and dynamic domains of membrane proteins

    Science.gov (United States)

    Gopinath, T.; Nelson, Sarah E. D.; Veglia, Gianluigi

    2017-12-01

    Magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy is emerging as a unique method for the atomic resolution structure determination of native membrane proteins in lipid bilayers. Although 13C-detected ssNMR experiments continue to play a major role, recent technological developments have made it possible to carry out 1H-detected experiments, boosting both sensitivity and resolution. Here, we describe a new set of 1H-detected hybrid pulse sequences that combine through-bond and through-space correlation elements into single experiments, enabling the simultaneous detection of rigid and dynamic domains of membrane proteins. As proof-of-principle, we applied these new pulse sequences to the membrane protein phospholamban (PLN) reconstituted in lipid bilayers under moderate MAS conditions. The cross-polarization (CP) based elements enabled the detection of the relatively immobile residues of PLN in the transmembrane domain using through-space correlations; whereas the most dynamic region, which is in equilibrium between folded and unfolded states, was mapped by through-bond INEPT-based elements. These new 1H-detected experiments will enable one to detect not only the most populated (ground) states of biomacromolecules, but also sparsely populated high-energy (excited) states for a complete characterization of protein free energy landscapes.

  20. Triple resonance 15N NMR relaxation experiments for studies of intrinsically disordered proteins

    Czech Academy of Sciences Publication Activity Database

    Srb, Pavel; Nováček, J.; Kadeřávek, P.; Rabatinová, Alžběta; Krásný, Libor; Žídková, Jitka; Bobálová, Janette; Sklenář, V.; Žídek, L.

    2017-01-01

    Roč. 69, č. 3 (2017), s. 133-146 ISSN 0925-2738 R&D Projects: GA ČR GA13-16842S; GA MŠk(CZ) LO1304 Institutional support: RVO:61388963 ; RVO:61388971 ; RVO:68081715 Keywords : nuclear magnetic resonance * relaxation * non-uniform sampling * intrinsically disordered proteins Subject RIV: CB - Analytical Chemistry, Separation; EE - Microbiology, Virology (MBU-M); CB - Analytical Chemistry, Separation (UIACH-O) OBOR OECD: Analytical chemistry; Microbiology (MBU-M); Analytical chemistry (UIACH-O) Impact factor: 2.410, year: 2016

  1. Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    Lopez del Amo, Juan-Miguel; Fink, Uwe; Reif, Bernd

    2010-01-01

    We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual 15 N-T 1 timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s -1 . Backbone amide 15 N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D 2 O is employed as a solvent for sample preparation. Due to the intrinsically long 15 N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

  2. Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method.

    Science.gov (United States)

    Takeda, Mitsuhiro; Chang, Chung-ke; Ikeya, Teppei; Güntert, Peter; Chang, Yuan-hsiang; Hsu, Yen-lan; Huang, Tai-huang; Kainosho, Masatsune

    2008-07-18

    The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform (13)C and (15)N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the beta-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.

  3. "Invisible" conformers of an antifungal disulfide protein revealed by constrained cold and heat unfolding, CEST-NMR experiments, and molecular dynamics calculations.

    Science.gov (United States)

    Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

    2015-03-23

    Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20-40 % at 298 K in a disulfide-rich protein. In addition, sensitive (15) N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR "dark matter". Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  4. “Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding, CEST-NMR Experiments, and Molecular Dynamics Calculations

    Science.gov (United States)

    Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

    2015-01-01

    Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide-rich protein. In addition, sensitive 15N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. PMID:25676351

  5. Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

    Energy Technology Data Exchange (ETDEWEB)

    Eddy, Matthew T. [Massachusetts Institute of Technology, Department of Chemistry (United States); Belenky, Marina [Brandeis University, Department of Chemistry (United States); Sivertsen, Astrid C. [Massachusetts Institute of Technology, Francis Bitter Magnet Laboratory (United States); Griffin, Robert G. [Massachusetts Institute of Technology, Department of Chemistry (United States); Herzfeld, Judith, E-mail: herzfeld@brandeis.edu [Brandeis University, Department of Chemistry (United States)

    2013-10-15

    The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new 'redox' approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-{sup 13}C] acetate does not label {alpha} carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-{sup 13}C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra.

  6. Proton NMR Studies of a Large Protein. pH, Substrate Titrations, and NOESY Experiments with Perdeuterated Yeast Phosphoglycerate Kinase Containing [ 1H]Histidine Residues

    Science.gov (United States)

    Pappu, K. M.; Serpersu, E. H.

    Fully deuterated yeast phosphoglycerate kinase ([ 2H]PGK) was prepared biosynthetically with only histidine side chains of normal ( 1H) isotopic composition. The 1H NMR spectrum of this enzyme([ 1H]His[ 2H]PGK) showed that the histidine side chains are clearly visible as sharp signals. Thus detailed structural studies by 1H NMR became feasible with isotope-hybrid phosphoglycerate kinase which is otherwise too large ( Mr ˜ 46,000) for conventional 1H NMR studies. Proton signals of bound substrates were visible in the 1H NMR spectrum even with a substrate-to-enzyme ratio of less than 1/2 (mol/mol). The 2D NOESY spectrum of enzyme-MgdATP-glycerol 3-phosphate complex showed that, although protein concentration was very high (1.5 m M), no intraprotein cross peaks were observed other than those of intraresidue histidine NOE cross peaks. In addition, intrasubstrate NOEs and intermolecular NOEs between histidine and substrate protons were visible at a 1.5/1 substrate/enzyme (mol/mol) ratio. Paramagnetic effects of a substrate analog, Cr(III)ATP, on some of the histidine side chains indicated that the formation of the ternary enzyme-substrate complex causes large conformational changes in the enzyme.

  7. Combining NMR ensembles and molecular dynamics simulations provides more realistic models of protein structures in solution and leads to better chemical shift prediction

    International Nuclear Information System (INIS)

    Lehtivarjo, Juuso; Tuppurainen, Kari; Hassinen, Tommi; Laatikainen, Reino; Peräkylä, Mikael

    2012-01-01

    While chemical shifts are invaluable for obtaining structural information from proteins, they also offer one of the rare ways to obtain information about protein dynamics. A necessary tool in transforming chemical shifts into structural and dynamic information is chemical shift prediction. In our previous work we developed a method for 4D prediction of protein 1 H chemical shifts in which molecular motions, the 4th dimension, were modeled using molecular dynamics (MD) simulations. Although the approach clearly improved the prediction, the X-ray structures and single NMR conformers used in the model cannot be considered fully realistic models of protein in solution. In this work, NMR ensembles (NMRE) were used to expand the conformational space of proteins (e.g. side chains, flexible loops, termini), followed by MD simulations for each conformer to map the local fluctuations. Compared with the non-dynamic model, the NMRE+MD model gave 6–17% lower root-mean-square (RMS) errors for different backbone nuclei. The improved prediction indicates that NMR ensembles with MD simulations can be used to obtain a more realistic picture of protein structures in solutions and moreover underlines the importance of short and long time-scale dynamics for the prediction. The RMS errors of the NMRE+MD model were 0.24, 0.43, 0.98, 1.03, 1.16 and 2.39 ppm for 1 Hα, 1 HN, 13 Cα, 13 Cβ, 13 CO and backbone 15 N chemical shifts, respectively. The model is implemented in the prediction program 4DSPOT, available at http://www.uef.fi/4dspothttp://www.uef.fi/4dspot.

  8. Combining NMR ensembles and molecular dynamics simulations provides more realistic models of protein structures in solution and leads to better chemical shift prediction

    Energy Technology Data Exchange (ETDEWEB)

    Lehtivarjo, Juuso, E-mail: juuso.lehtivarjo@uef.fi; Tuppurainen, Kari; Hassinen, Tommi; Laatikainen, Reino [University of Eastern Finland, School of Pharmacy (Finland); Peraekylae, Mikael [University of Eastern Finland, Institute of Biomedicine (Finland)

    2012-03-15

    While chemical shifts are invaluable for obtaining structural information from proteins, they also offer one of the rare ways to obtain information about protein dynamics. A necessary tool in transforming chemical shifts into structural and dynamic information is chemical shift prediction. In our previous work we developed a method for 4D prediction of protein {sup 1}H chemical shifts in which molecular motions, the 4th dimension, were modeled using molecular dynamics (MD) simulations. Although the approach clearly improved the prediction, the X-ray structures and single NMR conformers used in the model cannot be considered fully realistic models of protein in solution. In this work, NMR ensembles (NMRE) were used to expand the conformational space of proteins (e.g. side chains, flexible loops, termini), followed by MD simulations for each conformer to map the local fluctuations. Compared with the non-dynamic model, the NMRE+MD model gave 6-17% lower root-mean-square (RMS) errors for different backbone nuclei. The improved prediction indicates that NMR ensembles with MD simulations can be used to obtain a more realistic picture of protein structures in solutions and moreover underlines the importance of short and long time-scale dynamics for the prediction. The RMS errors of the NMRE+MD model were 0.24, 0.43, 0.98, 1.03, 1.16 and 2.39 ppm for {sup 1}H{alpha}, {sup 1}HN, {sup 13}C{alpha}, {sup 13}C{beta}, {sup 13}CO and backbone {sup 15}N chemical shifts, respectively. The model is implemented in the prediction program 4DSPOT, available at http://www.uef.fi/4dspothttp://www.uef.fi/4dspot.

  9. An NMR-based quenched hydrogen exchange investigation of model amyloid fibrils formed by cold shock protein A.

    Science.gov (United States)

    Alexandrescu, A T

    2001-01-01

    Acid-denatured cold shock protein A (CspA) self-assembles into polymers with properties typical of amyloid fibrils. In the present work, a quenched hydrogen exchange experiment was used to probe the interactions of CspA fibrils with solvent. Exchange was initiated by incubating suspensions of fibrils in D2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D2O (pH* 4.5) were sufficiently slow (approximately 1 x 10(-3) min-1) to enable quantification of NMR signal intensity decays due to H/D exchange in the fibrils. Hydrogen exchange rate constants for CspA fibrils were found to vary less than 3-fold from a mean value of 5 x 10(-5) min-1. The uniformity of rate constants suggests that exchange is in the EX1 limit, and that the mechanism of exchange involves a cooperative dissociation of CspA monomers from fibrils, concomitant with unfolding. Previous studies indicated that the highest protection factors in native CspA are approximately 10(3), and that protection factors for the acid-denatured monomer precursors of CspA fibrils are close to unity. Because exchange in is in the EX1 regime, it is only possible to place a lower limit of at least 10(5) on protection factors in CspA fibrils. The observation that all amide protons are protected from exchange indicates that the entire CspA polypeptide chain is structured in the fibrils.

  10. Utilization of paramagnetic relaxation enhancements for high-resolution NMR structure determination of a soluble loop-rich protein with sparse NOE distance restraints

    International Nuclear Information System (INIS)

    Furuita, Kyoko; Kataoka, Saori; Sugiki, Toshihiko; Hattori, Yoshikazu; Kobayashi, Naohiro; Ikegami, Takahisa; Shiozaki, Kazuhiro; Fujiwara, Toshimichi; Kojima, Chojiro

    2015-01-01

    NMR structure determination of soluble proteins depends in large part on distance restraints derived from NOE. In this study, we examined the impact of paramagnetic relaxation enhancement (PRE)-derived distance restraints on protein structure determination. A high-resolution structure of the loop-rich soluble protein Sin1 could not be determined by conventional NOE-based procedures due to an insufficient number of NOE restraints. By using the 867 PRE-derived distance restraints obtained from the NOE-based structure determination procedure, a high-resolution structure of Sin1 could be successfully determined. The convergence and accuracy of the determined structure were improved by increasing the number of PRE-derived distance restraints. This study demonstrates that PRE-derived distance restraints are useful in the determination of a high-resolution structure of a soluble protein when the number of NOE constraints is insufficient

  11. Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: Application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins

    Energy Technology Data Exchange (ETDEWEB)

    Liu Aizhuo; Hu Weidong; Qamar, Seema; Majumdar, Ananya [Memorial Sloan-Kettering Cancer Center, Cellular Biochemistry and Biophysics Program (United States)

    2000-05-15

    In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in {sup 13}C/{sup 15}N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen-carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond {sup 3h}J{sub NC'} couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.

  12. A general assignment method for oriented sample (OS) solid-state NMR of proteins based on the correlation of resonances through heteronuclear dipolar couplings in samples aligned parallel and perpendicular to the magnetic field.

    Science.gov (United States)

    Lu, George J; Son, Woo Sung; Opella, Stanley J

    2011-04-01

    A general method for assigning oriented sample (OS) solid-state NMR spectra of proteins is demonstrated. In principle, this method requires only a single sample of a uniformly ¹⁵N-labeled membrane protein in magnetically aligned bilayers, and a previously assigned isotropic chemical shift spectrum obtained either from solution NMR on micelle or isotropic bicelle samples or from magic angle spinning (MAS) solid-state NMR on unoriented proteoliposomes. The sequential isotropic resonance assignments are transferred to the OS solid-state NMR spectra of aligned samples by correlating signals from the same residue observed in protein-containing bilayers aligned with their normals parallel and perpendicular to the magnetic field. The underlying principle is that the resonances from the same residue have heteronuclear dipolar couplings that differ by exactly a factor of two between parallel and perpendicular alignments. The method is demonstrated on the membrane-bound form of Pf1 coat protein in phospholipid bilayers, whose assignments have been previously made using an earlier generation of methods that relied on the preparation of many selectively labeled (by residue type) samples. The new method provides the correct resonance assignments using only a single uniformly ¹⁵N-labeled sample, two solid-state NMR spectra, and a previously assigned isotropic spectrum. Significantly, this approach is equally applicable to residues in alpha helices, beta sheets, loops, and any other elements of tertiary structure. Moreover, the strategy bridges between OS solid-state NMR of aligned samples and solution NMR or MAS solid-state NMR of unoriented samples. In combination with the development of complementary experimental methods, it provides a step towards unifying these apparently different NMR approaches. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

    Science.gov (United States)

    Reddy, Jithender G; Kumar, Dinesh; Hosur, Ramakrishna V

    2015-02-01

    Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments – (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH – which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins. Copyright © 2014 John Wiley & Sons, Ltd.

  14. NMR spectroscopy and drug development

    International Nuclear Information System (INIS)

    Craik, D.; Munro, S.

    1990-01-01

    The use of nuclear magnetic resonance (NMR) spectroscopy for structural and conformational studies on drug molecules, the three-dimensional investigation of proteins structure and their interactions with ligands are discussed. In-vivo NMR studies of the effects of drugs on metabolism in perfused organs and whole animals are also briefly presented. 5 refs., ills

  15. Progress in proton-detected solid-state NMR (SSNMR): Super-fast 2D SSNMR collection for nano-mole-scale proteins

    Science.gov (United States)

    Ishii, Yoshitaka; Wickramasinghe, Ayesha; Matsuda, Isamu; Endo, Yuki; Ishii, Yuji; Nishiyama, Yusuke; Nemoto, Takahiro; Kamihara, Takayuki

    2018-01-01

    Proton-detected solid-state NMR (SSNMR) spectroscopy has attracted much attention due to its excellent sensitivity and effectiveness in the analysis of trace amounts of amyloid proteins and other important biological systems. In this perspective article, we present the recent sensitivity limit of 1H-detected SSNMR using "ultra-fast" magic-angle spinning (MAS) at a spinning rate (νR) of 80-100 kHz. It was demonstrated that the high sensitivity of 1H-detected SSNMR at νR of 100 kHz and fast recycling using the paramagnetic-assisted condensed data collection (PACC) approach permitted "super-fast" collection of 1H-detected 2D protein SSNMR. A 1H-detected 2D 1H-15N correlation SSNMR spectrum for ∼27 nmol of a uniformly 13C- and 15N-labeled GB1 protein sample in microcrystalline form was acquired in only 9 s with 50% non-uniform sampling and short recycle delays of 100 ms. Additional data suggests that it is now feasible to detect as little as 1 nmol of the protein in 5.9 h by 1H-detected 2D 1H-15N SSNMR at a nominal signal-to-noise ratio of five. The demonstrated sensitivity is comparable to that of modern solution protein NMR. Moreover, this article summarizes the influence of ultra-fast MAS and 1H-detection on the spectral resolution and sensitivity of protein SSNMR. Recent progress in signal assignment and structural elucidation by 1H-detected protein SSNMR is outlined with both theoretical and experimental aspects.

  16. Improvement of hydrogen bond geometry in protein NMR structures by residual dipolar couplings - an assessment of the interrelation of NMR restraints

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, Pernille Rose; Axelsen, Jacob Bock [University of Copenhagen, Institute of Molecular Biology (Denmark); Lerche, Mathilde Hauge [Amersham Health (Sweden); Poulsen, Flemming M. [University of Copenhagen, Institute of Molecular Biology (Denmark)], E-mail: fmp@apk.molbio.ku.dk

    2004-01-15

    We have examined how the hydrogen bond geometry in three different proteins is affected when structural restraints based on measurements of residual dipolar couplings are included in the structure calculations. The study shows, that including restraints based solely on {sup 1}H{sup N}-{sup 15}N residual dipolar couplings has pronounced impact on the backbone rmsd and Ramachandran plot but does not improve the hydrogen bond geometry. In the case of chymotrypsin inhibitor 2 the addition of {sup 13}CO-{sup 13}C{sup {alpha}} and {sup 15}N-{sup 13}CO one bond dipolar couplings as restraints in the structure calculations improved the hydrogen bond geometry to a quality comparable to that obtained in the 1.8 A resolution X-ray structure of this protein. A systematic restraint study was performed, in which four types of restraints, residual dipolar couplings, hydrogen bonds, TALOS angles and NOEs, were allowed in two states. This study revealed the importance of using several types of residual dipolar couplings to get good hydrogen bond geometry. The study also showed that using a small set of NOEs derived only from the amide protons, together with a full set of residual dipolar couplings resulted in structures of very high quality. When reducing the NOE set, it is mainly the side-chain to side-chain NOEs that are removed. Despite of this the effect on the side-chain packing is very small when a reduced NOE set is used, which implies that the over all fold of a protein structure is mainly determined by correct folding of the backbone.

  17. Solution NMR Structure of Hypothetical Protein CV_2116 Encoded by a Viral Prophage Element in Chromobacterium violaceum

    Directory of Open Access Journals (Sweden)

    Yunhuang Yang

    2012-06-01

    Full Text Available CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e−07 corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and 13C- and 15N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + β fold containing two anti-parallel β -sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum.

  18. The dynamics of the G protein-coupled neuropeptide Y2 receptor in monounsaturated membranes investigated by solid-state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Lars; Kahr, Julian; Schmidt, Peter; Krug, Ulrike; Scheidt, Holger A.; Huster, Daniel, E-mail: daniel.huster@medizin.uni-leipzig.de [University of Leipzig, Institute of Medical Physics and Biophysics (Germany)

    2015-04-15

    In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors’ function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static {sup 15}N NMR spectra and quantitative determination of {sup 1}H–{sup 13}C order parameters through measurement of the {sup 1}H–{sup 13}C dipolar couplings of the CH, CH{sub 2} and CH{sub 3} groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S{sub backbone} = 0.59–0.67, S{sub CH2} = 0.41–0.51 and S{sub CH3} = 0.22) obtained in directly polarized {sup 13}C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

  19. EZ-ASSIGN, a program for exhaustive NMR chemical shift assignments of large proteins from complete or incomplete triple-resonance data

    Energy Technology Data Exchange (ETDEWEB)

    Zuiderweg, Erik R. P., E-mail: zuiderwe@umich.edu; Bagai, Ireena [The University of Michigan Medical School, Department of Biological Chemistry (United States); Rossi, Paolo [Rutgers University, Center for Integrative Proteomics Research (United States); Bertelsen, Eric B. [Arbor Communications, Inc. (United States)

    2013-10-15

    For several of the proteins in the BioMagResBank larger than 200 residues, 60 % or fewer of the backbone resonances were assigned. But how reliable are those assignments? In contrast to complete assignments, where it is possible to check whether every triple-resonance Generalized Spin System (GSS) is assigned once and only once, with incomplete data one should compare all possible assignments and pick the best one. But that is not feasible: For example, for 200 residues and an incomplete set of 100 GSS, there are 1.6 Multiplication-Sign 10{sup 260} possible assignments. In 'EZ-ASSIGN', the protein sequence is divided in smaller unique fragments. Combined with intelligent search approaches, an exhaustive comparison of all possible assignments is now feasible using a laptop computer. The program was tested with experimental data of a 388-residue domain of the Hsp70 chaperone protein DnaK and for a 351-residue domain of a type III secretion ATPase. EZ-ASSIGN reproduced the hand assignments. It did slightly better than the computer program PINE (Bahrami et al. in PLoS Comput Biol 5(3):e1000307, 2009) and significantly outperformed SAGA (Crippen et al. in J Biomol NMR 46:281-298, 2010), AUTOASSIGN (Zimmerman et al. in J Mol Biol 269:592-610, 1997), and IBIS (Hyberts and Wagner in J Biomol NMR 26:335-344, 2003). Next, EZ-ASSIGN was used to investigate how well NMR data of decreasing completeness can be assigned. We found that the program could confidently assign fragments in very incomplete data. Here, EZ-ASSIGN dramatically outperformed all the other assignment programs tested.

  20. EZ-ASSIGN, a program for exhaustive NMR chemical shift assignments of large proteins from complete or incomplete triple-resonance data

    International Nuclear Information System (INIS)

    Zuiderweg, Erik R. P.; Bagai, Ireena; Rossi, Paolo; Bertelsen, Eric B.

    2013-01-01

    For several of the proteins in the BioMagResBank larger than 200 residues, 60 % or fewer of the backbone resonances were assigned. But how reliable are those assignments? In contrast to complete assignments, where it is possible to check whether every triple-resonance Generalized Spin System (GSS) is assigned once and only once, with incomplete data one should compare all possible assignments and pick the best one. But that is not feasible: For example, for 200 residues and an incomplete set of 100 GSS, there are 1.6 × 10 260 possible assignments. In “EZ-ASSIGN”, the protein sequence is divided in smaller unique fragments. Combined with intelligent search approaches, an exhaustive comparison of all possible assignments is now feasible using a laptop computer. The program was tested with experimental data of a 388-residue domain of the Hsp70 chaperone protein DnaK and for a 351-residue domain of a type III secretion ATPase. EZ-ASSIGN reproduced the hand assignments. It did slightly better than the computer program PINE (Bahrami et al. in PLoS Comput Biol 5(3):e1000307, 2009) and significantly outperformed SAGA (Crippen et al. in J Biomol NMR 46:281–298, 2010), AUTOASSIGN (Zimmerman et al. in J Mol Biol 269:592–610, 1997), and IBIS (Hyberts and Wagner in J Biomol NMR 26:335–344, 2003). Next, EZ-ASSIGN was used to investigate how well NMR data of decreasing completeness can be assigned. We found that the program could confidently assign fragments in very incomplete data. Here, EZ-ASSIGN dramatically outperformed all the other assignment programs tested

  1. PFG-NMR self-diffusion in casein dispersions: effect of probe size and protein aggregate size

    NARCIS (Netherlands)

    Salami, S.; Rondeau, C.; Duynhoven, van J.P.M.; Mariette, F.

    2013-01-01

    The self-diffusion coefficients of different molecular weight PEGs (Polyethylene glycol) and casein particles were measured, using a pulsed-gradient nuclear magnetic resonance technique (PFG-NMR), in native phosphocaseinate (NPC) and sodium caseinate (SC) dispersions where caseins are not structured

  2. Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR.

    Science.gov (United States)

    Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira M; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, Jungoo; Güntert, Peter; Aceti, David J; Markley, John L; Kainosho, Masatsune

    2008-12-01

    The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

  3. BioMagResBank database with sets of experimental NMR constraints corresponding to the structures of over 1400 biomolecules deposited in the Protein Data Bank

    International Nuclear Information System (INIS)

    Doreleijers, Jurgen F.; Mading, Steve; Maziuk, Dimitri; Sojourner, Kassandra; Yin Lei; Zhu Jun; Markley, John L.; Ulrich, Eldon L.

    2003-01-01

    Experimental constraints associated with NMR structures are available from the Protein Data Bank (PDB) in the form of 'Magnetic Resonance' (MR) files. These files contain multiple types of data concatenated without boundary markers and are difficult to use for further research. Reported here are the results of a project initiated to annotate, archive, and disseminate these data to the research community from a searchable resource in a uniform format. The MR files from a set of 1410 NMR structures were analyzed and their original constituent data blocks annotated as to data type using a semi-automated protocol. A new software program called Wattos was then used to parse and archive the data in a relational database. From the total number of MR file blocks annotated as constraints, it proved possible to parse 84% (3337/3975). The constraint lists that were parsed correspond to three data types (2511 distance, 788 dihedral angle, and 38 residual dipolar couplings lists) from the three most popular software packages used in NMR structure determination: XPLOR/CNS (2520 lists), DISCOVER (412 lists), and DYANA/DIANA (405 lists). These constraints were then mapped to a developmental version of the BioMagResBank (BMRB) data model. A total of 31 data types originating from 16 programs have been classified, with the NOE distance constraint being the most commonly observed. The results serve as a model for the development of standards for NMR constraint deposition in computer-readable form. The constraints are updated regularly and are available from the BMRB web site (http://www.bmrb.wisc.edu)

  4. 13CHD2–CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins

    International Nuclear Information System (INIS)

    Rennella, Enrico; Huang, Rui; Velyvis, Algirdas; Kay, Lewis E.

    2015-01-01

    An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of 13 CHD 2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360 kDa half-proteasome, establish that the sensitivity of this 13 CHD 2 13 C–CEST technique can be upwards of a factor of 5 times higher than for a previously published 13 CH 3 13 C–CEST approach (Bouvignies and Kay in J Biomol NMR 53:303–310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins

  5. The "long tail" of the protein tumbling correlation function: observation by (1)H NMR relaxometry in a wide frequency and concentration range.

    Science.gov (United States)

    Roos, Matthias; Hofmann, Marius; Link, Susanne; Ott, Maria; Balbach, Jochen; Rössler, Ernst; Saalwächter, Kay; Krushelnitsky, Alexey

    2015-12-01

    Inter-protein interactions in solution affect the auto-correlation function of Brownian tumbling not only in terms of a simple increase of the correlation time, they also lead to the appearance of a weak slow component ("long tail") of the correlation function due to a slowly changing local anisotropy of the microenvironment. The conventional protocol of correlation time estimation from the relaxation rate ratio R1/R2 assumes a single-component tumbling correlation function, and thus can provide incorrect results as soon as the "long tail" is of relevance. This effect, however, has been underestimated in many instances. In this work we present a detailed systematic study of the tumbling correlation function of two proteins, lysozyme and bovine serum albumin, at different concentrations and temperatures using proton field-cycling relaxometry combined with R1ρ and R2 measurements. Unlike high-field NMR relaxation methods, these techniques enable a detailed study of dynamics on a time scale longer than the normal protein tumbling correlation time and, thus, a reliable estimate of the parameters of the "long tail". In this work we analyze the concentration dependence of the intensity and correlation time of the slow component and perform simulations of high-field (15)N NMR relaxation data demonstrating the importance of taking the "long tail" in the analysis into account.

  6. relaxGUI: a new software for fast and simple NMR relaxation data analysis and calculation of ps-ns and μs motion of proteins

    International Nuclear Information System (INIS)

    Bieri, Michael; D’Auvergne, Edward J.; Gooley, Paul R.

    2011-01-01

    Investigation of protein dynamics on the ps-ns and μs-ms timeframes provides detailed insight into the mechanisms of enzymes and the binding properties of proteins. Nuclear magnetic resonance (NMR) is an excellent tool for studying protein dynamics at atomic resolution. Analysis of relaxation data using model-free analysis can be a tedious and time consuming process, which requires good knowledge of scripting procedures. The software relaxGUI was developed for fast and simple model-free analysis and is fully integrated into the software package relax. It is written in Python and uses wxPython to build the graphical user interface (GUI) for maximum performance and multi-platform use. This software allows the analysis of NMR relaxation data with ease and the generation of publication quality graphs as well as color coded images of molecular structures. The interface is designed for simple data analysis and management. The software was tested and validated against the command line version of relax.

  7. relaxGUI: a new software for fast and simple NMR relaxation data analysis and calculation of ps-ns and μs motion of proteins.

    Science.gov (United States)

    Bieri, Michael; d'Auvergne, Edward J; Gooley, Paul R

    2011-06-01

    Investigation of protein dynamics on the ps-ns and μs-ms timeframes provides detailed insight into the mechanisms of enzymes and the binding properties of proteins. Nuclear magnetic resonance (NMR) is an excellent tool for studying protein dynamics at atomic resolution. Analysis of relaxation data using model-free analysis can be a tedious and time consuming process, which requires good knowledge of scripting procedures. The software relaxGUI was developed for fast and simple model-free analysis and is fully integrated into the software package relax. It is written in Python and uses wxPython to build the graphical user interface (GUI) for maximum performance and multi-platform use. This software allows the analysis of NMR relaxation data with ease and the generation of publication quality graphs as well as color coded images of molecular structures. The interface is designed for simple data analysis and management. The software was tested and validated against the command line version of relax.

  8. Effects of solvent concentration and composition on protein dynamics: 13C MAS NMR studies of elastin in glycerol-water mixtures.

    Science.gov (United States)

    Demuth, Dominik; Haase, Nils; Malzacher, Daniel; Vogel, Michael

    2015-08-01

    We use (13)C CP MAS NMR to investigate the dependence of elastin dynamics on the concentration and composition of the solvent at various temperatures. For elastin in pure glycerol, line-shape analysis shows that larger-scale fluctuations of the protein backbone require a minimum glycerol concentration of ~0.6 g/g at ambient temperature, while smaller-scale fluctuations are activated at lower solvation levels of ~0.2 g/g. Immersing elastin in various glycerol-water mixtures, we observe at room temperature that the protein mobility is higher for lower glycerol fractions in the solvent and, thus, lower solvent viscosity. When decreasing the temperature, the elastin spectra approach the line shape for the rigid protein at 245 K for all studied samples, indicating that the protein ceases to be mobile on the experimental time scale of ~10(-5) s. Our findings yield evidence for a strong coupling between elastin fluctuations and solvent dynamics and, hence, such interaction is not restricted to the case of protein-water mixtures. Spectral resolution of different carbon species reveals that the protein-solvent couplings can, however, be different for side chain and backbone units. We discuss these results against the background of the slaving model for protein dynamics. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. NMR imaging

    International Nuclear Information System (INIS)

    Andrew, E.R.

    1983-01-01

    Since hydrogen is the most abundant element in all living organisms, proton NMR lends itself well as a method of investigation in biology and medicine. NMR imaging has some special advantages as a diagnostic tool: no ionizing radiation is used, it is noninvasive; it provides a safer means of imaging than the use of x-rays, gamma rays, positrons, or heavy ions. In contrast with ultrasound, the radiation penetrates the bony structures without attenuation. In additional to morphological information, NMR imaging provides additional diagnostic insights through relaxation parameters, which are not available from other imaging methods. In the decade since the first primitive NMR images were obtained, the quality of images now obtained approaches those from CT x-ray scanners. Prototype instruments are being constructed for clinical evaluation and the first whole-body scanners are beginning to appear on the market at costs comparable to CT scanners. Primary differences in equipment for conventional NMR and NMR imaging are the much larger aperture magnets that are required for the examination of human subjects and the addition of coils to generate field gradients and facilities for manipulating the gradients. Early results from clinical trials in many parts of the world are encouraging, and in a few years, the usefuleness of this modality of medical imaging to the medical profession in diagnosis and treatment of disease will be defined. 10 figures

  10. Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy*

    Science.gov (United States)

    Zandarashvili, Levani; Sahu, Debashish; Lee, Kwanbok; Lee, Yong Sun; Singh, Pomila; Rajarathnam, Krishna; Iwahara, Junji

    2013-01-01

    Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized 15N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear 1H-15N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ∼17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules. PMID:23447529

  11. NMR characterisation of the minimal interacting regions of centrosomal proteins 4.1R and NuMA1: effect of phosphorylation

    Directory of Open Access Journals (Sweden)

    Bruix Marta

    2010-01-01

    Full Text Available Abstract Background Some functions of 4.1R in non-erythroid cells are directly related with its distinct sub-cellular localisation during cell cycle phases. During mitosis, 4.1R is implicated in cell cycle progression and spindle pole formation, and co-localizes with NuMA1. However, during interphase 4.1R is located in the nucleus and only partially co-localizes with NuMA1. Results We have characterized by NMR the structural features of the C-terminal domain of 4.1R and those of the minimal region (the last 64 residues involved in the interaction with NuMA1. This subdomain behaves as an intrinsically unfolded protein containing a central region with helical tendency. The specific residues implicated in the interaction with NuMA1 have been mapped by NMR titrations and involve the N-terminal and central helical regions. The segment of NuMA1 that interacts with 4.1R is phosphorylated during mitosis. Interestingly, NMR data indicates that the phosphorylation of NuMA1 interacting peptide provokes a change in the interaction mechanism. In this case, the recognition occurs through the central helical region as well as through the C-terminal region of the subdomain meanwhile the N-terminal region do not interact. Conclusions These changes in the interaction derived from the phosphorylation state of NuMA1 suggest that phosphorylation can act as subtle mechanism of temporal and spatial regulation of the complex 4.1R-NuMA1 and therefore of the processes where both proteins play a role.

  12. NMR structure of navel orangeworm moth pheromone-binding protein (AtraPBP1): implications for pH-sensitive pheromone detection.

    Science.gov (United States)

    Xu, Xianzhong; Xu, Wei; Rayo, Josep; Ishida, Yuko; Leal, Walter S; Ames, James B

    2010-02-23

    The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through the aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present the three-dimensional structure of a PBP from A. transitella (AtraPBP1) in solution at pH 4.5 determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments, multiangle light scattering, and (15)N NMR relaxation analysis indicate that AtraPBP1 forms a stable monomer in solution at pH 4.5 in contrast to forming mostly dimers at pH 7. The NMR structure of AtraPBP1 at pH 4.5 contains seven alpha-helices (alpha1, L8-L23; alpha2, D27-F36; alpha3, R46-V62; alpha4, A73-M78; alpha5, D84-S100; alpha6, R107-L125; alpha7, M131-E141) that adopt an overall main-chain fold similar to that of PBPs found in Antheraea polyphemus and Bombyx mori. The AtraPBP1 structure is stabilized by three disulfide bonds formed by C19/C54, C50/C108, and C97/C117 and salt bridges formed by H69/E60, H70/E57, H80/E132, H95/E141, and H123/D40. All five His residues are cationic at pH 4.5, whereas H80 and H95 become neutral at pH 7.0. The C-terminal helix (alpha7) contains hydrophobic residues (M131, V133, V134, V135, V138, L139, and A140) that contact conserved residues (W37, L59, A73, F76, A77, I94, V111, and V115) suggested to interact with bound pheromone. Our NMR studies reveal that acid-induced formation of the C-terminal helix at pH 4.5 is triggered by a histidine protonation switch that promotes rapid release of bound pheromone under acidic conditions.

  13. Simultaneous acquisition of 2D and 3D solid-state NMR experiments for sequential assignment of oriented membrane protein samples

    Energy Technology Data Exchange (ETDEWEB)

    Gopinath, T. [University of Minnesota, Department of Biochemistry, Molecular Biology, and Biophysics (United States); Mote, Kaustubh R. [University of Minnesota, Department of Chemistry (United States); Veglia, Gianluigi, E-mail: vegli001@umn.edu [University of Minnesota, Department of Biochemistry, Molecular Biology, and Biophysics (United States)

    2015-05-15

    We present a new method called DAISY (Dual Acquisition orIented ssNMR spectroScopY) for the simultaneous acquisition of 2D and 3D oriented solid-state NMR experiments for membrane proteins reconstituted in mechanically or magnetically aligned lipid bilayers. DAISY utilizes dual acquisition of sine and cosine dipolar or chemical shift coherences and long living {sup 15}N longitudinal polarization to obtain two multi-dimensional spectra, simultaneously. In these new experiments, the first acquisition gives the polarization inversion spin exchange at the magic angle (PISEMA) or heteronuclear correlation (HETCOR) spectra, the second acquisition gives PISEMA-mixing or HETCOR-mixing spectra, where the mixing element enables inter-residue correlations through {sup 15}N–{sup 15}N homonuclear polarization transfer. The analysis of the two 2D spectra (first and second acquisitions) enables one to distinguish {sup 15}N–{sup 15}N inter-residue correlations for sequential assignment of membrane proteins. DAISY can be implemented in 3D experiments that include the polarization inversion spin exchange at magic angle via I spin coherence (PISEMAI) sequence, as we show for the simultaneous acquisition of 3D PISEMAI–HETCOR and 3D PISEMAI–HETCOR-mixing experiments.

  14. Simultaneous acquisition of 2D and 3D solid-state NMR experiments for sequential assignment of oriented membrane protein samples.

    Science.gov (United States)

    Gopinath, T; Mote, Kaustubh R; Veglia, Gianluigi

    2015-05-01

    We present a new method called DAISY (Dual Acquisition orIented ssNMR spectroScopY) for the simultaneous acquisition of 2D and 3D oriented solid-state NMR experiments for membrane proteins reconstituted in mechanically or magnetically aligned lipid bilayers. DAISY utilizes dual acquisition of sine and cosine dipolar or chemical shift coherences and long living (15)N longitudinal polarization to obtain two multi-dimensional spectra, simultaneously. In these new experiments, the first acquisition gives the polarization inversion spin exchange at the magic angle (PISEMA) or heteronuclear correlation (HETCOR) spectra, the second acquisition gives PISEMA-mixing or HETCOR-mixing spectra, where the mixing element enables inter-residue correlations through (15)N-(15)N homonuclear polarization transfer. The analysis of the two 2D spectra (first and second acquisitions) enables one to distinguish (15)N-(15)N inter-residue correlations for sequential assignment of membrane proteins. DAISY can be implemented in 3D experiments that include the polarization inversion spin exchange at magic angle via I spin coherence (PISEMAI) sequence, as we show for the simultaneous acquisition of 3D PISEMAI-HETCOR and 3D PISEMAI-HETCOR-mixing experiments.

  15. Dante-Z sequence as selective impulsion in high field mono and multidimensional NMR. Application to the study of proteins, peptides and their interactions

    International Nuclear Information System (INIS)

    Roumestand, C.; Toma, F.

    1992-01-01

    DANTE-Z is a simple and efficient way for NMR spectral selection. We present here different applications of DANTE-Z in high-resolution NMR of peptides and proteins. We have been using proton selective excitation by DANTE-Z to perform 1D-correlated (homo- or heteronuclear) experiments corresponding to one line of either 2D or 3D experiments. Following the same scheme, we could also edit planes of 3D experiments by concatenating 1D-correlated experiments with conventional 2D experiments. In the heteronuclear case (i.e. 1 H, 31 P), we could also edit planes of a 4D experiment by the simultaneous selection of 1 H and the X nucleus. Owing to the favourable excitation profile of DANTE-Z, we used it successfully for topological excitations (spectral width from 150 Hz up to 1500 Hz) in 'semi-soft'-2D experiments and 'soft'-2D experiment. These applications are illustrated by the results obtained at 600 MHz on a protein and a phosphonamide peptide

  16. Reconstitution of FMN-free NADPH-cytochrome P-450 reductase with a phosphorothioate analog of FMN: 31P NMR studies of the reconstituted protein

    International Nuclear Information System (INIS)

    Krum, D.P.; Otvos, J.D.; Calhoun, J.P.; Miziorko, H.M.; Masters, B.S.S.

    1987-01-01

    A phosphorothioate analog of FMN (FMNS) has been synthesized and shown to be completely competent in reconstituting the FMN-free form of NADPH-cytochrome P-450 reductase as evidenced by flavin determinations and cytochrome c reductase activity assays. The FMNS-reconstituted FMN-free reductase gives rise to an air-stable semiquinone, and the fluorescence of FMNS is quenched upon addition of FMN-free reductase. 31 P NMR spectra of the FMN-free reductase reveal only two resonances (-7.3 and -11.3 ppm), which are attributable to FAD. This result confirms the assignments of Otvos et al, and demonstrates unequivocally that there are no phosphate residues other than those of FMN and FAD attached to the steapsin-solubilized reductase. The addition of FMN to the FMN-free reductase resulted in the appearance of one additional resonance at 3.9 ppm. Addition of FMNS to the FMN-free reductase caused no change, surprisingly, in the 31 P NMR spectrum until Mn(II) was added, after which a peak centered at ∼ 45 ppm was observed. This unexpected result may be explained if the T 1 for the phosphate of FMNS is significantly longer than that of FMN, and suggests that the sulfur atom of FMNS may perturb the interaction of the phosphate with its protein environment. These results demonstrate the utility of phosphorothioate analogs as mechanistic probes for proteins containing nucleotide cofactors

  17. Simultaneous acquisition of 2D and 3D solid-state NMR experiments for sequential assignment of oriented membrane protein samples

    International Nuclear Information System (INIS)

    Gopinath, T.; Mote, Kaustubh R.; Veglia, Gianluigi

    2015-01-01

    We present a new method called DAISY (Dual Acquisition orIented ssNMR spectroScopY) for the simultaneous acquisition of 2D and 3D oriented solid-state NMR experiments for membrane proteins reconstituted in mechanically or magnetically aligned lipid bilayers. DAISY utilizes dual acquisition of sine and cosine dipolar or chemical shift coherences and long living 15 N longitudinal polarization to obtain two multi-dimensional spectra, simultaneously. In these new experiments, the first acquisition gives the polarization inversion spin exchange at the magic angle (PISEMA) or heteronuclear correlation (HETCOR) spectra, the second acquisition gives PISEMA-mixing or HETCOR-mixing spectra, where the mixing element enables inter-residue correlations through 15 N– 15 N homonuclear polarization transfer. The analysis of the two 2D spectra (first and second acquisitions) enables one to distinguish 15 N– 15 N inter-residue correlations for sequential assignment of membrane proteins. DAISY can be implemented in 3D experiments that include the polarization inversion spin exchange at magic angle via I spin coherence (PISEMAI) sequence, as we show for the simultaneous acquisition of 3D PISEMAI–HETCOR and 3D PISEMAI–HETCOR-mixing experiments

  18. An economical method for production of (2H, (13CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome.

    Directory of Open Access Journals (Sweden)

    Algirdas Velyvis

    Full Text Available NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby (13CH(3 methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-(2H; β-(2H;γ-(13C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr (13C,(1H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[(13C,(1H]-Thr.

  19. Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers

    International Nuclear Information System (INIS)

    Mote, Kaustubh R.; Gopinath, T.; Traaseth, Nathaniel J.; Kitchen, Jason; Gor’kov, Peter L.; Brey, William W.; Veglia, Gianluigi

    2011-01-01

    Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1 H- 15 N dipolar couplings (DC) and 15 N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([ 1 H, 15 N]-SE-PISEMA-PDSD). The incorporation of 2D 15 N/ 15 N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15 N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.

  20. Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers.

    Science.gov (United States)

    Mote, Kaustubh R; Gopinath, T; Traaseth, Nathaniel J; Kitchen, Jason; Gor'kov, Peter L; Brey, William W; Veglia, Gianluigi

    2011-11-01

    Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring (1)H-(15)N dipolar couplings (DC) and (15)N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles' heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([(1)H,(15)N]-SE-PISEMA-PDSD). The incorporation of 2D (15)N/(15)N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the (15)N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers. © Springer Science+Business Media B.V. 2011

  1. Interaction between Wine Phenolic Acids and Salivary Proteins by Saturation-Transfer Difference Nuclear Magnetic Resonance Spectroscopy (STD-NMR) and Molecular Dynamics Simulations.

    Science.gov (United States)

    Ferrer-Gallego, Raúl; Hernández-Hierro, José Miguel; Brás, Natércia F; Vale, Nuno; Gomes, Paula; Mateus, Nuno; de Freitas, Victor; Heredia, Francisco J; Escribano-Bailón, María Teresa

    2017-08-09

    The interaction between phenolic compounds and salivary proteins is highly related to the astringency perception. Recently, it has been proven the existence of synergisms on the perceived astringency when phenolic acids were tested as mixtures in comparison to individual compounds, maintaining constant the total amount of the stimulus. The interactions between wine phenolic acids and the peptide fragment IB7 12 have been studied by saturation-transfer difference (STD) NMR spectroscopy. This technique provided the dissociation constants and the percentage of interaction between both individual and mixtures of hydroxybenzoic and hydroxycinnamic acids and the model peptide. It is noteworthy that hydroxybenzoic acids showed higher affinity for the peptide than hydroxycinnamic acids. To obtain further insights into the mechanisms of interaction, molecular dynamics simulations have been performed. Results obtained not only showed the ability of these compounds to interact with salivary proteins but also may justify the synergistic effect observed in previous sensory studies.

  2. Compact NMR

    Energy Technology Data Exchange (ETDEWEB)

    Bluemich, Bernhard; Haber-Pohlmeier, Sabina; Zia, Wasif [RWTH Aachen Univ. (Germany). Inst. fuer Technische und Makromolekulare Chemie (ITMC)

    2014-06-01

    Nuclear Magnetic Resonance (NMR) spectroscopy is the most popular method for chemists to analyze molecular structures, while Magnetic Resonance Imaging (MRI) is a non-invasive diagnostic tool for medical doctors that provides high-contrast images of biological tissue. In both applications, the sample (or patient) is positioned inside a large, superconducting magnet to magnetize the atomic nuclei. Interrogating radio-frequency pulses result in frequency spectra that provide the chemist with molecular information, the medical doctor with anatomic images, and materials scientist with NMR relaxation parameters. Recent advances in magnet technology have led to a variety of small permanent magnets to allow compact and low-cost instruments. The goal of this book is to provide an introduction to the practical use of compact NMR at a level nearly as basic as the operation of a smart phone.

  3. Sequence-specific assignments in the 1H NMR spectrum of the human inflammatory protein C5a

    International Nuclear Information System (INIS)

    Zuiderweg, E.R.P.; Mollison, K.W.; Henkin, J.; Carter, G.W.

    1988-01-01

    Full sequence-specific assignments for the 1 H NMR lines of the backbone protons of the human complement factor C5a are described and documented. The results were obtained by largely following the methodology developed by Wuethrich et al. Assignments for the majority of the amino acid side chain protons were obtained by using a comparison of double- and triple-quantum-filtered two-dimensional correlated experiments together with the analysis of relayed coherence transfer spectra. The assignments provide the basis for the determination of the thus far unknown three-dimensional structure of C5a from nuclear Overhauser enhancement distance constraints

  4. Structural biology of the sequestration and transport of heavy metal toxins: NMR structure determination of proteins containing the -Cys-X-Y-Cys-metal binding motifs. 1998 annual progress report

    International Nuclear Information System (INIS)

    Opella, S.J.

    1998-01-01

    'The overall goal of the research is to apply the methods of structural biology, which have been previously used primarily in biomedical applications, to bioremediation. The authors are doing this by using NMR spectroscopy to determine the structures of proteins involved in the bacterial mercury detoxification system. The research is based on the premise that the proteins encoded in the genes of the bacterial detoxification system are an untapped source of reagents and, more fundamentally, chemical strategies that can be used to remove heavy metal toxins from the environment. The initial goals are to determine the structures of the proteins of the bacterial mercury detoxification systems responsible for the sequestration and transport of the Hg(II) ions in to the cell where reduction to Hg(O) occurs. These proteins are meP, which is water soluble and can be investigated with multidimensional solution NMR methods, and merT, the transport protein in the membrane that requires solid-state NMR methods. As of June 1998, this report summarizes work after about one and half years of the three-year award. The authors have made significant accomplishments in three aspects of the NMR studies of the proteins of the bacterial mercury detoxification system.'

  5. Structure of the Putative 32 kDa Myrosinase Binding Protein from Arabidopsis (At3g16450.1) Determined by SAIL-NMR

    Science.gov (United States)

    Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira Mei; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, JunGoo; Güntert, Peter; Aceti, David J.; Markley, John L.; Kainosho, Masatsune

    2009-01-01

    The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme, myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniformly 13C/15N labeling methods, we used our stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected with the SAIL-protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at the low concentration (0.2 mM) of the protein product. We collected additional NOESY data and solved the three-dimensional structure with the CYANA software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent, but similar, lectin-fold domains composed of three β-sheets. PMID:19021763

  6. Study of the interactions between a proline-rich protein and a flavan-3-ol by NMR: residual structures in the natively unfolded protein provides anchorage points for the ligands.

    Science.gov (United States)

    Pascal, Christine; Paté, Franck; Cheynier, Véronique; Delsuc, Marc-André

    2009-09-01

    Astringency is one of the major organoleptic properties of food and beverages that are made from plants, such as tea, chocolate, beer, or red wine. This sensation is thought to be due to interactions between tannins and salivary proline-rich proteins, which are natively unfolded proteins. A human salivary proline-rich protein, namely IB-5, was produced by the recombinant method. Its interactions with a model tannin, epigallocatechin gallate (EGCG), the major flavan-3-ol in green tea, were studied here. Circular dichroism experiments showed that IB-5 presents residual structures (PPII helices) when the ionic strength is close to that in saliva. In the presence of these residual structures, IB-5 undergoes an increase in structural content upon binding to EGCG. NMR data corroborated the presence of preformed structural elements within the protein prior to binding and a partial assignment was proposed, showing partial structuration. TOCSY experiments showed that amino acids that are involved in PPII helices are more likely to interact with EGCG than those in random coil regions, as if they were anchorage points for the ligand. The signal from IB-5 in the DOSY NMR spectrum revealed an increase in polydispersity upon addition of EGCG while the mean hydrodynamic radius remained unchanged. This strongly suggests the formation of IB-5/EGCG aggregates.

  7. 2D NMR studies of biomolecules

    International Nuclear Information System (INIS)

    Lamerichs, R.M.J.N.

    1989-01-01

    The work described in this thesis comprises two related subjects. The first part describes methods to derive high-resolution structures of proteins in solution using two-dimensional (2-D) NMR. The second part describes 2-D NMR studies on the interaction between proteins and DNA. (author). 261 refs.; 52 figs.; 23 tabs

  8. 15N NMR relaxation studies of calcium-loaded parvalbumin show tight dynamics compared to those of other EF-hand proteins

    DEFF Research Database (Denmark)

    Baldellon, C; Alattia, J R; Strub, M P

    1998-01-01

    Dynamics of the rat alpha-parvalbumin calcium-loaded form have been determined by measurement of 15N nuclear relaxation using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed using spectral density functions and the Lipari-Szabo formalism. The major dynamic features...... for the rat alpha-parvalbumin calcium-loaded form are (1) the extreme rigidity of the helix-loop-helix EF-hand motifs and the linker segment connecting them, (2) the N and C termini of the protein being restricted in their mobility, (3) a conformational exchange occurring at the kink of helix D, and (4...... properties which are conserved in the EF-hand domains from different members of this superfamily: (1) a tendency toward higher mobility of NH vectors at relative position 2 in the Ca2+-binding loop, (2) a restricted mobility for the other residues in the binding loop, and (3) an overall rigidity...

  9. Theoretical analysis of geometry and NMR isotope shift in hydrogen-bonding center of photoactive yellow protein by combination of multicomponent quantum mechanics and ONIOM scheme

    Energy Technology Data Exchange (ETDEWEB)

    Kanematsu, Yusuke; Tachikawa, Masanori [Quantum Chemistry Division, Yokohama City University, Seto 22-2, Kanazawa-ku, Yokohama 236-0027 (Japan)

    2014-11-14

    Multicomponent quantum mechanical (MC-QM) calculation has been extended with ONIOM (our own N-layered integrated molecular orbital + molecular mechanics) scheme [ONIOM(MC-QM:MM)] to take account of both the nuclear quantum effect and the surrounding environment effect. The authors have demonstrated the first implementation and application of ONIOM(MC-QM:MM) method for the analysis of the geometry and the isotope shift in hydrogen-bonding center of photoactive yellow protein. ONIOM(MC-QM:MM) calculation for a model with deprotonated Arg52 reproduced the elongation of O–H bond of Glu46 observed by neutron diffraction crystallography. Among the unique isotope shifts in different conditions, the model with protonated Arg52 with solvent effect reasonably provided the best agreement with the corresponding experimental values from liquid NMR measurement. Our results implied the availability of ONIOM(MC-QM:MM) to distinguish the local environment around hydrogen bonds in a biomolecule.

  10. Protein catabolism and high lipid metabolism associated with long-distance exercise are revealed by plasma NMR metabolomics in endurance horses.

    Directory of Open Access Journals (Sweden)

    Laurence Le Moyec

    Full Text Available During long distance endurance races, horses undergo high physiological and metabolic stresses. The adaptation processes involve the modulation of the energetic pathways in order to meet the energy demand. The aims were to evaluate the effects of long endurance exercise on the plasma metabolomic profiles and to investigate the relationships with the individual horse performances. The metabolomic profiles of the horses were analyzed using the non-dedicated methodology, NMR spectroscopy and statistical multivariate analysis. The advantage of this method is to investigate several metabolomic pathways at the same time in a single sample. The plasmas were obtained before exercise (BE and post exercise (PE from 69 horses competing in three endurance races at national level (130-160 km. Biochemical assays were also performed on the samples taken at PE. The proton NMR spectra were compared using the supervised orthogonal projection on latent structure method according to several factors. Among these factors, the race location was not significant whereas the effect of the race exercise (sample BE vs PE of same horse was highly discriminating. This result was confirmed by the projection of unpaired samples (only BE or PE sample of different horses. The metabolomic profiles proved that protein, energetic and lipid metabolisms as well as glycoproteins content are highly affected by the long endurance exercise. The BE samples from finisher horses could be discriminated according to the racing speed based on their metabolomic lipid content. The PE samples could be discriminated according to the horse ranking position at the end of the race with lactate as unique correlated metabolite. As a conclusion, the metabolomic profiles of plasmas taken before and after the race provided a better understanding of the high energy demand and protein catabolism pathway that could expose the horses to metabolic disorders.

  11. Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy

    International Nuclear Information System (INIS)

    Sun Hechao; Godoy-Ruiz, Raquel; Tugarinov, Vitali

    2012-01-01

    Relaxation violated coherence transfer NMR spectroscopy (Tugarinov et al. in J Am Chem Soc 129:1743–1750, 2007) is an established experimental tool for quantitative estimation of the amplitudes of side-chain motions in methyl-protonated, highly deuterated proteins. Relaxation violated coherence transfer experiments monitor the build-up of methyl proton multiple-quantum coherences that can be created in magnetically equivalent spin-systems as long as their transverse magnetization components relax with substantially different rates. The rate of this build-up is a reporter of the methyl-bearing side-chain mobility. Although the build-up of multiple-quantum 1 H coherences is monitored in these experiments, the decay of the methyl signal during relaxation delays occurs when methyl proton magnetization is in a single-quantum state. We describe a relaxation violated coherence transfer approach where the relaxation of multiple-quantum 1 H– 13 C methyl coherences during the relaxation delay period is quantified. The NMR experiment and the associated fitting procedure that models the time-dependence of the signal build-up, are applicable to the characterization of side-chain order in [ 13 CH 3 ]-methyl-labeled, highly deuterated protein systems up to ∼100 kDa in molecular weight. The feasibility of extracting reliable measures of side-chain order is experimentally verified on methyl-protonated, perdeuterated samples of an 8.5-kDa ubiquitin at 10°C and an 82-kDa Malate Synthase G at 37°C.

  12. Internal motion time scales of a small, highly stable and disulfide-rich protein: A 15N, 13C NMR and molecular dynamics study

    International Nuclear Information System (INIS)

    Guenneugues, Marc; Gilquin, Bernard; Wolff, Nicolas; Menez, Andre; Zinn-Justin, Sophie

    1999-01-01

    Motions of the backbone CαHα and threonine CβHβ bonds of toxin α were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H → 13C NOE were obtained, as well as the variations of R1ρ(90 deg.) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several CαHα and threonine CβHβ experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin α, a highly stable protein (Tm=75 deg. C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 μs to 10 ms

  13. Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: Use of the INEPT [insensitive nucleus enhancement by polarization transfer] experiment to follow individual amides in detergent-solubilized M13 coat protein

    International Nuclear Information System (INIS)

    Henry, G.D.; Sykes, B.D.

    1990-01-01

    The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1 H nuclear magnetic resonance (NMR) study, multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow kinetic sets containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10 5 -fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein the authors use 15 N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiments can be used to transfer magnetization to the 15 N nucleus from a coupled proton; when 15 N-labeled protonated protein is dissolved in 2 H 2 O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H + and OH - ions. The time-dependent exchange-out experiment is suitable for slow exchange rates (k ex ). The INEPT experiment was also adapted to measure some of the more rapidly exchanging amides in the coat protein using either saturation transfer from water or exchange effects on the polarization transfer step itself. The results of all of these experiments are consistent with previous models of the coat protein in which a stable segment extends from the hydrophobic membrane-spanning region through to the C-terminus, whereas the N-terminal region is undergoing more extensive dynamic fluctuations

  14. Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

    International Nuclear Information System (INIS)

    Henry, G.D.; Weiner, J.H.; Sykes, B.D.

    1987-01-01

    Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13 C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D 2 O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H 2 O solutions; in 1:1 H 2 O/D 2 O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13 C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects, the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule. A model of the detergent-solubilized coat protein is constructed from these H-exchange data which is consistent with circular dichroism and other NMR results

  15. Improving the Accuracy of NMR Structures of Large Proteins Using Pseudocontact Shifts as Long-Range Restraints

    Energy Technology Data Exchange (ETDEWEB)

    Gaponenko, Vadim [National Cancer Institute, Structural Biophysics Laboratory (United States); Sarma, Siddhartha P. [Indian Institute of Science, Molecular Biophysics Unit (India); Altieri, Amanda S. [National Cancer Institute, Structural Biophysics Laboratory (United States); Horita, David A. [Wake Forest University School of Medicine, Department of Biochemistry (United States); Li, Jess; Byrd, R. Andrew [National Cancer Institute, Structural Biophysics Laboratory (United States)], E-mail: rabyrd@ncifcrf.gov

    2004-03-15

    We demonstrate improved accuracy in protein structure determination for large ({>=}30 kDa), deuterated proteins (e.g. STAT4{sub NT}) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.

  16. NMR imaging

    International Nuclear Information System (INIS)

    Ouchi, Toshihiro; Steiner, R.E.

    1984-01-01

    Three epidermoid and two dermoid tumours, pathologically proven, were examined by NMR and CT scans. Although most brain tumours have a low signal with a long T 1 , a dermoid cyst and one of the two components of the other dermoid tumour had a high signal and therefore a short T 1 . All three epidermoid tumours had a low signal and a long T 1 . Because of the high level contrast between some of the tumours and cerebrospinal fluid, NMR is helpful to detect the lesion. Neither of the liquid fluid levels in the tumour cysts or floating fat in the subarachnoid space was recognized in one patients, but the fine leakage of the content from the epidermoid cyst into the lateral ventricle was detected on a saturation recovery 1000 image in one case. (author)

  17. 15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

    Science.gov (United States)

    Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

    2005-04-01

    This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

  18. Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: Assignment of aromatic resonances with application to protein folding, structure, and dynamics

    International Nuclear Information System (INIS)

    Weiss, M.A.; Shoelson, S.E.; Nguyen, D.T.; O'Shea, E.; Karplus, M.; Khait, I.; Neuringer, L.J.; Inouye, K.; Frank, B.H.; Beckage, M.

    1989-01-01

    The aromatic 1 H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25 → Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constrains in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures

  19. Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag

    Energy Technology Data Exchange (ETDEWEB)

    Sengupta, Ishita; Gao, Min; Arachchige, Rajith J.; Nadaud, Philippe S. [The Ohio State University, Department of Chemistry and Biochemistry (United States); Cunningham, Timothy F.; Saxena, Sunil [University of Pittsburgh, Department of Chemistry (United States); Schwieters, Charles D. [National Institutes of Health, Center for Information Technology (United States); Jaroniec, Christopher P., E-mail: jaroniec@chemistry.ohio-state.edu [The Ohio State University, Department of Chemistry and Biochemistry (United States)

    2015-01-15

    Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu{sup 2+} binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC–Cu{sup 2+} K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ∼10 Å of the Cu{sup 2+} center experience considerable transverse PREs leading to severely attenuated resonances in 2D {sup 15}N–{sup 13}C correlation spectra. For more distant residues, electron–nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for {sup 15}N–Cu{sup 2+} distances up to ∼20 Å.

  20. Frontiers of NMR in Molecular Biology

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-08-25

    NMR spectroscopy is expanding the horizons of structural biology by determining the structures and describing the dynamics of blobular proteins in aqueous solution, as well as other classes of proteins including membrane proteins and the polypeptides that form the aggregates diagnostic of prion and amyloid diseases. Significant results are also emerging on DNA and RNA oligomers and their complexes with proteins. This meeting focused attention on key structural questions emanating from molecular biology and how NMR spectroscopy can be used to answer them.

  1. Toward an integrated model of protein-DNA recognition as inferred from NMR studies on the Lac repressor system

    NARCIS (Netherlands)

    Kalodimos, Ch.; Boelens, R.|info:eu-repo/dai/nl/070151407; Kaptein, R.|info:eu-repo/dai/nl/074334603

    2004-01-01

    Sequence-specific protein-DNA interactions are responsible for the regulation of key biological functions such as replication of the genome, initiation of transcription, and repair of damaged DNA. All of these regulatory pathways are built on the foundation that proteins are able to bind selectively

  2. High resolution NMR theory and chemical applications

    CERN Document Server

    Becker, Edwin D

    1999-01-01

    High Resolution NMR provides a broad treatment of the principles and theory of nuclear magnetic resonance (NMR) as it is used in the chemical sciences. It is written at an "intermediate" level, with mathematics used to augment, rather than replace, clear verbal descriptions of the phenomena. The book is intended to allow a graduate student, advanced undergraduate, or researcher to understand NMR at a fundamental level, and to see illustrations of the applications of NMR to the determination of the structure of small organic molecules and macromolecules, including proteins. Emphasis is on the study of NMR in liquids, but the treatment also includes high resolution NMR in the solid state and the principles of NMR imaging and localized spectroscopy. Careful attention is given to developing and interrelating four approaches - steady state energy levels, the rotating vector picture, the density matrix, and the product operator formalism. The presentation is based on the assumption that the reader has an acquaintan...

  3. Ligand-receptor Interactions by NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Novak. P.

    2008-04-01

    Full Text Available Today NMR spectroscopy is a method of choice for elucidation of interactions between biomolecules and the potential ligands. Knowledge on these interactions is an essential prerequisite for the rational drug design. The most important contribution of NMR to drug design a few years ago was the 3D structure determination of proteins. Besides delivering the 3D structures of the free proteins as a raw material for the modeling studies on ligand binding, NMR can directly yield valuable experimental data on the biologically important protein-ligand complexes. In addition to X-ray diffraction, NMR spectroscopy can provide information on the internal protein dynamics ordynamics of intermolecular interactions. Changes in NMR parameters allow us to detect ("SAR by NMR" and quantitatively determine binding affinities (titration, diffusion NMR experiments, etc. of potential ligands. Also, it is possible to determine the binding site and conformations of ligands, receptors and receptor-ligand complexes with the help of NMR methods such as tr-NOESY. Epitopes or functional groups responsible for binding of ligands to the receptor can be identified by employing STD or WaterLOGSY experiments. In this review are described some of the most frequent NMR methods for the characterization of the interactions between biomolecules and ligands, together with their advantages and disadvantages.

  4. Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

    Science.gov (United States)

    Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2017-02-22

    Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

  5. Proton detection for signal enhancement in solid-state NMR experiments on mobile species in membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ward, Meaghan E.; Ritz, Emily [University of Guelph, Department of Physics (Canada); Ahmed, Mumdooh A. M. [Suez University, The Department of Physics, Faculty of Science (Egypt); Bamm, Vladimir V.; Harauz, George [University of Guelph, Biophysics Interdepartmental Group (Canada); Brown, Leonid S.; Ladizhansky, Vladimir, E-mail: vladizha@uoguelph.ca [University of Guelph, Department of Physics (Canada)

    2015-12-15

    Direct proton detection is becoming an increasingly popular method for enhancing sensitivity in solid-state nuclear magnetic resonance spectroscopy. Generally, these experiments require extensive deuteration of the protein, fast magic angle spinning (MAS), or a combination of both. Here, we implement direct proton detection to selectively observe the mobile entities in fully-protonated membrane proteins at moderate MAS frequencies. We demonstrate this method on two proteins that exhibit different motional regimes. Myelin basic protein is an intrinsically-disordered, peripherally membrane-associated protein that is highly flexible, whereas Anabaena sensory rhodopsin is composed of seven rigid transmembrane α-helices connected by mobile loop regions. In both cases, we observe narrow proton linewidths and, on average, a 10× increase in sensitivity in 2D insensitive nuclear enhancement of polarization transfer-based HSQC experiments when proton detection is compared to carbon detection. We further show that our proton-detected experiments can be easily extended to three dimensions and used to build complete amino acid systems, including sidechain protons, and obtain inter-residue correlations. Additionally, we detect signals which do not correspond to amino acids, but rather to lipids and/or carbohydrates which interact strongly with membrane proteins.

  6. Absolute nutrient concentration measurements in cell culture media: 1H q-NMR spectra and data to compare the efficiency of pH-controlled protein precipitation versus CPMG or post-processing filtering approaches

    Directory of Open Access Journals (Sweden)

    Luca Goldoni

    2016-09-01

    Full Text Available The NMR spectra and data reported in this article refer to the research article titled “A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using q-NMR” [1]. We provide the 1H q-NMR spectra of cell culture media (DMEM after removal of serum proteins, which show the different efficiency of various precipitating solvents, the solvent/DMEM ratios, and pH of the solution. We compare the data of the absolute nutrient concentrations, measured by PULCON external standard method, before and after precipitation of serum proteins and those obtained using CPMG (Carr-Purcell-Meiboom-Gill sequence or applying post-processing filtering algorithms to remove, from the 1H q-NMR spectra, the proteins signal contribution. For each of these approaches, the percent error in the absolute value of every measurement for all the nutrients is also plotted as accuracy assessment. Keywords: 1H NMR, pH-controlled serum removal, PULCON, Accuracy, CPMG, Deconvolution

  7. High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

    Science.gov (United States)

    Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

    2018-04-27

    Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-{sup 19}F-NMR

    Energy Technology Data Exchange (ETDEWEB)

    Dickinson, Elizabeth, E-mail: elizabeth.dickinson@york.ac.uk [University of York, Department of Chemistry (United Kingdom); Arnold, John R. P. [Selby College (United Kingdom); Fisher, Julie [University of Leeds, School of Chemistry (United Kingdom)

    2017-02-15

    The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using {sup 19}F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.

  9. Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-"1"9F-NMR

    International Nuclear Information System (INIS)

    Dickinson, Elizabeth; Arnold, John R. P.; Fisher, Julie

    2017-01-01

    The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using "1"9F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.

  10. A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

    International Nuclear Information System (INIS)

    Schubert, Mario; Labudde, Dirk; Leitner, Dietmar; Oschkinat, Hartmut; Schmieder, Peter

    2005-01-01

    The determination of the three-dimensional structure of a protein or the study of protein-ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p

  11. Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations

    Czech Academy of Sciences Publication Activity Database

    Paluch, P.; Pawlak, T.; Jeziorna, A.; Trébosc, J.; Hou, G.; Vega, A. J.; Amoureux, J. P.; Dračínský, Martin; Polenova, T.; Potrzebowski, M. J.

    2015-01-01

    Roč. 17, č. 43 (2015), s. 28789-28801 ISSN 1463-9076 R&D Projects: GA ČR GA15-11223S Institutional support: RVO:61388963 Keywords : solid-state NMR * angle spinning NMR * NMR Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.449, year: 2015 http://pubs.rsc.org/en/content/articlepdf/2015/cp/c5cp04475h

  12. NMR spectroscopy

    International Nuclear Information System (INIS)

    Gruenert, J.

    1989-01-01

    The book reviews the applications of NMR-spectroscopy in medicine and biology. The first chapter of about 40 pages summarizes the history of development and explains the chemical and physical fundamentals of this new and non-invasive method in an easily comprehensible manner. The other chapters summarize diagnostic results obtained with this method in organs and tissues, so that the reader will find a systematic overview of the available findings obtained in the various organ systems. It must be noted, however, that ongoing research work and new insight quite naturally will necessitate corrections to be done, as is the case here with some biochemical interpretations which would need adjustment to latest research results. NMR-spectroscopy is able to measure very fine energy differences on the molecular level, and thus offers insight into metabolic processes, with the advantage that there is no need of applying ionizing radiation in order to qualitatively or quantitatively analyse the metabolic processes in the various organ systems. (orig./DG) With 40 figs., 4 tabs [de

  13. Myowater dynamics and protein secondary structural changes as affected by heating rate in three pork qualities: a combined FT-IR microspectroscopic and 1H NMR relaxometry study.

    Science.gov (United States)

    Wu, Zhiyun; Bertram, Hanne Christine; Böcker, Ulrike; Ofstad, Ragni; Kohler, Achim

    2007-05-16

    The objective of this study was to investigate the influence of heating rate on myowater dynamics and protein secondary structures in three pork qualities by proton NMR T2 relaxation and Fourier transform infrared (FT-IR) microspectroscopy measurements. Two oven temperatures at 100 degrees C and 200 degrees C corresponding to slow and fast heating rates were applied on three pork qualities (DFD, PSE, and normal) to an internal center temperature of 65 degrees C. The fast heating induced a higher cooking loss, particularly for PSE meat. The water proton T21 distribution representing water entrapped within the myofibrillar network was influenced by heating rate and meat quality. Fast heating broadened the T21 distribution and decreased the relaxation times of the T21 peak position for three meat qualities. The changes in T21 relaxation times in meat can be interpreted in terms of chemical and diffusive exchange. FT-IR showed that fast heating caused a higher gain of random structures and aggregated beta-sheets at the expense of native alpha-helixes, and these changes dominate the fast-heating-induced broadening of T21 distribution and reduction in T21 times. Furthermore, of the three meat qualities, PSE meat had the broadest T21 distribution and the lowest T21 times for both heating rates, reflecting that the protein aggregation of PSE caused by heating is more extensive than those of DFD and normal, which is consistent with the IR data. The present study demonstrated that the changes in T2 relaxation times of water protons affected by heating rate and raw meat quality are well related to the protein secondary structural changes as probed by FT-IR microspectroscopy.

  14. NMR in structure-based drug design.

    Science.gov (United States)

    Carneiro, Marta G; Ab, Eiso; Theisgen, Stephan; Siegal, Gregg

    2017-11-08

    NMR spectroscopy is a powerful technique that can provide valuable structural information for drug discovery endeavors. Here, we discuss the strengths (and limitations) of NMR applications to structure-based drug discovery, highlighting the different levels of resolution and throughput obtainable. Additionally, the emerging field of paramagnetic NMR in drug discovery and recent developments in approaches to speed up and automate protein-observed NMR data collection and analysis are discussed. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  15. Synergic Investigation Of The Self-Assembly Structure And Mechanism Of Retroviral Capsid Proteins By Solid State NMR, Transmission Electron Microscopy And Multiscale simulation

    Science.gov (United States)

    2017-03-29

    18], a question naturally arises: if our ssNMR constraints actually impart any meaningful differences to the final model. To answer this question...Mitra at University of Auckland. Xin Qiao, Dr. Chen’s student, presented the ssNMR assignment strategy as a poster presentation titled “Methods

  16. Spectral editing at ultra-fast magic-angle-spinning in solid-state NMR: facilitating protein sequential signal assignment by HIGHLIGHT approach

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Songlin; Matsuda, Isamu; Long, Fei; Ishii, Yoshitaka, E-mail: yishii@uic.edu [University of Illinois at Chicago, Department of Chemistry (United States)

    2016-02-15

    This study demonstrates a novel spectral editing technique for protein solid-state NMR (SSNMR) to simplify the spectrum drastically and to reduce the ambiguity for protein main-chain signal assignments in fast magic-angle-spinning (MAS) conditions at a wide frequency range of 40–80 kHz. The approach termed HIGHLIGHT (Wang et al., in Chem Comm 51:15055–15058, 2015) combines the reverse {sup 13}C, {sup 15}N-isotope labeling strategy and selective signal quenching using the frequency-selective REDOR pulse sequence under fast MAS. The scheme allows one to selectively observe the signals of “highlighted” labeled amino-acid residues that precede or follow unlabeled residues through selectively quenching {sup 13}CO or {sup 15}N signals for a pair of consecutively labeled residues by recoupling {sup 13}CO–{sup 15}N dipolar couplings. Our numerical simulation results showed that the scheme yielded only ∼15 % loss of signals for the highlighted residues while quenching as much as ∼90 % of signals for non-highlighted residues. For lysine-reverse-labeled micro-crystalline GB1 protein, the 2D {sup 15}N/{sup 13}C{sub α} correlation and 2D {sup 13}C{sub α}/{sup 13}CO correlation SSNMR spectra by the HIGHLIGHT approach yielded signals only for six residues following and preceding the unlabeled lysine residues, respectively. The experimental dephasing curves agreed reasonably well with the corresponding simulation results for highlighted and quenched residues at spinning speeds of 40 and 60 kHz. The compatibility of the HIGHLIGHT approach with fast MAS allows for sensitivity enhancement by paramagnetic assisted data collection (PACC) and {sup 1}H detection. We also discuss how the HIGHLIGHT approach facilitates signal assignments using {sup 13}C-detected 3D SSNMR by demonstrating full sequential assignments of lysine-reverse-labeled micro-crystalline GB1 protein (∼300 nmol), for which data collection required only 11 h. The HIGHLIGHT approach offers valuable

  17. Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by (1)H-NMR-based metabonomics.

    Science.gov (United States)

    Dan Yue; Zhang, Yuwei; Cheng, Liuliu; Ma, Jinhu; Xi, Yufeng; Yang, Liping; Su, Chao; Shao, Bin; Huang, Anliang; Xiang, Rong; Cheng, Ping

    2016-04-14

    Hepatitis B virus X protein (HBx) plays an important role in HBV-related hepatocarcinogenesis; however, mechanisms underlying HBx-mediated carcinogenesis remain unclear. In this study, an NMR-based metabolomics approach was applied to systematically investigate the effects of HBx on cell metabolism. EdU incorporation assay was conducted to examine the effects of HBx on DNA synthesis, an important feature of nucleic acid metabolism. The results revealed that HBx disrupted metabolism of glucose, lipids, and amino acids, especially nucleic acids. To understand the potential mechanism of HBx-induced abnormalities of nucleic acid metabolism, gene expression profiles of HepG2 cells expressing HBx were investigated. The results showed that 29 genes involved in DNA damage and DNA repair were differentially expressed in HBx-expressing HepG2 cells. HBx-induced DNA damage was further demonstrated by karyotyping, comet assay, Western blotting, immunofluorescence and immunohistochemistry analyses. Many studies have previously reported that DNA damage can induce abnormalities of nucleic acid metabolism. Thus, our results implied that HBx initially induces DNA damage, and then disrupts nucleic acid metabolism, which in turn blocks DNA repair and induces the occurrence of hepatocellular carcinoma (HCC). These findings further contribute to our understanding of the occurrence of HCC.

  18. NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23-230)

    International Nuclear Information System (INIS)

    Liu Aizhuo; Riek, Roland; Wider, Gerhard; Schroetter, Christine von; Zahn, Ralph; Wuethrich, Kurt

    2000-01-01

    A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly 15 N, 13 C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable 15 N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse 15 N relaxation rates of unfolded polypeptides in high resolution constant-time [ 1 H, 15 N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the 15 N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence

  19. Advanced NMR technology for bioscience and biotechnology

    Energy Technology Data Exchange (ETDEWEB)

    Hammel, P.C.; Hernandez, G.; Trewhella, J.; Unkefer, C.J. [Los Alamos National Lab., NM (US); Boumenthal, D.K. [Univ. of Utah, Salt Lake City, UT (US); Kennedy, M.A. [Pacific Northwest National Lab., Richland, WA (US); Moore, G.J. [Wayne State Univ., Detroit, MI (US)

    1998-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). NMR plays critical roles in bioscience and biotechnology in both imaging and structure determination. NMR is limited, however, by the inherent low sensitivity of the NMR experiment and the demands for spectral resolution required to study biomolecules. The authors addressed both of these issues by working on the development of NMR force microscopy for molecular imaging, and high field NMR with isotope labeling to overcome limitations in the size of biomolecules that can be studied using NMR. A novel rf coil design for NMR force microscopy was developed that increases the limits of sensitivity in magnetic resonance detection for imaging, and the authors demonstrated sub-surface spatial imaging capabilities. The authors also made advances in the miniaturization of two critical NMR force microscope components. They completed high field NMR and isotope labeling studies of a muscle protein complex which is responsible for regulating muscle contraction and is too large for study using conventional NMR approaches.

  20. Application of high resolution NMR, ESR, and gamma-ray scintillation spectroscopy to the study of ligand binding in proteins

    International Nuclear Information System (INIS)

    Lancione, G.V.

    1982-01-01

    Electron spin resonance spectroscopy has been employed to study the nature of the ligand binding site of alpha-1-antitrypsin. Spectra of spin-labeled alpha-1-antitrypsin were recorded at pH's ranging from 2.4 to 12.5. This data demonstrates the tight binding of the spin-label to the protease, and the sensitivity of the bound spin-label to informational changes in the protease inhibitor. A molecular dipstick approach has also been applied to this system and has yielded information on the geometry of the cleft accommodating the spin-label. 160 Terbium(III) exchange experiments have been performed on the acetylcholine receptor protein isolated from Torpedo californica, employing a specially designed flow dialysis apparatus constructed in the laboratory. The apparatus is designed to allow continuous monitoring of 160 Tb(III) gamma-ray emission from the protein compartment of the flow dialysis cell. Nicotinic ligand-induced displacement of 160 Tb(III) from the nicotinic binding site of the receptor was monitored as a funtion of (1) the concentration of nicotinic ligand in the washout buffer, and (2) the nature of the nicotinic ligand in the buffer. Measured 160 Tb(III) exchange half-lives indicate (1) a direct relationship between 160 Tb(III) displacement and nicotinic ligand concentration in the wash-out buffer, and (2) an enhanced 160 Tb(III) displacement for nicotinic agents possessing quaternary ammonium functions

  1. Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dong-Hwa; Ha, Ji-Hyang [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Kim, Yul [Department of Bio and Brain Engineering, KAIST, Daejeon 305-701 (Korea, Republic of); Bae, Kwang-Hee [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Park, Jae-Yong [Department of Physiology, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju, Gyeongnam 660-751 (Korea, Republic of); Choi, Wan Sung [Department of Anatomy and Neurobiology, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju, Gyeongnam 660-751 (Korea, Republic of); Yoon, Ho Sup [Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637511 (Singapore); Park, Sung Goo; Park, Byoung Chul [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Yi, Gwan-Su, E-mail: gsyi@kaist.ac.kr [Department of Bio and Brain Engineering, KAIST, Daejeon 305-701 (Korea, Republic of); Chi, Seung-Wook, E-mail: swchi@kribb.re.kr [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of)

    2011-05-20

    Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.

  2. Bayesian Peak Picking for NMR Spectra

    KAUST Repository

    Cheng, Yichen

    2014-02-01

    Protein structure determination is a very important topic in structural genomics, which helps people to understand varieties of biological functions such as protein-protein interactions, protein–DNA interactions and so on. Nowadays, nuclear magnetic resonance (NMR) has often been used to determine the three-dimensional structures of protein in vivo. This study aims to automate the peak picking step, the most important and tricky step in NMR structure determination. We propose to model the NMR spectrum by a mixture of bivariate Gaussian densities and use the stochastic approximation Monte Carlo algorithm as the computational tool to solve the problem. Under the Bayesian framework, the peak picking problem is casted as a variable selection problem. The proposed method can automatically distinguish true peaks from false ones without preprocessing the data. To the best of our knowledge, this is the first effort in the literature that tackles the peak picking problem for NMR spectrum data using Bayesian method.

  3. Structure determination of uniformly {sup 13}C, {sup 15}N labeled protein using qualitative distance restraints from MAS solid-state {sup 13}C-NMR observed paramagnetic relaxation enhancement

    Energy Technology Data Exchange (ETDEWEB)

    Tamaki, Hajime [Hokkaido University, Graduate School of Life Science (Japan); Egawa, Ayako [Osaka University, Institute for Protein Research (Japan); Kido, Kouki [Hokkaido University, Graduate School of Life Science (Japan); Kameda, Tomoshi [National Institute of Advanced Industrial Science and Technology, Biotechnology Research Institute for Drug Discovery (Japan); Kamiya, Masakatsu; Kikukawa, Takashi; Aizawa, Tomoyasu [Hokkaido University, Faculty of Advanced Life Science (Japan); Fujiwara, Toshimichi [Osaka University, Institute for Protein Research (Japan); Demura, Makoto, E-mail: demura@sci.hokudai.ac.jp [Hokkaido University, Faculty of Advanced Life Science (Japan)

    2016-01-15

    Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn{sup 2+} mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.

  4. Integration of NMR And SAXS with Atomistic Simulations for Characterizing the Structure and Dynamics of Multi-Domain Proteins

    Science.gov (United States)

    Debiec, Karl Thomas

    In the seven decades since the first atomic-level structures of biomolecules were determined, the development and application of novel research methods has led to an advanced understanding of biological functions at the molecular level. In addition to experimental methods, key advances have been spurred by computer simulations, which provide an in silico representation of accumulated prior knowledge of biomolecular structure and dynamics. These models can be used both (i) as a complement to experimental results, filling in the gaps where experimental information is not accessible, and (ii) as complete representations, directing future research. Critically, the validity of either application depends on the accuracy of the models used. In this work, I aspired to combine computational and experimental methods to characterize the structure and dynamics of the flexibly linked two-domain protein MoCVNH3. In Chapter 1 I describe my motivation, and the suspected simulation artifacts observed in our preliminary simulations, which led me to investigate how accurately simulation models represent salt bridge interactions. Chapter 2 details my comparison of current models ("force fields"), for which significant variation but consistent overstabilization of salt bridges was discovered. This work motivated the development of a new force field, AMBER ff15ipq, which corrects, to some degree, the overstabilization and introduces extensive improvements, described in Chapter 3. Finally, in Chapter 4, I applied this new force field in simulations of MoCVNH3, for which I collected extensive experimental data leading to the determination of a structural ensemble. I validated the simulations against the experimental data set, and identified further directions for improvement. Overall, the work presented here demonstrates the power of integrating experimental and computational methods.

  5. NMR Analysis of Amide Hydrogen Exchange Rates in a Pentapeptide-Repeat Protein from A. thaliana.

    Science.gov (United States)

    Xu, Shenyuan; Ni, Shuisong; Kennedy, Michael A

    2017-05-23

    At2g44920 from Arabidopsis thaliana is a pentapeptide-repeat protein (PRP) composed of 25 repeats capped by N- and C-terminal α-helices. PRP structures are dominated by four-sided right-handed β-helices typically consisting of mixtures of type II and type IV β-turns. PRPs adopt repeated five-residue (Rfr) folds with an Rfr consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Unlike other PRPs, At2g44920 consists exclusively of type II β-turns. At2g44920 is predicted to be located in the thylakoid lumen although its biochemical function remains unknown. Given its unusual structure, we investigated the biophysical properties of At2g44920 as a representative of the β-helix family to determine if it had exceptional global stability, backbone dynamics, or amide hydrogen exchange rates. Circular dichroism measurements yielded a melting point of 62.8°C, indicating unexceptional global thermal stability. Nuclear spin relaxation measurements indicated that the Rfr-fold core was rigid with order parameters ranging from 0.7 to 0.9. At2g44920 exhibited a striking range of amide hydrogen exchange rates spanning 10 orders of magnitude, with lifetimes ranging from minutes to several months. A weak correlation was found among hydrogen exchange rates, hydrogen bonding energies, and amino acid solvent-accessible areas. Analysis of contributions from fast (approximately picosecond to nanosecond) backbone dynamics to amide hydrogen exchange rates revealed that the average order parameter of amides undergoing fast exchange was significantly smaller compared to those undergoing slow exchange. Importantly, the activation energies for amide hydrogen exchange were found to be generally higher for the slowest exchanging amides in the central Rfr coil and decreased toward the terminal coils. This could be explained by assuming that the concerted motions of two preceding or following coils required for hydrogen bond disruption and amide hydrogen exchange have a higher activation energy

  6. NMR investigations of molecular dynamics

    Science.gov (United States)

    Palmer, Arthur

    2011-03-01

    NMR spectroscopy is a powerful experimental approach for characterizing protein conformational dynamics on multiple time scales. The insights obtained from NMR studies are complemented and by molecular dynamics (MD) simulations, which provide full atomistic details of protein dynamics. Homologous mesophilic (E. coli) and thermophilic (T. thermophilus) ribonuclease H (RNase H) enzymes serve to illustrate how changes in protein sequence and structure that affect conformational dynamic processes can be monitored and characterized by joint analysis of NMR spectroscopy and MD simulations. A Gly residue inserted within a putative hinge between helices B and C is conserved among thermophilic RNases H, but absent in mesophilic RNases H. Experimental spin relaxation measurements show that the dynamic properties of T. thermophilus RNase H are recapitulated in E. coli RNase H by insertion of a Gly residue between helices B and C. Additional specific intramolecular interactions that modulate backbone and sidechain dynamical properties of the Gly-rich loop and of the conserved Trp residue flanking the Gly insertion site have been identified using MD simulations and subsequently confirmed by NMR spin relaxation measurements. These results emphasize the importance of hydrogen bonds and local steric interactions in restricting conformational fluctuations, and the absence of such interactions in allowing conformational adaptation to substrate binding.

  7. Antifreeze poisoning

    Science.gov (United States)

    ... and breathing machine Chest x-ray CT scan (advanced brain imaging) ECG (electrocardiogram or heart tracing) Intravenous ... Nelson ME. Toxic alcohols. In: Walls RM, Hockberger RS, Gausche-Hill M, eds. Rosen's Emergency Medicine: Concepts ...

  8. NMR in the SPINE Structural Proteomics project.

    Science.gov (United States)

    Ab, E; Atkinson, A R; Banci, L; Bertini, I; Ciofi-Baffoni, S; Brunner, K; Diercks, T; Dötsch, V; Engelke, F; Folkers, G E; Griesinger, C; Gronwald, W; Günther, U; Habeck, M; de Jong, R N; Kalbitzer, H R; Kieffer, B; Leeflang, B R; Loss, S; Luchinat, C; Marquardsen, T; Moskau, D; Neidig, K P; Nilges, M; Piccioli, M; Pierattelli, R; Rieping, W; Schippmann, T; Schwalbe, H; Travé, G; Trenner, J; Wöhnert, J; Zweckstetter, M; Kaptein, R

    2006-10-01

    This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

  9. Structural analysis of alanine tripeptide with antiparallel and parallel beta-sheet structures in relation to the analysis of mixed beta-sheet structures in Samia cynthia ricini silk protein fiber using solid-state NMR spectroscopy.

    Science.gov (United States)

    Asakura, Tetsuo; Okonogi, Michi; Nakazawa, Yasumoto; Yamauchi, Kazuo

    2006-05-10

    The structural analysis of natural protein fibers with mixed parallel and antiparallel beta-sheet structures by solid-state NMR is reported. To obtain NMR parameters that can characterize these beta-sheet structures, (13)C solid-state NMR experiments were performed on two alanine tripeptide samples: one with 100% parallel beta-sheet structure and the other with 100% antiparallel beta-sheet structure. All (13)C resonances of the tripeptides could be assigned by a comparison of the methyl (13)C resonances of Ala(3) with different [3-(13)C]Ala labeling schemes and also by a series of RFDR (radio frequency driven recoupling) spectra observed by changing mixing times. Two (13)C resonances observed for each Ala residue could be assigned to two nonequivalent molecules per unit cell. Differences in the (13)C chemical shifts and (13)C spin-lattice relaxation times (T(1)) were observed between the two beta-sheet structures. Especially, about 3 times longer T(1) values were obtained for parallel beta-sheet structure as compared to those of antiparallel beta-sheet structure, which could be explicable by the difference in the hydrogen-bond networks of both structures. This very large difference in T(1) becomes a good measure to differentiate between parallel or antiparallel beta-sheet structures. These differences in the NMR parameters found for the tripeptides may be applied to assign the parallel and antiparallel beta-sheet (13)C resonances in the asymmetric and broad methyl spectra of [3-(13)C]Ala silk protein fiber of a wild silkworm, Samia cynthia ricini.

  10. Carbon-13 NMR spectroscopy of biological systems

    CERN Document Server

    Beckmann, Nicolau

    1995-01-01

    This book is intended to provide an in-depth understanding of 13C NMR as a tool in biological research. 13C NMR has provided unique information concerning complex biological systems, from proteins and nucleic acids to animals and humans. The subjects addressed include multidimensional heteronuclear techniques for structural studies of molecules in the liquid and solid states, the investigation of interactions in model membranes, the elucidation of metabolic pathwaysin vitro and in vivo on animals, and noninvasive metabolic studies performed on humans. The book is a unique mix of NMR methods and biological applications which makes it a convenient reference for those interested in research in this interdisciplinary area of physics, chemistry, biology, and medicine.Key Features* An interdisciplinary text with emphasis on both 13C NMR methodology and the relevant biological and biomedical issues* State-of-the-art 13C NMR techniques are described; Whenever possible, their advantages over other approaches are empha...

  11. Recommendations of the wwPDB NMR Validation Task Force

    Science.gov (United States)

    Montelione, Gaetano T.; Nilges, Michael; Bax, Ad; Güntert, Peter; Herrmann, Torsten; Richardson, Jane S.; Schwieters, Charles; Vranken, Wim F.; Vuister, Geerten W.; Wishart, David S.; Berman, Helen M.; Kleywegt, Gerard J.; Markley, John L.

    2013-01-01

    As methods for analysis of biomolecular structure and dynamics using nuclear magnetic resonance spectroscopy (NMR) continue to advance, the resulting 3D structures, chemical shifts, and other NMR data are broadly impacting biology, chemistry, and medicine. Structure model assessment is a critical area of NMR methods development, and is an essential component of the process of making these structures accessible and useful to the wider scientific community. For these reasons, the Worldwide Protein Data Bank (wwPDB) has convened an NMR Validation Task Force (NMR-VTF) to work with the wwPDB partners in developing metrics and policies for biomolecular NMR data harvesting, structure representation, and structure quality assessment. This paper summarizes the recommendations of the NMR-VTF, and lays the groundwork for future work in developing standards and metrics for biomolecular NMR structure quality assessment. PMID:24010715

  12. NMR of lignins

    Science.gov (United States)

    John Ralph; Larry L. Landucci

    2010-01-01

    This chapter will consider the basic aspects and findings of several forms of NMR spectroscopy, including separate discussions of proton, carbon, heteronuclear, and multidimensional NMR. Enhanced focus will be on 13C NMR, because of its qualitative and quantitative importance, followed by NMR’s contributions to our understanding of lignin...

  13. Heteronuclear 2D NMR studies on an engineered insulin monomer: Assignments and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design

    International Nuclear Information System (INIS)

    Weiss, M.A.; Hua, Qingxin; Lynch, C.S.; Shoelson, S.E.; Frank, B.H.

    1991-01-01

    Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. The authors demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10 → Asp, ProB28 → Lys, and LysB29 → Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1 H NMR studies of native human insulin and a series of three related analogues-(i) the singly substituted analogue [HisB10→Asp], (ii) the doubly substituted analogue [ProB28→Lys; LysB29→Pro], and (iii) DKP-insulin-demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2 H and 13 C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues

  14. Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes.

    Science.gov (United States)

    Liang, Shuang; Yuan, Bao; Kwon, Jeong-Woo; Ahn, Mija; Cui, Xiang-Shun; Bang, Jeong Kyu; Kim, Nam-Hyung

    2016-07-15

    The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. A chemical approach for site-specific identification of NMR signals from protein side-chain NH{sub 3}{sup +} groups forming intermolecular ion pairs in protein–nucleic acid complexes

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Kurtis M. [University of Texas Health Science Center at Houston, Department of NanoMedicine and Biomedical Engineering and Institute of Molecular Medicine (United States); Nguyen, Dan; Esadze, Alexandre; Zandrashvili, Levani [University of Texas Medical Branch, Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics (United States); Gorenstein, David G. [University of Texas Health Science Center at Houston, Department of NanoMedicine and Biomedical Engineering and Institute of Molecular Medicine (United States); Iwahara, Junji, E-mail: juiwahar@utmb.edu, E-mail: j.iwahara@utmb.edu [University of Texas Medical Branch, Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics (United States)

    2015-05-15

    Protein–nucleic acid interactions involve intermolecular ion pairs of protein side-chain and DNA or RNA phosphate groups. Using three protein–DNA complexes, we demonstrate that site-specific oxygen-to-sulfur substitution in phosphate groups allows for identification of NMR signals from the protein side-chain NH{sub 3}{sup +} groups forming the intermolecular ion pairs. A characteristic change in their {sup 1}H and {sup 15}N resonances upon this modification (i.e., substitution of phosphate to phosphorodithioate) can represent a signature of an intermolecular ion pair. Hydrogen-bond scalar coupling between protein side-chain {sup 15}N and DNA phosphorodithiaote {sup 31}P nuclei provides direct confirmation of the intermolecular ion pair. The same approach is likely applicable to protein–RNA complexes as well.

  16. A chemical approach for site-specific identification of NMR signals from protein side-chain NH3+ groups forming intermolecular ion pairs in protein–nucleic acid complexes

    International Nuclear Information System (INIS)

    Anderson, Kurtis M.; Nguyen, Dan; Esadze, Alexandre; Zandrashvili, Levani; Gorenstein, David G.; Iwahara, Junji

    2015-01-01

    Protein–nucleic acid interactions involve intermolecular ion pairs of protein side-chain and DNA or RNA phosphate groups. Using three protein–DNA complexes, we demonstrate that site-specific oxygen-to-sulfur substitution in phosphate groups allows for identification of NMR signals from the protein side-chain NH 3 + groups forming the intermolecular ion pairs. A characteristic change in their 1 H and 15 N resonances upon this modification (i.e., substitution of phosphate to phosphorodithioate) can represent a signature of an intermolecular ion pair. Hydrogen-bond scalar coupling between protein side-chain 15 N and DNA phosphorodithiaote 31 P nuclei provides direct confirmation of the intermolecular ion pair. The same approach is likely applicable to protein–RNA complexes as well

  17. Characterization of mu s-ms dynamics of proteins using a combined analysis of N-15 NMR relaxation and chemical shift: Conformational exchange in plastocyanin induced by histidine protonations

    DEFF Research Database (Denmark)

    Hass, M. A. S.; Thuesen, Marianne Hallberg; Christensen, Hans Erik Mølager

    2004-01-01

    of the exchanging species can be determined independently of the relaxation rates. The applicability of the approach is demonstrated by a detailed analysis of the conformational exchange processes previously observed in the reduced form of the blue copper protein, plastocyanin from the cyanobacteria Anabaena......An approach is presented that allows a detailed, quantitative characterization of conformational exchange processes in proteins on the mus-ms time scale. The approach relies on a combined analysis of NMR relaxation rates and chemical shift changes and requires that the chemical shift...... quantitatively by the correlation between the R-ex terms and the corresponding chemical shift differences of the exchanging species. By this approach, the R-ex terms of N-15 nuclei belonging to contiguous regions in the protein could be assigned to the same exchange process. Furthermore, the analysis...

  18. Amplitudes and time scales of picosecond-to-microsecond motion in proteins studied by solid-state NMR: a critical evaluation of experimental approaches and application to crystalline ubiquitin

    International Nuclear Information System (INIS)

    Haller, Jens D.; Schanda, Paul

    2013-01-01

    Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps–μs motions in proteins by solid-state NMR

  19. Amplitudes and time scales of picosecond-to-microsecond motion in proteins studied by solid-state NMR: a critical evaluation of experimental approaches and application to crystalline ubiquitin

    Energy Technology Data Exchange (ETDEWEB)

    Haller, Jens D.; Schanda, Paul, E-mail: paul.schanda@ibs.fr [Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) (France)

    2013-10-09

    Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps–μs motions in proteins by solid-state NMR.

  20. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

    Science.gov (United States)

    Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

    2016-05-01

    Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells. © 2016 The Protein Society.

  1. Prediction of peak overlap in NMR spectra

    International Nuclear Information System (INIS)

    Hefke, Frederik; Schmucki, Roland; Güntert, Peter

    2013-01-01

    Peak overlap is one of the major factors complicating the analysis of biomolecular NMR spectra. We present a general method for predicting the extent of peak overlap in multidimensional NMR spectra and its validation using both, experimental data sets and Monte Carlo simulation. The method is based on knowledge of the magnetization transfer pathways of the NMR experiments and chemical shift statistics from the Biological Magnetic Resonance Data Bank. Assuming a normal distribution with characteristic mean value and standard deviation for the chemical shift of each observable atom, an analytic expression was derived for the expected overlap probability of the cross peaks. The analytical approach was verified to agree with the average peak overlap in a large number of individual peak lists simulated using the same chemical shift statistics. The method was applied to eight proteins, including an intrinsically disordered one, for which the prediction results could be compared with the actual overlap based on the experimentally measured chemical shifts. The extent of overlap predicted using only statistical chemical shift information was in good agreement with the overlap that was observed when the measured shifts were used in the virtual spectrum, except for the intrinsically disordered protein. Since the spectral complexity of a protein NMR spectrum is a crucial factor for protein structure determination, analytical overlap prediction can be used to identify potentially difficult proteins before conducting NMR experiments. Overlap predictions can be tailored to particular classes of proteins by preparing statistics from corresponding protein databases. The method is also suitable for optimizing recording parameters and labeling schemes for NMR experiments and improving the reliability of automated spectra analysis and protein structure determination.

  2. An introduction to biological NMR spectroscopy

    International Nuclear Information System (INIS)

    Marion, Dominique

    2013-01-01

    NMR spectroscopy is a powerful tool for biologists interested in the structure, dynamics, and interactions of biological macromolecules. This review aims at presenting in an accessible manner the requirements and limitations of this technique. As an introduction, the history of NMR will highlight how the method evolved from physics to chemistry and finally to biology over several decades. We then introduce the NMR spectral parameters used in structural biology, namely the chemical shift, the J-coupling, nuclear Overhauser effects, and residual dipolar couplings. Resonance assignment, the required step for any further NMR study, bears a resemblance to jigsaw puzzle strategy. The NMR spectral parameters are then converted into angle and distances and used as input using restrained molecular dynamics to compute a bundle of structures. When interpreting a NMR-derived structure, the biologist has to judge its quality on the basis of the statistics provided. When the 3D structure is a priori known by other means, the molecular interaction with a partner can be mapped by NMR: information on the binding interface as well as on kinetic and thermodynamic constants can be gathered. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function. In the last section of this review, intrinsically disordered proteins, which have escaped the attention of classical structural biology, are discussed in the perspective of NMR, one of the rare available techniques able to describe structural ensembles. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 16 MCP). (authors)

  3. The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

    DEFF Research Database (Denmark)

    Teilum, Kaare; Poulsen, F. M.; Akke, M.

    2006-01-01

    those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements allowed determination of k f and k u in the range from 0.48 M GuHCl to 1.28 M GuHCl. Individually, the stopped-flow and NMR data fit two-state models...... for folding. However, although the values of k f determined by the two methods agree, the values of k u do not. As a result, a combined analysis of all data does not comply with a two-state model but indicates that an unfolding intermediate exists on the native side of the dominant energy barrier...

  4. WaVPeak: Picking NMR peaks through wavelet-based smoothing and volume-based filtering

    KAUST Repository

    Liu, Zhi; Abbas, Ahmed; Jing, Bing-Yi; Gao, Xin

    2012-01-01

    Motivation: Nuclear magnetic resonance (NMR) has been widely used as a powerful tool to determine the 3D structures of proteins in vivo. However, the post-spectra processing stage of NMR structure determination usually involves a tremendous amount

  5. Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: Potential for NMR structure determination of large proteins

    International Nuclear Information System (INIS)

    Kelly, Mark J.S.; Krieger, Cornelia; Ball, Linda J.; Yu Yihua; Richter, Gerald; Schmieder, Peter; Bacher, Adelbert; Oschkinat, Hartmut

    1999-01-01

    NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins

  6. NMR-CT scanner

    International Nuclear Information System (INIS)

    Kose, Katsumi; Sato, Kozo; Sugimoto, Hiroshi; Sato, Masataka.

    1983-01-01

    A brief explanation is made on the imaging methods for a practical diagnostic NMR-CT scanner : A whole-body NMR-CT scanner utilizing a resistive magnet has been developed by Toshiba in cooperation with the Institute for Solid State Physics, the University of Tokyo. Typical NMR-CT images of volunteers and patients obtained in the clinical experiments using this device are presented. Detailed specifications are also shown about the practical NMR-CTs which are to be put on the market after obtaining the government approval. (author)

  7. NMR imaging and pharmaceutical sciences

    International Nuclear Information System (INIS)

    Beall, P.T.; Good, W.R.

    1986-01-01

    Described is the technique of NMR-imaging in diagnostic medicine. Proton and phosphorus NMR in diagnosis of abnormal tissue pathology. Discussed is the value of NMR to the pharmaceutical sciences. NMR may play an important role in monitoring the response of tissues to drugs, determining the localization of drugs, performing real time pharmacokinetics and testing the use of NMR contrast pharmaceuticals

  8. Identifying inter-residue resonances in crowded 2D {sup 13}C-{sup 13}C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Miao Yimin; Cross, Timothy A. [Florida State University, Department of Chemistry and Biochemistry (United States); Fu Riqiang, E-mail: rfu@magnet.fsu.edu [National High Magnet Field Lab (United States)

    2013-07-15

    The feasibility of using difference spectroscopy, i.e. subtraction of two correlation spectra at different mixing times, for substantially enhanced resolution in crowded two-dimensional {sup 13}C-{sup 13}C chemical shift correlation spectra is presented. With the analyses of {sup 13}C-{sup 13}C spin diffusion in simple spin systems, difference spectroscopy is proposed to partially separate the spin diffusion resonances of relatively short intra-residue distances from the longer inter-residue distances, leading to a better identification of the inter-residue resonances. Here solid-state magic-angle-spinning NMR spectra of the full length M2 protein embedded in synthetic lipid bilayers have been used to illustrate the resolution enhancement in the difference spectra. The integral membrane M2 protein of Influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Based on known amino acid resonance assignments from amino acid specific labeled samples of truncated M2 sequences or from time-consuming 3D experiments of uniformly labeled samples, some inter-residue resonances of the full length M2 protein can be identified in the difference spectra of uniformly {sup 13}C labeled protein that are consistent with the high resolution structure of the M2 (22-62) protein (Sharma et al., Science 330(6003):509-512, 2010)

  9. Rhodopsin-lipid interactions studied by NMR.

    Science.gov (United States)

    Soubias, Olivier; Gawrisch, Klaus

    2013-01-01

    The biophysical properties of the lipid matrix are known to influence function of integral membrane proteins. We report on a sample preparation method for reconstitution of membrane proteins which uses porous anodic aluminum oxide (AAO) filters with 200-nm-wide pores of high density. The substrate permits formation of tubular, single membranes that line the inner surface of pores. One square centimeter of filter with a thickness of 60μm yields on the order of 500cm(2) of solid-supported single bilayer surface, sufficient for NMR studies. The tubular bilayers are free of detergent, fully hydrated, and accessible for ligands from one side of the membrane. The use of AAO filters greatly improves reproducibility of the reconstitution process such that the influence of protein on lipid order parameters can be studied with high resolution. As an example, results for the G protein-coupled receptor of class A, bovine rhodopsin, are shown. By (2)H NMR order parameter measurements, it is detected that rhodopsin insertion elastically deforms membranes near the protein. Furthermore, by (1)H saturation-transfer NMR under conditions of magic angle spinning, we demonstrate detection of preferences in interactions of rhodopsin with particular lipid species. It is assumed that function of integral membrane proteins depends on both protein-induced elastic deformations of the lipid matrix and preferences for interaction of the protein with particular lipid species in the first layer of lipids surrounding the protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Lectures on pulsed NMR

    International Nuclear Information System (INIS)

    Pines, A.

    1988-08-01

    These lectures discuss some recent developments in pulsed NMR, emphasizing fundamental principles with selected illustrative applications. Major topics covered include multiple-quantum spectroscopy, spin decoupling, the interaction of spins with a quantized field, adiabatic rapid passage, spin temperature and statistics of cross-polarization, coherent averaging, and zero field NMR. 32 refs., 56 figs

  11. Lectures on pulsed NMR

    International Nuclear Information System (INIS)

    Pines, A.

    1986-09-01

    These lectures discuss some recent developments in pulsed NMR, emphasizing fundamental principles with selected illustrative applications. Major topics covered include multiple-quantum spectroscopy, spin decoupling, the interaction of spins with a quantized field, adiabatic rapid passage, spin temperature and statistics of cross-polarization, coherent averaging, and zero field NMR. 55 figs

  12. NMR data-driven structure determination using NMR-I-TASSER in the CASD-NMR experiment

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Richard [Huazhong University of Science and Technology, School of Software Engineering (China); Wang, Yan [Huazhong University of Science and Technology, School of Life Science and Technology (China); Xue, Zhidong, E-mail: zdxue@hust.edu.cn [Huazhong University of Science and Technology, School of Software Engineering (China); Zhang, Yang, E-mail: zhng@umich.edu [University of Michigan, Department of Computational Medicine and Bioinformatics (United States)

    2015-08-15

    NMR-I-TASSER, an adaption of the I-TASSER algorithm combining NMR data for protein structure determination, recently joined the second round of the CASD-NMR experiment. Unlike many molecular dynamics-based methods, NMR-I-TASSER takes a molecular replacement-like approach to the problem by first threading the target through the PDB to identify structural templates which are then used for iterative NOE assignments and fragment structure assembly refinements. The employment of multiple templates allows NMR-I-TASSER to sample different topologies while convergence to a single structure is not required. Retroactive and blind tests of the CASD-NMR targets from Rounds 1 and 2 demonstrate that even without using NOE peak lists I-TASSER can generate correct structure topology with 15 of 20 targets having a TM-score above 0.5. With the addition of NOE-based distance restraints, NMR-I-TASSER significantly improved the I-TASSER models with all models having the TM-score above 0.5. The average RMSD was reduced from 5.29 to 2.14 Å in Round 1 and 3.18 to 1.71 Å in Round 2. There is no obvious difference in the modeling results with using raw and refined peak lists, indicating robustness of the pipeline to the NOE assignment errors. Overall, despite the low-resolution modeling the current NMR-I-TASSER pipeline provides a coarse-grained structure folding approach complementary to traditional molecular dynamics simulations, which can produce fast near-native frameworks for atomic-level structural refinement.

  13. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    Science.gov (United States)

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

  14. Contact replacement for NMR resonance assignment.

    Science.gov (United States)

    Xiong, Fei; Pandurangan, Gopal; Bailey-Kellogg, Chris

    2008-07-01

    Complementing its traditional role in structural studies of proteins, nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in functional studies. NMR dynamics experiments characterize motions involved in target recognition, ligand binding, etc., while NMR chemical shift perturbation experiments identify and localize protein-protein and protein-ligand interactions. The key bottleneck in these studies is to determine the backbone resonance assignment, which allows spectral peaks to be mapped to specific atoms. This article develops a novel approach to address that bottleneck, exploiting an available X-ray structure or homology model to assign the entire backbone from a set of relatively fast and cheap NMR experiments. We formulate contact replacement for resonance assignment as the problem of computing correspondences between a contact graph representing the structure and an NMR graph representing the data; the NMR graph is a significantly corrupted, ambiguous version of the contact graph. We first show that by combining connectivity and amino acid type information, and exploiting the random structure of the noise, one can provably determine unique correspondences in polynomial time with high probability, even in the presence of significant noise (a constant number of noisy edges per vertex). We then detail an efficient randomized algorithm and show that, over a variety of experimental and synthetic datasets, it is robust to typical levels of structural variation (1-2 AA), noise (250-600%) and missings (10-40%). Our algorithm achieves very good overall assignment accuracy, above 80% in alpha-helices, 70% in beta-sheets and 60% in loop regions. Our contact replacement algorithm is implemented in platform-independent Python code. The software can be freely obtained for academic use by request from the authors.

  15. Isotope labeling for NMR studies of macromolecular structure and interactions

    International Nuclear Information System (INIS)

    Wright, P.E.

    1994-01-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform 13 C, 15 N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific 13 C and 15 N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions

  16. Isotope labeling for NMR studies of macromolecular structure and interactions

    Energy Technology Data Exchange (ETDEWEB)

    Wright, P.E. [Scripps Research Institute, La Jolla, CA (United States)

    1994-12-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.

  17. Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps

    International Nuclear Information System (INIS)

    Mishra, Subrata H.; Frueh, Dominique P.

    2015-01-01

    Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to C α and C β separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups

  18. Detection of the sulfhydryl groups in proteins with slow hydrogen exchange rates and determination of their proton/deuteron fractionation factors using the deuterium-induced effects on the 13C(beta) NMR signals.

    Science.gov (United States)

    Takeda, Mitsuhiro; Jee, JunGoo; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-05-05

    A method for identifying cysteine (Cys) residues with sulfhydryl (SH) groups exhibiting slow hydrogen exchange rates has been developed for proteins in aqueous media. The method utilizes the isotope shifts of the C(beta) chemical shifts induced by the deuteration of the SH groups. The 18.2 kDa E. coli peptidyl prolyl cis-trans isomerase b (EPPIb), which was selectively labeled with [3-(13)C;3,3-(2)H(2)]Cys, showed much narrower line widths for the (13)C(beta) NMR signals, as compared to those of the proteins labeled with either [3-(13)C]Cys or (3R)-[3-(13)C;3-(2)H]Cys. The (13)C(beta) signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-(13)C;3,3-(2)H(2)]Cys, split into four signals in H(2)O/D(2)O (1:1) at 40 degrees C and pH 7.5, indicating that the exchange rates of the side-chain SH's and the backbone amides are too slow to average the chemical shift differences of the (13)C(beta) signals, due to the two- and three-bond isotope shifts. By virtue of the well-separated signals, the proton/deuteron fractional factors for both the SH and amide groups of the two Cys residues in EPPIb could be directly determined, as approximately 0.4-0.5 for [SD]/[SH] and 0.9-1.0 for [ND]/[NH], by the relative intensities of the NMR signals for the isotopomers. The proton NOE's of the two slowly exchanging SH's were clearly identified in the NOESY spectra and were useful for the determining the local structure of EPPIb around the Cys residues.

  19. Nano-Mole Scale Side-Chain Signal Assignment by 1H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling

    KAUST Repository

    Wang, Songlin

    2015-04-09

    We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.

  20. Nano-Mole Scale Side-Chain Signal Assignment by 1H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling

    KAUST Repository

    Wang, Songlin; Parthasarathy, Sudhakar; Nishiyama, Yusuke; Endo, Yuki; Nemoto, Takahiro; Yamauchi, Kazuo; Asakura, Tetsuo; Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune; Ishii, Yoshitaka

    2015-01-01

    We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.

  1. Solution NMR Spectroscopy in Target-Based Drug Discovery.

    Science.gov (United States)

    Li, Yan; Kang, Congbao

    2017-08-23

    Solution NMR spectroscopy is a powerful tool to study protein structures and dynamics under physiological conditions. This technique is particularly useful in target-based drug discovery projects as it provides protein-ligand binding information in solution. Accumulated studies have shown that NMR will play more and more important roles in multiple steps of the drug discovery process. In a fragment-based drug discovery process, ligand-observed and protein-observed NMR spectroscopy can be applied to screen fragments with low binding affinities. The screened fragments can be further optimized into drug-like molecules. In combination with other biophysical techniques, NMR will guide structure-based drug discovery. In this review, we describe the possible roles of NMR spectroscopy in drug discovery. We also illustrate the challenges encountered in the drug discovery process. We include several examples demonstrating the roles of NMR in target-based drug discoveries such as hit identification, ranking ligand binding affinities, and mapping the ligand binding site. We also speculate the possible roles of NMR in target engagement based on recent processes in in-cell NMR spectroscopy.

  2. Functional studies using NMR

    International Nuclear Information System (INIS)

    McCready, V.R.; Leach, M.O.; Sutton; Ell, P.

    1986-01-01

    The object of this book is to discuss and evaluate an area of Nuclear Magnetic Resonance which to date has been less emphasized than it might be, namely the use of NMR for functional studies. The book commences with a discussion of the areas in which the NMR techniques might be needed due to deficiencies in other techniques. The physics of NMR especially relating to functional measurement are then explained. Technical factors in producing functional images are discussed and the use of paramagnetic substances for carrying out flow studies are detailed. Particular attention is paid to specific studies in the various organs. The book ends with a survey of imaging in each organ and the relation of NMR images to other techniques such as ultrasound, nuclear medicine and X-rays

  3. Functional studies using NMR

    International Nuclear Information System (INIS)

    McCready, V.R.; Leach, M.; Ell, P.J.

    1987-01-01

    This volume is based on a series of lectures delivered at a one-day teaching symposium on functional and metabolic aspects of NMR measurements held at the Middlesex Hospital Medical School on 1st September 1985 as a part of the European Nuclear Medicine Society Congress. Currently the major emphasis in medical NMR in vivo is on its potential to image and display abnormalities in conventional radiological images, providing increased contrast between normal and abnormal tissue, improved definition of vasculature, and possibly an increased potential for differential diagnosis. Although these areas are undeniably of major importance, it is probable that NMR will continue to complement conventional measurement methods. The major potential benefits to be derived from in vivo NMR measurements are likely to arise from its use as an instrument for functional and metabolic studies in both clinical research and in the everyday management of patients. It is to this area that this volume is directed

  4. Applications of high resolution 3H NMR spectroscopy

    International Nuclear Information System (INIS)

    Williams, P.G.

    1987-10-01

    The advantages of tritium as an NMR nucleus are pointed out. Examples of its use are given, including labelled toluene, hydrogenation of β-methylstyrene, and maltose and its binding proteins. 7 refs., 2 figs

  5. Effects of antifreezes and bundled material on the stability and optical limiting in aqueous suspensions of carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Vlasov, Andrey Yu.; Venediktova, Anastasia V.; Sokolova, Ekaterina P. [Department of Chemistry, St. Petersburg State University, Universitetsky Pr. 26, St. Petersburg 198504 (Russian Federation); Videnichev, Dmitry A. [S.I. Vavilov State Optical Institute, Birzhevaya line 12, St. Petersburg 199034 (Russian Federation); St. Petersburg National Research University ITMO, Kronverksky pr. 49, St. Petersburg 197101 (Russian Federation); Lasers and Optical Systems JSC, Birzhevaya line 12, St. Petersburg 199034 (Russian Federation); Kislyakov, Ivan M. [S.I. Vavilov State Optical Institute, Birzhevaya line 12, St. Petersburg 199034 (Russian Federation); St. Petersburg National Research University ITMO, Kronverksky pr. 49, St. Petersburg 197101 (Russian Federation); Obraztsova, Elena D. [A.M. Prokhorov Institute of General Physics, Russian Academy of Sciences, Vavilov Str. 38, Moscow 119991 (Russian Federation)

    2012-12-15

    This work gives data on the stability of dispersions of single wall carbon nanotubes stabilized by sodium dodecylbenzenesulfonate in binary polar solvents ''water + antifreeze'' (glycerol, polyethyleneglycole) with eutectic compositions. The absorption spectra of the suspensions demonstrate no changes during 1-year storage with temperature spanning from -40 to +40 C. The systems provide relevant optical power limiting properties, the one with glycerol showing good resistance to optical bleaching effects. We also demonstrate that aqueous dispersions of nanotubes exhibit considerable enhancement of optical limiting parameters alongside an increase of the bundled material populace. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  6. Theory of NMR probe design

    International Nuclear Information System (INIS)

    Schnall, M.D.

    1988-01-01

    The NMR probe is the intrinsic part of the NMR system which allows transmission of a stimulus to a sample and the reception of a resulting signal from a sample. NMR probes are used in both imaging and spectroscopy. Optimal probe design is important to the production of adequate signal/moise. It is important for anyone using NMR techniques to understand how NMR probes work and how to optimize probe design

  7. Data supporting beta-amyloid dimer structural transitions and protein–lipid interactions on asymmetric lipid bilayer surfaces using MD simulations on experimentally derived NMR protein structures

    Directory of Open Access Journals (Sweden)

    Sara Y. Cheng

    2016-06-01

    Full Text Available This data article supports the research article entitled “Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface” [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes.

  8. Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.

    Science.gov (United States)

    Rizzo, Alessandro A; Suhanovsky, Margaret M; Baker, Matthew L; Fraser, LaTasha C R; Jones, Lisa M; Rempel, Don L; Gross, Michael L; Chiu, Wah; Alexandrescu, Andrei T; Teschke, Carolyn M

    2014-06-10

    Some capsid proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 coat protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to improve cryoEM models of coat protein. The I-domain has an antiparallel six-stranded β-barrel fold, not previously observed in HK97-fold accessory domains. The D-loop, which is dynamic in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. The S-loop is important for capsid size determination, likely through intrasubunit interactions. Ten of 18 coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Dynamics in photosynthetic transient complexes studied by paramagnetic NMR spectroscopy

    NARCIS (Netherlands)

    Scanu, Sandra

    2013-01-01

    This PhD thesis focuses on fundamental aspects of protein-protein interactions. A multidisciplinary methodology for the detection and visualization of transient, lowly-populated encounter protein complexes is described. The new methodology combined paramagnetic NMR spectroscopy with computational

  10. Microprocessorized NMR measurement

    International Nuclear Information System (INIS)

    Rijllart, A.

    1984-01-01

    An MC68000 CAMAC microprocessor system for fast and accurate NMR signal measurement will be presented. A stand-alone CAMAC microprocessor system (MC68000 STAC) with a special purpose interface sweeps a digital frequency synthesizer and digitizes the NMR signal with a 16-bit ADC of 17 μs conversion time. It averages the NMR signal data over many sweeps and then transfers it through CAMAC to a computer for calculation of the signal parameters. The computer has full software control over the timing and sweep settings of this signal averager, and thus allows optimization of noise suppression. Several of these processor systems can be installed in the same crate for parallel processing, and the flexibility of the STAC also allows easy adaptation to other applications such as transient recording or phase-sensitive detection. (orig.)

  11. Structural Studies of Biological Solids Using NMR

    Science.gov (United States)

    Ramamoorthy, Ayyalusamy

    2011-03-01

    High-resolution structure and dynamics of biological molecules are important in understanding their function. While studies have been successful in solving the structures of water-soluble biomolecules, it has been proven difficult to determine the structures of membrane proteins and fibril systems. Recent studies have shown that solid-state NMR is a promising technique and could be highly valuable in studying such non-crystalline and non-soluble biosystems. I will present strategies to study the structures of such challenging systems and also about the applications of solid-state NMR to study the modes of membrane-peptide interactions for a better assessment of the prospects of antimicrobial peptides as substitutes to antibiotics in the control of human disease. Our studies on the mechanism of membrane disruption by LL-37 (a human antimicrobial peptide), analogs of the naturally occurring antimicrobial peptide magainin2 extracted from the skin of the African frog Xenopus Laevis, and pardaxin will be presented. Solid-state NMR experiments were used to determine the secondary structure, dynamics and topology of these peptides in lipid bilayers. Similarities and difference in the cell-lysing mechanism, and their dependence on the membrane composition, of these peptides will be discussed. Atomic-level resolution NMR structures of amyloidogenic proteins revealing the misfolding pathway and early intermediates that play key roles in amyloid toxicity will also be presented.

  12. Fourier transform NMR

    International Nuclear Information System (INIS)

    Hallenga, K.

    1991-01-01

    This paper discusses the concept of Fourier transformation one of the many precious legacies of the French mathematician Jean Baptiste Joseph Fourier, essential for understanding the link between continuous-wave (CW) and Fourier transform (FT) NMR. Although in modern FT NMR the methods used to obtain a frequency spectrum from the time-domain signal may vary greatly, from the efficient Cooley-Tukey algorithm to very elaborate iterative least-square methods based other maximum entropy method or on linear prediction, the principles for Fourier transformation are unchanged and give invaluable insight into the interconnection of many pairs of physical entities called Fourier pairs

  13. Relaxation-compensated difference spin diffusion NMR for detecting {sup 13}C–{sup 13}C long-range correlations in proteins and polysaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tuo; Williams, Jonathan K. [Massachusetts Institute of Technology, Department of Chemistry (United States); Schmidt-Rohr, Klaus [Brandeis University, Department of Chemistry (United States); Hong, Mei, E-mail: meihong@mit.edu [Massachusetts Institute of Technology, Department of Chemistry (United States)

    2015-02-15

    The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly {sup 13}C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular {sup 13}C–{sup 13}C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D {sup 1}H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for {sup 13}C T{sub 1} relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ∼200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T{sub 1} relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T{sub 1} relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter

  14. Relaxation-compensated difference spin diffusion NMR for detecting 13C–13C long-range correlations in proteins and polysaccharides

    International Nuclear Information System (INIS)

    Wang, Tuo; Williams, Jonathan K.; Schmidt-Rohr, Klaus; Hong, Mei

    2015-01-01

    The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly 13 C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular 13 C– 13 C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D 1 H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for 13 C T 1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ∼200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T 1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T 1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks

  15. Comparing pharmacophore models derived from crystallography and NMR ensembles

    Science.gov (United States)

    Ghanakota, Phani; Carlson, Heather A.

    2017-11-01

    NMR and X-ray crystallography are the two most widely used methods for determining protein structures. Our previous study examining NMR versus X-Ray sources of protein conformations showed improved performance with NMR structures when used in our Multiple Protein Structures (MPS) method for receptor-based pharmacophores (Damm, Carlson, J Am Chem Soc 129:8225-8235, 2007). However, that work was based on a single test case, HIV-1 protease, because of the rich data available for that system. New data for more systems are available now, which calls for further examination of the effect of different sources of protein conformations. The MPS technique was applied to Growth factor receptor bound protein 2 (Grb2), Src SH2 homology domain (Src-SH2), FK506-binding protein 1A (FKBP12), and Peroxisome proliferator-activated receptor-γ (PPAR-γ). Pharmacophore models from both crystal and NMR ensembles were able to discriminate between high-affinity, low-affinity, and decoy molecules. As we found in our original study, NMR models showed optimal performance when all elements were used. The crystal models had more pharmacophore elements compared to their NMR counterparts. The crystal-based models exhibited optimum performance only when pharmacophore elements were dropped. This supports our assertion that the higher flexibility in NMR ensembles helps focus the models on the most essential interactions with the protein. Our studies suggest that the "extra" pharmacophore elements seen at the periphery in X-ray models arise as a result of decreased protein flexibility and make very little contribution to model performance.

  16. The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, 31P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition

    Directory of Open Access Journals (Sweden)

    Müller Karin

    2009-11-01

    Full Text Available Abstract Background Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. Results Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. Conclusion The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the

  17. Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

    International Nuclear Information System (INIS)

    Bellstedt, Peter; Herbst, Christian; Häfner, Sabine; Leppert, Jörg; Görlach, Matthias; Ramachandran, Ramadurai

    2012-01-01

    We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC′C and 3D C′NCA with sequential 13 C acquisitions, 3D NHH and 3D NC′H with sequential 1 H acquisitions and 3D CANH and 3D C’NH with broadband 13 C– 15 N mixing are demonstrated using microcrystalline samples of the β1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.

  18. Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

    Energy Technology Data Exchange (ETDEWEB)

    Bellstedt, Peter [Fritz Lipmann Institute, Biomolecular NMR spectroscopy, Leibniz Institute for Age Research (Germany); Herbst, Christian [Ubon Ratchathani University, Department of Physics, Faculty of Science (Thailand); Haefner, Sabine; Leppert, Joerg; Goerlach, Matthias; Ramachandran, Ramadurai, E-mail: raman@fli-leibniz.de [Fritz Lipmann Institute, Biomolecular NMR spectroscopy, Leibniz Institute for Age Research (Germany)

    2012-12-15

    We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC Prime C and 3D C Prime NCA with sequential {sup 13}C acquisitions, 3D NHH and 3D NC Prime H with sequential {sup 1}H acquisitions and 3D CANH and 3D C'NH with broadband {sup 13}C-{sup 15}N mixing are demonstrated using microcrystalline samples of the {beta}1 immunoglobulin binding domain of protein G (GB1) and the chicken {alpha}-spectrin SH3 domain.

  19. International symposium on NMR spectroscopy

    International Nuclear Information System (INIS)

    The publication consists of 32 papers and presentations from the field of NMR spectroscopy applications submitted to the International Symposium on NMR Spectroscopy held at Smolenice between 29 Sep and 3 Oct, 1980. (B.S.)

  20. Single-sided NMR

    CERN Document Server

    Casanova, Federico; Blümich, Bernhard

    2011-01-01

    Single-Sided NMR describes the design of the first functioning single-sided tomograph, the related measurement methods, and a number of applications. One of the key advantages to this method is the speed at which the images are obtained.

  1. Autonomous driving in NMR.

    Science.gov (United States)

    Perez, Manuel

    2017-01-01

    The automatic analysis of NMR data has been a much-desired endeavour for the last six decades, as it is the case with any other analytical technique. This need for automation has only grown as advances in hardware; pulse sequences and automation have opened new research areas to NMR and increased the throughput of data. Full automatic analysis is a worthy, albeit hard, challenge, but in a world of artificial intelligence, instant communication and big data, it seems that this particular fight is happening with only one technique at a time (let this be NMR, MS, IR, UV or any other), when the reality of most laboratories is that there are several types of analytical instrumentation present. Data aggregation, verification and elucidation by using complementary techniques (e.g. MS and NMR) is a desirable outcome to pursue, although a time-consuming one if performed manually; hence, the use of automation to perform the heavy lifting for users is required to make the approach attractive for scientists. Many of the decisions and workflows that could be implemented under automation will depend on the two-way communication with databases that understand analytical data, because it is desirable not only to query these databases but also to grow them in as much of an automatic manner as possible. How these databases are designed, set up and the data inside classified will determine what workflows can be implemented. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

    Directory of Open Access Journals (Sweden)

    Nusrat Yasmin

    2017-01-01

    Full Text Available A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+ and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

  3. NMR for chemists and biologists

    CERN Document Server

    Carbajo, Rodrigo J

    2013-01-01

    This book offers a concise introduction to the field of nuclear magnetic resonance or NMR. It presents the basic foundations of NMR in a non-mathematical way and provides an overview of both recent and important biological applications of NMR.

  4. NMR studies of the sporulation protein SpoIIAA: Implications for the regulation of the transcription factor σF in Bacillus subtilis

    International Nuclear Information System (INIS)

    Kovacs, Helena; Comfort, David; Lord, Matthew; Yudkin, Michael; Campbell, Iain D.; Nilges, Michael

    2001-01-01

    SpoIIAA participates in a four-component mechanism for phosphorylation-dependent transcription control at the outset of sporulation. We report the refinement of the solution structure of SpoIIAA by using the automated iterative NOE assignment method ARIA. To complement the structural data, the protein dynamics were determined by measuring the T 1 , T 2 and NOE of the backbone 15 N-nuclei. The refined structure permits a discussion of the structural features that are important for the function of SpoIIAA in the regulation of the sporulation sigma factor σ F , and for homologous regulatory pathways present in B. subtilis and in other bacilli

  5. Internal protein dynamics on ps to μs timescales as studied by multi-frequency {sup 15}N solid-state NMR relaxation

    Energy Technology Data Exchange (ETDEWEB)

    Zinkevich, Tatiana [Martin-Luther-Universität Halle-Wittenberg, NMRGroup, Faculty of Natural Sciences II, Institut für Physik (Germany); Chevelkov, Veniamin [Max-Planck-Institut für Biophysikalische Chemie (Germany); Reif, Bernd [Technische Universität München, Department Chemie (Germany); Saalwächter, Kay; Krushelnitsky, Alexey, E-mail: krushelnitsky@physik.uni-halle.de [Martin-Luther-Universität Halle-Wittenberg, NMRGroup, Faculty of Natural Sciences II, Institut für Physik (Germany)

    2013-09-19

    A comprehensive analysis of the dynamics of the SH3 domain of chicken alpha-spectrin is presented, based upon {sup 15}N T{sub 1} and on- and off-resonance T{sub 1ρ} relaxation times obtained on deuterated samples with a partial back-exchange of labile protons under a variety of the experimental conditions, taking explicitly into account the dipolar order parameters calculated from {sup 15}N–{sup 1}H dipole–dipole couplings. It is demonstrated that such a multi-frequency approach enables access to motional correlation times spanning about 6 orders of magnitude. We asses the validity of different motional models based upon orientation autocorrelation functions with a different number of motional components. We find that for many residues a “two components” model is not sufficient for a good description of the data and more complicated fitting models must be considered. We show that slow motions with correlation times on the order of 1–10 μs can be determined reliably in spite of rather low apparent amplitudes (below 1 %), and demonstrate that the distribution of the protein backbone mobility along the time scale axis is pronouncedly non-uniform and non-monotonic: two domains of fast (τ < 10{sup −10} s) and intermediate (10{sup −9} s < τ < 10{sup −7} s) motions are separated by a gap of one order of magnitude in time with almost no motions. For slower motions (τ > 10{sup −6} s) we observe a sharp ∼1 order of magnitude decrease of the apparent motional amplitudes. Such a distribution obviously reflects different nature of backbone motions on different time scales, where the slow end may be attributed to weakly populated “excited states.” Surprisingly, our data reveal no clearly evident correlations between secondary structure of the protein and motional parameters. We also could not notice any unambiguous correlations between motions in different time scales along the protein backbone emphasizing the importance of the inter

  6. Aqueous geochemistry in icy world interiors: Equilibrium fluid, rock, and gas compositions, and fate of antifreezes and radionuclides

    Science.gov (United States)

    Neveu, Marc; Desch, Steven J.; Castillo-Rogez, Julie C.

    2017-09-01

    The geophysical evolution of many icy moons and dwarf planets seems to have provided opportunities for interaction between liquid water and rock (silicate and organic solids). Here, we explore two ways by which water-rock interaction can feed back on geophysical evolution: the production or consumption of antifreeze compounds, which affect the persistence and abundance of cold liquid; and the potential leaching into the fluid of lithophile radionuclides, affecting the distribution of a long-term heat source. We compile, validate, and use a numerical model, implemented with the PHREEQC code, of the interaction of chondritic rock with pure water and with C, N, S-bearing cometary fluid, thought to be the materials initially accreted by icy worlds, and describe the resulting equilibrium fluid and rock assemblages at temperatures, pressures, and water-to-rock ratios of 0-200 ° C, 1-1000 bar, and 0.1-10 by mass, respectively. Our findings suggest that water-rock interaction can strongly alter the nature and amount of antifreezes, resulting in solutions rich in reduced nitrogen and carbon, and sometimes dissolved H2, with additional sodium, calcium, chlorine, and/or oxidized carbon. Such fluids can remain partially liquid down to 176 K if NH3 is present. The prominence of Cl in solution seems to hinge on its primordial supply in ices, which is unconstrained by the meteoritical record. Equilibrium assemblages, rich in serpentine and saponite clays, retain thorium and uranium radionuclides unless U-Cl or U-HCO3 complexing, which was not modeled, significantly enhances U solubility. However, the radionuclide 40 K can be leached at high water:rock ratio and/or low temperature at which K is exchanged with ammonium in minerals. We recommend the inclusion of these effects in future models of the geophysical evolution of ocean-bearing icy worlds. Our simulation products match observations of chloride salts on Europa and Enceladus; CI chondrites mineralogies; the observation of

  7. Isotope labeling strategies for NMR studies of RNA

    International Nuclear Information System (INIS)

    Lu, Kun; Miyazaki, Yasuyuki; Summers, Michael F.

    2010-01-01

    The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of long-range 1 H- 1 H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, may provide the best opportunities for obtaining structural information by NMR. Here we review methods that have been developed for preparing and purifying isotopically labeled RNAs, as well as NMR strategies that have been employed for signal assignment and structure determination.

  8. NMR studies of the sporulation protein SpoIIAA: Implications for the regulation of the transcription factor {sigma}F in Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Kovacs, Helena; Comfort, David; Lord, Matthew; Yudkin, Michael; Campbell, Iain D.; Nilges, Michael

    2001-04-15

    SpoIIAA participates in a four-component mechanism for phosphorylation-dependent transcription control at the outset of sporulation. We report the refinement of the solution structure of SpoIIAA by using the automated iterative NOE assignment method ARIA. To complement the structural data, the protein dynamics were determined by measuring the T{sub 1}, T{sub 2} and NOE of the backbone {sup 15}N-nuclei. The refined structure permits a discussion of the structural features that are important for the function of SpoIIAA in the regulation of the sporulation sigma factor {sigma}{sup F}, and for homologous regulatory pathways present in B. subtilis and in other bacilli.

  9. Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information