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Sample records for sampling alpha-helical protein

  1. On the diffusion of alpha-helical proteins in solvents

    Science.gov (United States)

    Barredo, Wilson I.; Bornales, Jinky B.; Bernido, Christopher C.; Aringa, Henry P.

    2015-01-01

    The winding probability function for a biopolymer diffusing in a crowded cell is obtained with the drift coefficient f(s) involving Bessel functions of general form f(s) = kJ2p+1 (νs). The variable s is the length along the chain and ν is a constant which can be used to simulate the frequency of appearance of a certain type of amino acid. Application of a particular case p = 3 to protein chains is carried out for different alpha helical proteins found in the Protein Data Bank (PDB). Analysis of our results leads us to an empirical formula that can be used to conveniently predict k/D and ν, where D is the diffusion coefficient of various α-helical proteins in solvents.

  2. Two exciton states in discrete and continuum alpha-helical proteins

    International Nuclear Information System (INIS)

    Latha, M.M.; Merlin, G.

    2012-01-01

    The dynamics of alpha-helical proteins is described by proposing a model Hamiltonian representing two exciton bound states. The dynamics is studied by constructing the equations of motion using a two exciton eigen-function in the discrete level. A numerical analysis shows the existence of two excitons in alpha-helical proteins and its propagation as solitons along the hydrogen bonding spines. The lattice model is also treated in the continuum limit which is a valid approximation in the low temperature, long wavelength limit. The resulting equation is studied using the multiple scale perturbation analysis which also shows the transfer of two exciton energy through alpha-helical proteins in the form of solitons with no change in velocity and amplitude. -- Highlights: ► The dynamics of alpha-helical proteins with two exciton states is studied. ► The dynamics is studied both in the discrete and continuum levels. ► The resulting equations are solved numerically and analytically. ► The solution supports the propagation of the energy in the form of solitons.

  3. Energy landscape, structure and rate effects on strength properties of alpha-helical proteins

    International Nuclear Information System (INIS)

    Bertaud, Jeremie; Hester, Joshua; Jimenez, Daniel D; Buehler, Markus J

    2010-01-01

    The strength of protein domains is crucial to identify the mechanical role of protein domains in biological processes such as mechanotransduction, tissue mechanics and tissue remodeling. Whereas the concept of strength has been widely investigated for engineered materials, the strength of fundamental protein material building blocks and how it depends on structural parameters such as the chemical bonding, the protein filament length and the timescale of observation or deformation velocity remains poorly understood. Here we report a systematic analysis of the influence of key parameters that define the energy landscape of the strength properties of alpha-helical protein domains, including energy barriers, unfolding and refolding distances, the locations of folded and unfolded states, as well as variations of the length and pulling velocity of alpha-helical protein filaments. The analysis is facilitated by the development of a double-well mesoscale potential formulation, utilized here to carry out a systematic numerical analysis of the behavior of alpha-helices. We compare the results against widely used protein strength models based on the Bell model, one of the simplest models used to characterize the strength of protein filaments. We find that, whereas Bell-type models are a reasonable approximation to describe the rupture of alpha-helical protein domains for a certain range of pulling speeds and values of energy barriers, the model ceases to hold for very large energy barriers and for very small pulling speeds, in agreement with earlier findings. We conclude with an application of our mesoscale model to investigate the effect of the length of alpha-helices on their mechanical strength. We find a weakening effect as the length of alpha-helical proteins increases, followed by an asymptotic regime in which the strength remains constant. We compare strand lengths found in biological proteins with the scaling law of strength versus alpha-helix filament length. The

  4. Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

    Science.gov (United States)

    Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

    2007-12-03

    The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

  5. Markov analysis of alpha-helical, beta-sheet and random coil regions of proteins

    International Nuclear Information System (INIS)

    Macchiato, M.; Tramontano, A.

    1983-01-01

    The rules up to now used to predict the spatial configuration of proteins from their primary structure are mostly based on the recurrence analysis of some doublets, triplets and so on of contiguous amino acids, but they do not take into account the correlation characteristics of the whole amino acid sequence. A statistical analysis of amino acid sequences for the alpha-helical, beta-sheet and random coil regions of about twenty proteins with known secondary structure by considering correlations effects has been carried out. The obtained results demonstrate that these sequences are at least a second-order Markov chain, i.e. they appear as if they were generated by a source that remembers at least the two aminoacids before the one being generated and that these two previous symbols influence the present choice

  6. Supramolecular Architectures and Mimics of Complex Natural Folds Derived from Rationally Designed alpha-Helical Protein Structures

    Science.gov (United States)

    Tavenor, Nathan Albert

    Protein-based supramolecular polymers (SMPs) are a class of biomaterials which draw inspiration from and expand upon the many examples of complex protein quaternary structures observed in nature: collagen, microtubules, viral capsids, etc. Designing synthetic supramolecular protein scaffolds both increases our understanding of natural superstructures and allows for the creation of novel materials. Similar to small-molecule SMPs, protein-based SMPs form due to self-assembly driven by intermolecular interactions between monomers, and monomer structure determines the properties of the overall material. Using protein-based monomers takes advantage of the self-assembly and highly specific molecular recognition properties encodable in polypeptide sequences to rationally design SMP architectures. The central hypothesis underlying our work is that alpha-helical coiled coils, a well-studied protein quaternary folding motif, are well-suited to SMP design through the addition of synthetic linkers at solvent-exposed sites. Through small changes in the structures of the cross-links and/or peptide sequence, we have been able to control both the nanoscale organization and the macroscopic properties of the SMPs. Changes to the linker and hydrophobic core of the peptide can be used to control polymer rigidity, stability, and dimensionality. The gaps in knowledge that this thesis sought to fill on this project were 1) the relationship between the molecular structure of the cross-linked polypeptides and the macroscopic properties of the SMPs and 2) a means of creating materials exhibiting multi-dimensional net or framework topologies. Separate from the above efforts on supramolecular architectures was work on improving backbone modification strategies for an alpha-helix in the context of a complex protein tertiary fold. Earlier work in our lab had successfully incorporated unnatural building blocks into every major secondary structure (beta-sheet, alpha-helix, loops and beta

  7. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

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    Nora Céspedes

    Full Text Available Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

  8. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif

    DEFF Research Database (Denmark)

    Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary

    2014-01-01

    Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Pla...

  9. Temperature-dependent structural changes in intrinsically disordered proteins: formation of alpha-helices or loss of polyproline II?

    DEFF Research Database (Denmark)

    Kjærgaard, Magnus; Nørholm, Ann-Beth; Hendus-Altenburger, Ruth

    2010-01-01

    temperature, which most likely reflects formation of transient alpha-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble...... with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs...

  10. Integrability and soliton solutions for an inhomogeneous generalized fourth-order nonlinear Schrödinger equation describing the inhomogeneous alpha helical proteins and Heisenberg ferromagnetic spin chains

    International Nuclear Information System (INIS)

    Wang, Pan; Tian, Bo; Jiang, Yan; Wang, Yu-Feng

    2013-01-01

    For describing the dynamics of alpha helical proteins with internal molecular excitations, nonlinear couplings between lattice vibrations and molecular excitations, and spin excitations in one-dimensional isotropic biquadratic Heisenberg ferromagnetic spin with the octupole–dipole interactions, we consider an inhomogeneous generalized fourth-order nonlinear Schrödinger equation. Based on the Ablowitz–Kaup–Newell–Segur system, infinitely many conservation laws for the equation are derived. Through the auxiliary function, bilinear forms and N-soliton solutions for the equation are obtained. Interactions of solitons are discussed by means of the asymptotic analysis. Effects of linear inhomogeneity on the interactions of solitons are also investigated graphically and analytically. Since the inhomogeneous coefficient of the equation h=α x+β, the soliton takes on the parabolic profile during the evolution. Soliton velocity is related to the parameter α, distance scale coefficient and biquadratic exchange coefficient, but has no relation with the parameter β. Soliton amplitude and width are only related to α. Soliton position is related to β

  11. Computational design and elaboration of a de novo heterotetrameric alpha-helical protein that selectively binds an emissive abiological (porphinato)zinc chromophore.

    Science.gov (United States)

    Fry, H Christopher; Lehmann, Andreas; Saven, Jeffery G; DeGrado, William F; Therien, Michael J

    2010-03-24

    The first example of a computationally de novo designed protein that binds an emissive abiological chromophore is presented, in which a sophisticated level of cofactor discrimination is pre-engineered. This heterotetrameric, C(2)-symmetric bundle, A(His):B(Thr), uniquely binds (5,15-di[(4-carboxymethyleneoxy)phenyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The A(2)B(2) heterotetrameric protein reflects ligand-directed elements of both positive and negative design, including hydrogen bonds to second-shell ligands. Experimental support for the appropriate formulation of [(DPP)Zn:A(His):B(Thr)](2) is provided by UV/visible and circular dichroism spectroscopies, size exclusion chromatography, and analytical ultracentrifugation. Time-resolved transient absorption and fluorescence spectroscopic data reveal classic excited-state singlet and triplet PZn photophysics for the A(His):B(Thr):(DPP)Zn protein (k(fluorescence) = 4 x 10(8) s(-1); tau(triplet) = 5 ms). The A(2)B(2) apoprotein has immeasurably low binding affinities for related [porphinato]metal chromophores that include a (DPP)Fe(III) cofactor and the zinc metal ion hemin derivative [(PPIX)Zn], underscoring the exquisite active-site binding discrimination realized in this computationally designed protein. Importantly, elements of design in the A(His):B(Thr) protein ensure that interactions within the tetra-alpha-helical bundle are such that only the heterotetramer is stable in solution; corresponding homomeric bundles present unfavorable ligand-binding environments and thus preclude protein structural rearrangements that could lead to binding of (porphinato)iron cofactors.

  12. Introduction of a proline residue into position 31 of the loop of the dimeric 4-alpha-helical protein ROP causes a drastic destabilization.

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    Peters, K; Hinz, H J; Cesareni, G

    1997-10-01

    The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993). The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.

  13. Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions [v3; ref status: indexed, http://f1000r.es/50u

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    Sandeep Chakraborty

    2015-01-01

    Full Text Available Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52 derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html.

  14. General architecture of the alpha-helical globule.

    Science.gov (United States)

    Murzin, A G; Finkelstein, A V

    1988-12-05

    A model is presented for the arrangement of alpha-helices in globular proteins. In the model, helices are placed on certain ribs of "quasi-spherical" polyhedra. The polyhedra are chosen so as to allow the close packing of helices around a hydrophobic core and to stress the collective interactions of the individual helices. The model predicts a small set of stable architectures for alpha-helices in globular proteins and describes the geometries of the helix packings. Some of the predicted helix arrangements have already been observed in known protein structures; others are new. An analysis of the three-dimensional structures of all proteins for which co-ordinates are available shows that the model closely approximates the arrangements and packing of helices actually observed. The average deviations of the real helix axes from those in the model polyhedra is +/- 20 degrees in orientation and +/- 2 A in position (1 A = 0.1 nm). We also show that for proteins that are not homologous, but whose helix arrangements are described by the same polyhedron, the root-mean-square difference in the position of the C alpha atoms in the helices is 1.6 to 3.0 A.

  15. Bilinear forms and soliton solutions for a fourth-order variable-coefficient nonlinear Schrödinger equation in an inhomogeneous Heisenberg ferromagnetic spin chain or an alpha helical protein

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jin-Wei; Gao, Yi-Tian, E-mail: gaoyt163@163.com; Wang, Qi-Min; Su, Chuan-Qi; Feng, Yu-Jie; Yu, Xin

    2016-01-15

    In this paper, a fourth-order variable-coefficient nonlinear Schrödinger equation is studied, which might describe a one-dimensional continuum anisotropic Heisenberg ferromagnetic spin chain with the octuple–dipole interaction or an alpha helical protein with higher-order excitations and interactions under continuum approximation. With the aid of auxiliary function, we derive the bilinear forms and corresponding constraints on the variable coefficients. Via the symbolic computation, we obtain the Lax pair, infinitely many conservation laws, one-, two- and three-soliton solutions. We discuss the influence of the variable coefficients on the solitons. With different choices of the variable coefficients, we obtain the parabolic, cubic, and periodic solitons, respectively. We analyse the head-on and overtaking interactions between/among the two and three solitons. Interactions between a bound state and a single soliton are displayed with different choices of variable coefficients. We also derive the quasi-periodic formulae for the three cases of the bound states.

  16. Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

    Science.gov (United States)

    Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

    2015-12-02

    The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Mutations in the alpha-helical region of the amino terminus of the Maize rayado fino virus capsid protein and CP:RNA ratios affect virus-like particle encapsidation of RNAs.

    Science.gov (United States)

    Natilla, Angela; Murphy, Charles; Hammond, Rosemarie W

    2015-01-22

    Viral-based nanoplatforms rely on balancing the delicate array of virus properties to optimally achieve encapsidation of foreign materials with various potential objectives. We investigated the use of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) as a multifunctional nanoplatform and their potential application as protein cages. MRFV-VLPs are composed of two serologically related, carboxy co-terminal coat proteins (CP1 and CP2) which are capable of self-assembling in Nicotiana benthamiana plants into 30nm particles with T=3 symmetry. The N-terminus of CP1 was targeted for genetic modification to exploit the driving forces for VLP assembly, packaging and retention of RNA in vivo and in vitro. The N-terminus of MRFV-CP1 contains a peptide sequence of 37 amino acids which has been predicted to have an alpha-helical structure, is rich in hydrophobic amino acids, facilitates CP-RNA interactions, and is not required for self-assembly. Amino acid substitutions were introduced in the 37 amino acid N-terminus by site-directed mutagenesis and the mutant VLPs produced in plants by a Potato virus X (PVX)-based vector were tested for particle stability and RNA encapsidation. All mutant CPs resulted in production of VLPs which encapsidated non-viral RNAs, including PVX genomic and subgenomic (sg) RNAs, 18S rRNA and cellular and viral mRNAs. In addition, MRFV-VLPs encapsidated GFP mRNA when was expressed in plant cells from the pGD vector. These results suggest that RNA packaging in MRFV-VLPs is predominantly driven by electrostatic interactions between the N-terminal 37 amino acid extension of CP1 and RNA, and that the overall species concentration of RNA in the cellular pool may determine the abundance and species of the RNAs packaged into the VLPs. Furthermore, RNA encapsidation is not required for VLPs stability, VLPs formed from MRFV-CP1 were stable at temperatures up to 70°C, and can be disassembled into CP monomers, which can then reassemble in vitro into

  18. Examining the Conservation of Kinks in Alpha Helices.

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    Eleanor C Law

    Full Text Available Kinks are a structural feature of alpha-helices and many are known to have functional roles. Kinks have previously tended to be defined in a binary fashion. In this paper we have deliberately moved towards defining them on a continuum, which given the unimodal distribution of kink angles is a better description. From this perspective, we examine the conservation of kinks in proteins. We find that kink angles are not generally a conserved property of homologs, pointing either to their not being functionally critical or to their function being related to conformational flexibility. In the latter case, the different structures of homologs are providing snapshots of different conformations. Sequence identity between homologous helices is informative in terms of kink conservation, but almost equally so is the sequence identity of residues in spatial proximity to the kink. In the specific case of proline, which is known to be prevalent in kinked helices, loss of a proline from a kinked helix often also results in the loss of a kink or reduction in its kink angle. We carried out a study of the seven transmembrane helices in the GPCR family and found that changes in kinks could be related both to subfamilies of GPCRs and also, in a particular subfamily, to the binding of agonists or antagonists. These results suggest conformational change upon receptor activation within the GPCR family. We also found correlation between kink angles in different helices, and the possibility of concerted motion could be investigated further by applying our method to molecular dynamics simulations. These observations reinforce the belief that helix kinks are key, functional, flexible points in structures.

  19. Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers.

    Science.gov (United States)

    Kreplak, L; Doucet, J; Briki, F

    2001-04-15

    Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.

  20. alpha-helical structural elements within the voltage-sensing domains of a K(+) channel.

    Science.gov (United States)

    Li-Smerin, Y; Hackos, D H; Swartz, K J

    2000-01-01

    Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.

  1. Fifty years of coiled-coils and alpha-helical bundles: a close relationship between sequence and structure.

    Science.gov (United States)

    Parry, David A D; Fraser, R D Bruce; Squire, John M

    2008-09-01

    alpha-Helical coiled coils are remarkable for the diversity of related conformations that they adopt in both fibrous and globular proteins, and for the range of functions that they exhibit. The coiled coils are based on a heptad (7-residue), hendecad (11-residue) or a related quasi-repeat of apolar residues in the sequences of the alpha-helical regions involved. Most of these, however, display one or more sequence discontinuities known as stutters or stammers. The resulting coiled coils vary in length, in the number of chains participating, in the relative polarity of the contributing alpha-helical regions (parallel or antiparallel), and in the pitch length and handedness of the supercoil (left- or right-handed). Functionally, the concept that a coiled coil can act only as a static rod is no longer valid, and the range of roles that these structures have now been shown to exhibit has expanded rapidly in recent years. An important development has been the recognition that the delightful simplicity that exists between sequence and structure, and between structure and function, allows coiled coils with specialized features to be designed de novo.

  2. Probing alpha-helical and beta-sheet structures of peptides at solid/liquid interfaces with SFG.

    Science.gov (United States)

    Chen, Xiaoyun; Wang, Jie; Sniadecki, Jason J; Even, Mark A; Chen, Zhan

    2005-03-29

    We demonstrated that sum frequency generation (SFG) vibrational spectroscopy can distinguish different secondary structures of proteins or peptides adsorbed at solid/liquid interfaces. The SFG spectrum for tachyplesin I at the polystyrene (PS)/solution interface has a fingerprint peak corresponding to the B1/B3 mode of the antiparallel beta-sheet. This peak disappeared upon the addition of dithiothreitol, which can disrupt the beta-sheet structure. The SFG spectrum indicative of the MSI594 alpha-helical structure was observed at the PS/MSI594 solution interface. This research validates SFG as a powerful technique for revealing detailed secondary structures of interfacial proteins and peptides.

  3. Antimicrobial peptides: the role of hydrophobicity in the alpha helical structure

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    Pandurangan Perumal

    2013-12-01

    Full Text Available The antimicrobial peptides (AMPs are a class of molecule obtained from plants, insects, animals, and humans. These peptides have been classified into five categories: 1. Anionic peptide, 2. Linear alpha helical cationic peptide, 3. Cationic peptide, 4. Anionic and cationic peptides with disulphide bonds, and 5. Anionic and cationic peptide fragments of larger proteins. Factors affecting AMPs are sequence, size, charge, hydrophobicity, amphipathicity, structure and conformation. Synthesis of these peptides is convenient by using solid phase peptide synthesis by using FMOC chemistry protocol. The secondary structures of three synthetic peptides were determined by circular dichroism. Also, it was compared the stability of the α-helical structure and confirmed the percentage of helix of these peptides by using circular dichroism. Some of these AMPs show therapeutic properties like antimicrobial, antiviral, contraceptive, and anticancer. The formulations of some peptides have been entered into the phase I, II, or III of clinical trials. This article to review briefly the sources, classification, factors affecting AMPs activity, synthesis, characterization, mechanism of action and therapeutic concern of AMPs and mainly focussed on percentage of α-helical structure in various medium.

  4. The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner.

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    Kazi, Aslamuzzaman; Sun, Jiazhi; Doi, Kenichiro; Sung, Shen-Shu; Takahashi, Yoshinori; Yin, Hang; Rodriguez, Johanna M; Becerril, Jorge; Berndt, Norbert; Hamilton, Andrew D; Wang, Hong-Gang; Sebti, Saïd M

    2011-03-18

    A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

  5. Chain length dependence of the helix orientation in Langmuir-Blodgett monolayers of alpha-helical diblock copolypeptides

    NARCIS (Netherlands)

    Nguyen, Le-Thu T.; Ardana, Aditya; Vorenkamp, Eltjo J.; ten Brinke, Gerrit; Schouten, Arend J.

    2010-01-01

    The effect of chain length on the helix orientation of alpha-helical diblock copolypeptides in Langmuir and Langmuir-Blodgett monolayers is reported for the first time. Amphiphilic diblock copolypeptides (PLGA-b-PMLGSLGs) of poly(alpha-L-glutamic acid) (PLGA) and

  6. Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

    Science.gov (United States)

    Oliver, A. E.; Deamer, D. W.

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two

  7. Voltage dependence of a stochastic model of activation of an alpha helical S4 sensor in a K channel membrane

    Science.gov (United States)

    Vaccaro, S. R.

    2011-09-01

    The voltage dependence of the ionic and gating currents of a K channel is dependent on the activation barriers of a voltage sensor with a potential function which may be derived from the principal electrostatic forces on an S4 segment in an inhomogeneous dielectric medium. By variation of the parameters of a voltage-sensing domain model, consistent with x-ray structures and biophysical data, the lowest frequency of the survival probability of each stationary state derived from a solution of the Smoluchowski equation provides a good fit to the voltage dependence of the slowest time constant of the ionic current in a depolarized membrane, and the gating current exhibits a rising phase that precedes an exponential relaxation. For each depolarizing potential, the calculated time dependence of the survival probabilities of the closed states of an alpha helical S4 sensor are in accord with an empirical model of the ionic and gating currents recorded during the activation process.

  8. Consequences of non-uniformity in the stoichiometry of component fractions within one and two loops models of alpha-helical peptides

    Science.gov (United States)

    Atoms in biomolecular structures like alpha helices contain an array of distances and angles which include abundant multiple patterns of redundancies. Thus all peptides backbones contain the three atom sequence N-C*C, whereas the repeating set of a four atom sequences (N-C*C-N, C*-C-N-C*, and C-N-C...

  9. Studies of alpha-helicity and intersegmental interactions in voltage-gated Na+ channels: S2D4.

    Directory of Open Access Journals (Sweden)

    Zhongming Ma

    2009-11-01

    Full Text Available Much data, including crystallographic, support structural models of sodium and potassium channels consisting of S1-S4 transmembrane segments (the "voltage-sensing domain" clustered around a central pore-forming region (S5-S6 segments and the intervening loop. Voltage gated sodium channels have four non-identical domains which differentiates them from the homotetrameric potassium channels that form the basis for current structural models. Since potassium and sodium channels also exhibit many different functional characteristics and the fourth domain (D4 of sodium channels differs in function from other domains (D1-D3, we have explored its structure in order to determine whether segments in D4 of sodium channels differ significantly from that determined for potassium channels. We have probed the secondary and tertiary structure and the role of the individual amino acid residues of the S2D4 of Na(v1.4 by employing cysteine-scanning mutagenesis (with tryptophan and glutamine substituted for native cysteine. A Fourier transform power spectrum of perturbations in free energy of steady-state inactivation gating (using midpoint potentials and slopes of Boltzmann equation fits of channel availability, h(infinity-V plots indicates a substantial amount of alpha-helical structure in S2D4 (peak at 106 degrees, alpha-Periodicity Index (alpha-PI of 3.10, This conclusion is supported by alpha-PI values of 3.28 and 2.84 for the perturbations in rate constants of entry into (beta and exit from (alpha fast inactivation at 0 mV for mutant channels relative to WT channels assuming a simple two-state model for transition from the open to inactivated state. The results of cysteine substitution at the two most sensitive sites of the S2D4 alpha-helix (N1382 and E1392C support the existence of electrostatic network interactions between S2 and other transmembrane segments within Na(v1.4D4 similar to but not identical to those proposed for K+ channels.

  10. 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.

  11. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

    Science.gov (United States)

    Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

    2013-06-14

    Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

  12. Focused conformational sampling in proteins

    Science.gov (United States)

    Bacci, Marco; Langini, Cassiano; Vymětal, Jiří; Caflisch, Amedeo; Vitalis, Andreas

    2017-11-01

    A detailed understanding of the conformational dynamics of biological molecules is difficult to obtain by experimental techniques due to resolution limitations in both time and space. Computer simulations avoid these in theory but are often too short to sample rare events reliably. Here we show that the progress index-guided sampling (PIGS) protocol can be used to enhance the sampling of rare events in selected parts of biomolecules without perturbing the remainder of the system. The method is very easy to use as it only requires as essential input a set of several features representing the parts of interest sufficiently. In this feature space, new states are discovered by spontaneous fluctuations alone and in unsupervised fashion. Because there are no energetic biases acting on phase space variables or projections thereof, the trajectories PIGS generates can be analyzed directly in the framework of transition networks. We demonstrate the possibility and usefulness of such focused explorations of biomolecules with two loops that are part of the binding sites of bromodomains, a family of epigenetic "reader" modules. This real-life application uncovers states that are structurally and kinetically far away from the initial crystallographic structures and are also metastable. Representative conformations are intended to be used in future high-throughput virtual screening campaigns.

  13. 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.

  14. Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex

    DEFF Research Database (Denmark)

    Westfield, Gerwin H; Rasmussen, Søren Gøgsig Faarup; Su, Min

    2011-01-01

    The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture...... of agonist-occupied β(2)-adrenoceptor (β(2)AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex...

  15. Alpha-Helical Fragaceatoxin C Nanopore Engineered for Double-Stranded and Single-Stranded Nucleic Acid Analysis

    NARCIS (Netherlands)

    Wloka, Carsten; Mutter, Natalie Lisa; Soskine, Misha; Maglia, Giovanni

    2016-01-01

    Nanopores are used in single-molecule DNA analysis and sequencing. Herein, we show that Fragaceatoxin C (FraC), an α-helical pore-forming toxin from an actinoporin protein family, can be reconstituted in sphingomyelin-free standard planar lipid bilayers. We engineered FraC for DNA analysis and show

  16. Convergence of sampling in protein simulations

    NARCIS (Netherlands)

    Hess, B

    With molecular dynamics protein dynamics can be simulated in atomic detail. Current computers are not fast enough to probe all available conformations, but fluctuations around one conformation can be sampled to a reasonable extent. The motions with the largest fluctuations can be filtered out of a

  17. The outer membrane protein assembly machinery of Neisseria meningitidis

    NARCIS (Netherlands)

    Volokhina, E.B.|info:eu-repo/dai/nl/304837202

    2009-01-01

    Gram-negative bacteria are characterized by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm. While the integral IM proteins are alpha-helical, all but one known integral OM proteins (OMPs) are

  18. Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

    Science.gov (United States)

    Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

    2006-11-28

    We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

  19. An efficient method for sampling the essential subspace of proteins

    NARCIS (Netherlands)

    Amadei, A; Linssen, A.B M; de Groot, B.L.; van Aalten, D.M.F.; Berendsen, H.J.C.

    A method is presented for a more efficient sampling of the configurational space of proteins as compared to conventional sampling techniques such as molecular dynamics. The method is based on the large conformational changes in proteins revealed by the ''essential dynamics'' analysis. A form of

  20. Topology of membrane proteins-predictions, limitations and variations.

    Science.gov (United States)

    Tsirigos, Konstantinos D; Govindarajan, Sudha; Bassot, Claudio; Västermark, Åke; Lamb, John; Shu, Nanjiang; Elofsson, Arne

    2017-10-26

    Transmembrane proteins perform a variety of important biological functions necessary for the survival and growth of the cells. Membrane proteins are built up by transmembrane segments that span the lipid bilayer. The segments can either be in the form of hydrophobic alpha-helices or beta-sheets which create a barrel. A fundamental aspect of the structure of transmembrane proteins is the membrane topology, that is, the number of transmembrane segments, their position in the protein sequence and their orientation in the membrane. Along these lines, many predictive algorithms for the prediction of the topology of alpha-helical and beta-barrel transmembrane proteins exist. The newest algorithms obtain an accuracy close to 80% both for alpha-helical and beta-barrel transmembrane proteins. However, lately it has been shown that the simplified picture presented when describing a protein family by its topology is limited. To demonstrate this, we highlight examples where the topology is either not conserved in a protein superfamily or where the structure cannot be described solely by the topology of a protein. The prediction of these non-standard features from sequence alone was not successful until the recent revolutionary progress in 3D-structure prediction of proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. High-resolution protein design with backbone freedom.

    Science.gov (United States)

    Harbury, P B; Plecs, J J; Tidor, B; Alber, T; Kim, P S

    1998-11-20

    Recent advances in computational techniques have allowed the design of precise side-chain packing in proteins with predetermined, naturally occurring backbone structures. Because these methods do not model protein main-chain flexibility, they lack the breadth to explore novel backbone conformations. Here the de novo design of a family of alpha-helical bundle proteins with a right-handed superhelical twist is described. In the design, the overall protein fold was specified by hydrophobic-polar residue patterning, whereas the bundle oligomerization state, detailed main-chain conformation, and interior side-chain rotamers were engineered by computational enumerations of packing in alternate backbone structures. Main-chain flexibility was incorporated through an algebraic parameterization of the backbone. The designed peptides form alpha-helical dimers, trimers, and tetramers in accord with the design goals. The crystal structure of the tetramer matches the designed structure in atomic detail.

  2. 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.

  3. 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.

  4. Advanced path sampling of the kinetic network of small proteins

    NARCIS (Netherlands)

    Du, W.

    2014-01-01

    This thesis is focused on developing advanced path sampling simulation methods to study protein folding and unfolding, and to build kinetic equilibrium networks describing these processes. In Chapter 1 the basic knowledge of protein structure and folding theories were introduced and a brief overview

  5. Sampling Realistic Protein Conformations Using Local Structural Bias

    DEFF Research Database (Denmark)

    Hamelryck, Thomas Wim; Kent, John T.; Krogh, A.

    2006-01-01

    The prediction of protein structure from sequence remains a major unsolved problem in biology. The most successful protein structure prediction methods make use of a divide-and-conquer strategy to attack the problem: a conformational sampling method generates plausible candidate structures, which...... are subsequently accepted or rejected using an energy function. Conceptually, this often corresponds to separating local structural bias from the long-range interactions that stabilize the compact, native state. However, sampling protein conformations that are compatible with the local structural bias encoded...... in a given protein sequence is a long-standing open problem, especially in continuous space. We describe an elegant and mathematically rigorous method to do this, and show that it readily generates native-like protein conformations simply by enforcing compactness. Our results have far-reaching implications...

  6. Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

    Science.gov (United States)

    Feist, Peter; Hummon, Amanda B.

    2015-01-01

    Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed. PMID:25664860

  7. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.

    Science.gov (United States)

    Feist, Peter; Hummon, Amanda B

    2015-02-05

    Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

  8. Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

    Directory of Open Access Journals (Sweden)

    Peter Feist

    2015-02-01

    Full Text Available Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

  9. Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

    Directory of Open Access Journals (Sweden)

    Giovanni Monaco

    Full Text Available The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R, the primary Ca(2+-release channel in the endoplasmic reticulum (ER. Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4 has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG. By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

  10. Rapid Sampling of Hydrogen Bond Networks for Computational Protein Design.

    Science.gov (United States)

    Maguire, Jack B; Boyken, Scott E; Baker, David; Kuhlman, Brian

    2018-05-08

    Hydrogen bond networks play a critical role in determining the stability and specificity of biomolecular complexes, and the ability to design such networks is important for engineering novel structures, interactions, and enzymes. One key feature of hydrogen bond networks that makes them difficult to rationally engineer is that they are highly cooperative and are not energetically favorable until the hydrogen bonding potential has been satisfied for all buried polar groups in the network. Existing computational methods for protein design are ill-equipped for creating these highly cooperative networks because they rely on energy functions and sampling strategies that are focused on pairwise interactions. To enable the design of complex hydrogen bond networks, we have developed a new sampling protocol in the molecular modeling program Rosetta that explicitly searches for sets of amino acid mutations that can form self-contained hydrogen bond networks. For a given set of designable residues, the protocol often identifies many alternative sets of mutations/networks, and we show that it can readily be applied to large sets of residues at protein-protein interfaces or in the interior of proteins. The protocol builds on a recently developed method in Rosetta for designing hydrogen bond networks that has been experimentally validated for small symmetric systems but was not extensible to many larger protein structures and complexes. The sampling protocol we describe here not only recapitulates previously validated designs with performance improvements but also yields viable hydrogen bond networks for cases where the previous method fails, such as the design of large, asymmetric interfaces relevant to engineering protein-based therapeutics.

  11. A periodic table of coiled-coil protein structures.

    Science.gov (United States)

    Moutevelis, Efrosini; Woolfson, Derek N

    2009-01-23

    Coiled coils are protein structure domains with two or more alpha-helices packed together via interlacing of side chains known as knob-into-hole packing. We analysed and classified a large set of coiled-coil structures using a combination of automated and manual methods. This led to a systematic classification that we termed a "periodic table of coiled coils," which we have made available at http://coiledcoils.chm.bris.ac.uk/ccplus/search/periodic_table. In this table, coiled-coil assemblies are arranged in columns with increasing numbers of alpha-helices and in rows of increased complexity. The table provides a framework for understanding possibilities in and limits on coiled-coil structures and a basis for future prediction, engineering and design studies.

  12. Calculating ensemble averaged descriptions of protein rigidity without sampling.

    Directory of Open Access Journals (Sweden)

    Luis C González

    Full Text Available Previous works have demonstrated that protein rigidity is related to thermodynamic stability, especially under conditions that favor formation of native structure. Mechanical network rigidity properties of a single conformation are efficiently calculated using the integer body-bar Pebble Game (PG algorithm. However, thermodynamic properties require averaging over many samples from the ensemble of accessible conformations to accurately account for fluctuations in network topology. We have developed a mean field Virtual Pebble Game (VPG that represents the ensemble of networks by a single effective network. That is, all possible number of distance constraints (or bars that can form between a pair of rigid bodies is replaced by the average number. The resulting effective network is viewed as having weighted edges, where the weight of an edge quantifies its capacity to absorb degrees of freedom. The VPG is interpreted as a flow problem on this effective network, which eliminates the need to sample. Across a nonredundant dataset of 272 protein structures, we apply the VPG to proteins for the first time. Our results show numerically and visually that the rigidity characterizations of the VPG accurately reflect the ensemble averaged [Formula: see text] properties. This result positions the VPG as an efficient alternative to understand the mechanical role that chemical interactions play in maintaining protein stability.

  13. Calculating ensemble averaged descriptions of protein rigidity without sampling.

    Science.gov (United States)

    González, Luis C; Wang, Hui; Livesay, Dennis R; Jacobs, Donald J

    2012-01-01

    Previous works have demonstrated that protein rigidity is related to thermodynamic stability, especially under conditions that favor formation of native structure. Mechanical network rigidity properties of a single conformation are efficiently calculated using the integer body-bar Pebble Game (PG) algorithm. However, thermodynamic properties require averaging over many samples from the ensemble of accessible conformations to accurately account for fluctuations in network topology. We have developed a mean field Virtual Pebble Game (VPG) that represents the ensemble of networks by a single effective network. That is, all possible number of distance constraints (or bars) that can form between a pair of rigid bodies is replaced by the average number. The resulting effective network is viewed as having weighted edges, where the weight of an edge quantifies its capacity to absorb degrees of freedom. The VPG is interpreted as a flow problem on this effective network, which eliminates the need to sample. Across a nonredundant dataset of 272 protein structures, we apply the VPG to proteins for the first time. Our results show numerically and visually that the rigidity characterizations of the VPG accurately reflect the ensemble averaged [Formula: see text] properties. This result positions the VPG as an efficient alternative to understand the mechanical role that chemical interactions play in maintaining protein stability.

  14. High-resolution neutron spectroscopy on protein solution samples

    International Nuclear Information System (INIS)

    Grimaldo, M.; Henning, M.; Roosen-Runge, F.; Seydel, T.; Jalarvo, N.; Zamponi, M.; Zanini, F.; Zhang, F.; Schreiber, F.

    2015-01-01

    Proteins in solution are subject to a complex superposition of global translational and rotational diffusion as well as internal relaxations covering a wide range of time scales. With the advent of new high-flux neutron spectrometers in combination with enhanced analysis frameworks it has become possible to separate these different contributions. We discuss new approaches to the analysis by presenting example spectra and fits from data recorded on the backscattering spectrometers IN16, IN16B, and BASIS on the same protein solution sample. We illustrate the separation of the rotational and translational diffusion contribution, the accurate treatment of the solvent contribution, and the extraction of information on internal fluctuations. We also highlight the progress made in passing from second- to third-generation backscattering spectrometers. (authors)

  15. Energy landscape of all-atom protein-protein interactions revealed by multiscale enhanced sampling.

    Directory of Open Access Journals (Sweden)

    Kei Moritsugu

    2014-10-01

    Full Text Available Protein-protein interactions are regulated by a subtle balance of complicated atomic interactions and solvation at the interface. To understand such an elusive phenomenon, it is necessary to thoroughly survey the large configurational space from the stable complex structure to the dissociated states using the all-atom model in explicit solvent and to delineate the energy landscape of protein-protein interactions. In this study, we carried out a multiscale enhanced sampling (MSES simulation of the formation of a barnase-barstar complex, which is a protein complex characterized by an extraordinary tight and fast binding, to determine the energy landscape of atomistic protein-protein interactions. The MSES adopts a multicopy and multiscale scheme to enable for the enhanced sampling of the all-atom model of large proteins including explicit solvent. During the 100-ns MSES simulation of the barnase-barstar system, we observed the association-dissociation processes of the atomistic protein complex in solution several times, which contained not only the native complex structure but also fully non-native configurations. The sampled distributions suggest that a large variety of non-native states went downhill to the stable complex structure, like a fast folding on a funnel-like potential. This funnel landscape is attributed to dominant configurations in the early stage of the association process characterized by near-native orientations, which will accelerate the native inter-molecular interactions. These configurations are guided mostly by the shape complementarity between barnase and barstar, and lead to the fast formation of the final complex structure along the downhill energy landscape.

  16. Sampling and energy evaluation challenges in ligand binding protein design.

    Science.gov (United States)

    Dou, Jiayi; Doyle, Lindsey; Jr Greisen, Per; Schena, Alberto; Park, Hahnbeom; Johnsson, Kai; Stoddard, Barry L; Baker, David

    2017-12-01

    The steroid hormone 17α-hydroxylprogesterone (17-OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17-OHP containing an extended, nonpolar, shape-complementary binding pocket for the four-ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17-OHP with micromolar affinity. A co-crystal structure of one of the designs revealed that 17-OHP is rotated 180° around a pseudo-two-fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same "flipped" orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two-fold symmetry of the molecule. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  17. Correlating the ability of VP24 protein from Ebola and Marburg viruses to bind human karyopherin to their immune suppression mechanism and pathogenicity using computational methods

    OpenAIRE

    Chakraborty, Sandeep; Rao, Basuthkar J.; Asgeirsson, Bjarni; Dandekar, Abhaya

    2015-01-01

    Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved ...

  18. Hierarchical Protein Free Energy Landscapes from Variationally Enhanced Sampling.

    Science.gov (United States)

    Shaffer, Patrick; Valsson, Omar; Parrinello, Michele

    2016-12-13

    In recent work, we demonstrated that it is possible to obtain approximate representations of high-dimensional free energy surfaces with variationally enhanced sampling ( Shaffer, P.; Valsson, O.; Parrinello, M. Proc. Natl. Acad. Sci. , 2016 , 113 , 17 ). The high-dimensional spaces considered in that work were the set of backbone dihedral angles of a small peptide, Chignolin, and the high-dimensional free energy surface was approximated as the sum of many two-dimensional terms plus an additional term which represents an initial estimate. In this paper, we build on that work and demonstrate that we can calculate high-dimensional free energy surfaces of very high accuracy by incorporating additional terms. The additional terms apply to a set of collective variables which are more coarse than the base set of collective variables. In this way, it is possible to build hierarchical free energy surfaces, which are composed of terms that act on different length scales. We test the accuracy of these free energy landscapes for the proteins Chignolin and Trp-cage by constructing simple coarse-grained models and comparing results from the coarse-grained model to results from atomistic simulations. The approach described in this paper is ideally suited for problems in which the free energy surface has important features on different length scales or in which there is some natural hierarchy.

  19. Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.

    Science.gov (United States)

    Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

    2015-03-27

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Functional and genomic analyses of alpha-solenoid proteins.

    Science.gov (United States)

    Fournier, David; Palidwor, Gareth A; Shcherbinin, Sergey; Szengel, Angelika; Schaefer, Martin H; Perez-Iratxeta, Carol; Andrade-Navarro, Miguel A

    2013-01-01

    Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/.

  1. Relative Abundance of Proteins in Blood Plasma Samples from Patients with Chronic Cerebral Ischemia.

    Science.gov (United States)

    Kaysheva, Anna L; Kopylov, Artur T; Ponomarenko, Elena A; Kiseleva, Olga I; Teryaeva, Nadezhda B; Potapov, Alexander A; Izotov, Alexander А; Morozov, Sergei G; Kudryavtseva, Valeria Yu; Archakov, Alexander I

    2018-03-01

    A comparative protein profile analysis of 17 blood plasma samples from patients with ischemia and 20 samples from healthy volunteers was carried out using ultra-high resolution mass spectrometry. The analysis of measurements was performed using the proteomics search engine OMSSA. Normalized spectrum abundance factor (NSAF) in the biological samples was assessed using SearchGUI. The findings of mass spectrometry analysis of the protein composition of blood plasma samples demonstrate that the depleted samples are quite similar in protein composition and relative abundance of proteins. By comparing them with the control samples, we have found a small group of 44 proteins characteristic of the blood plasma samples from patients with chronic cerebral ischemia. These proteins contribute to the processes of homeostasis maintenance, including innate immune response unfolding, the response of a body to stress, and contribution to the blood clotting cascade.

  2. Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

    Science.gov (United States)

    Chambers, Andrew G.; Percy, Andrew J.; Hardie, Darryl B.; Borchers, Christoph H.

    2013-09-01

    Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77 %-92 % within replicates and the majority of these repeated proteins (70 %) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

  3. Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

    Science.gov (United States)

    Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

    2006-05-19

    Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

  4. Sampling protein motion and solvent effect during ligand binding

    Science.gov (United States)

    Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele

    2012-01-01

    An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423

  5. A constraint logic programming approach to associate 1D and 3D structural components for large protein complexes.

    Science.gov (United States)

    Dal Palù, Alessandro; Pontelli, Enrico; He, Jing; Lu, Yonggang

    2007-01-01

    The paper describes a novel framework, constructed using Constraint Logic Programming (CLP) and parallelism, to determine the association between parts of the primary sequence of a protein and alpha-helices extracted from 3D low-resolution descriptions of large protein complexes. The association is determined by extracting constraints from the 3D information, regarding length, relative position and connectivity of helices, and solving these constraints with the guidance of a secondary structure prediction algorithm. Parallelism is employed to enhance performance on large proteins. The framework provides a fast, inexpensive alternative to determine the exact tertiary structure of unknown proteins.

  6. "Hot cores" in proteins: Comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms

    Directory of Open Access Journals (Sweden)

    Bossa Francesco

    2008-02-01

    Full Text Available Abstract Background A wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. These include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. It has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. In this work, protein crystal structures from hyper/thermophilic organisms and their mesophilic homologs have been compared, in order to quantify the difference of apolar contact area and to assess the role played by the hydrophobic contacts in the stabilization of the protein core, at high temperatures. Results The construction of two datasets was carried out so as to satisfy several restrictive criteria, such as minimum redundancy, resolution and R-value thresholds and lack of any structural defect in the collected structures. This approach allowed to quantify with relatively high precision the apolar contact area between interacting residues, reducing the uncertainty due to the position of atoms in the crystal structures, the redundancy of data and the size of the dataset. To identify the common core regions of these proteins, the study was focused on segments that conserve a similar main chain conformation in the structures analyzed, excluding the intervening regions whose structure differs markedly. The results indicated that hyperthermophilic proteins underwent a significant increase of the hydrophobic contact area contributed by those residues composing the alpha-helices of the structurally conserved regions. Conclusion This study indicates the decreased flexibility of alpha-helices in proteins core as a major factor contributing to the enhanced termostability of a number of hyperthermophilic proteins. This effect, in turn, may be due to an increased number of buried methyl groups in

  7. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

    Science.gov (United States)

    Weber, Daniela; Davies, Michael J; Grune, Tilman

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

  8. Protein expression based multimarker analysis of breast cancer samples

    International Nuclear Information System (INIS)

    Presson, Angela P; Horvath, Steve; Yoon, Nam K; Bagryanova, Lora; Mah, Vei; Alavi, Mohammad; Maresh, Erin L; Rajasekaran, Ayyappan K; Goodglick, Lee; Chia, David

    2011-01-01

    Tissue microarray (TMA) data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups. We apply weighted correlation network analysis (WGCNA) to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%), moderate (22%) and high (50%) mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-β1, and TGF β receptor II) that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis. We find that the rule-based grouping variable (referred to as WGCNA*) is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data), it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance). We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach. We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes

  9. Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

    OpenAIRE

    Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

    2001-01-01

    Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic ...

  10. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

    Directory of Open Access Journals (Sweden)

    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  11. Determination of phosphorus in small amounts of protein samples by ICP-MS.

    Science.gov (United States)

    Becker, J Sabine; Boulyga, Sergei F; Pickhardt, Carola; Becker, J; Buddrus, Stefan; Przybylski, Michael

    2003-02-01

    Inductively coupled plasma mass spectrometry (ICP-MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 micro g) or digest (1-10 micro L) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-SFMS) and quadrupole-based ICP-MS (ICP-QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8+/-0.4 mg g(-1), was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP-QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP-MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1-100 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP-SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.The detection limits for phosphorus in biological samples were determined by ICP-SFMS down to the ng g(-1) level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP-SFMS in comparison to ICP-QMS.

  12. Effects of Long-Term Storage Time and Original Sampling Month on Biobank Plasma Protein Concentrations

    Directory of Open Access Journals (Sweden)

    Stefan Enroth

    2016-10-01

    Full Text Available The quality of clinical biobank samples is crucial to their value for life sciences research. A number of factors related to the collection and storage of samples may affect the biomolecular composition. We have studied the effect of long-time freezer storage, chronological age at sampling, season and month of the year and on the abundance levels of 108 proteins in 380 plasma samples collected from 106 Swedish women. Storage time affected 18 proteins and explained 4.8–34.9% of the observed variance. Chronological age at sample collection after adjustment for storage-time affected 70 proteins and explained 1.1–33.5% of the variance. Seasonal variation had an effect on 15 proteins and month (number of sun hours affected 36 proteins and explained up to 4.5% of the variance after adjustment for storage-time and age. The results show that freezer storage time and collection date (month and season exerted similar effect sizes as age on the protein abundance levels. This implies that information on the sample handling history, in particular storage time, should be regarded as equally prominent covariates as age or gender and need to be included in epidemiological studies involving protein levels.

  13. [Interconnection between architecture of protein globule and disposition of conformational conservative oligopeptides in proteins from one protein family].

    Science.gov (United States)

    Batianovskiĭ, A V; Filatov, I V; Namiot, V A; Esipova, N G; Volotovskiĭ, I D

    2012-01-01

    It was shown that selective interactions between helical segments of macromolecules can realize in globular proteins in the segments characterized by the same periodicities of charge distribution i.e. between conformationally conservative oligopeptides. It was found that in the macromolecules of alpha-helical proteins conformationally conservative oligopeptides are disposed at a distance being characteristic of direct interactions. For representatives of many structural families of alpha-type proteins specific disposition of conformationally conservative segments is observed. This disposition is inherent to a particular structural family. Disposition of conformationally conservative segments is not related to homology of the amino acid sequence but reflects peculiarities of native 3D-architectures of protein globules.

  14. Protein Profile study of clinical samples using Laser Induced Fluorescence as the detection method

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Raja, Sujatha N.; Rai, Lavanya

    2009-01-01

      Protein profiles of tissue homogenates were recorded using HPLC separation and LIF detection method. The samples were collected from volunteers with clinically normal or cervical cancer conditions. It is shown that the protein profile can be classified as belonging to malignant or normal state ...

  15. A sample holder for in-house X-ray powder diffraction studies of protein powders

    DEFF Research Database (Denmark)

    Frankær, Christian Grundahl; Harris, Pernille; Ståhl, Kenny

    2011-01-01

    A sample holder for handling samples of protein for in-house X-ray powder diffraction (XRPD) analysis has been made and tested on lysozyme. The use of an integrated pinhole reduced the background, and good signal-to-noise ratios were obtained from only 7 l of sample, corresponding to approximatel...... 2-3 mg of dry protein. The sample holder is further adaptable to X-ray absorption spectroscopy (XAS) measurements. Both XRPD and XAS at the Zn K-edge were tested with hexameric Zn insulin....

  16. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

  17. Sequence and expression pattern of a novel human orphan G-protein-coupled receptor, GPRC5B, a family C receptor with a short amino-terminal domain

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Krogsgaard-Larsen, P

    2000-01-01

    Query of GenBank with the amino acid sequence of human metabotropic glutamate receptor subtype 2 (mGluR2) identified a predicted gene product of unknown function on BAC clone CIT987SK-A-69G12 (located on chromosome band 16p12) as a homologous protein. The transcript, entitled GPRC5B, was cloned f...... from an expressed sequence tag clone that contained the entire open reading frame of the transcript encoding a protein of 395 amino acids. Analysis of the protein sequence reveal that GPRC5B contains a signal peptide and seven transmembrane alpha-helices, which is a hallmark of G...

  18. How reverse turns may mediate the formation of helical segments in proteins: an x-ray model.

    OpenAIRE

    Perczel, A; Foxman, B M; Fasman, G D

    1992-01-01

    The three-dimensional structure of a protein is the assembly of different secondary structural elements, such as alpha-helices, beta-pleated sheets, and beta-turns. Although the conformation of hundreds of proteins has been elaborated in the solid state, only a vague understanding of the mechanism of their conformational folding is known. One facet of this topic is the conformational interconversion of one or more beta-turns to a helical structure (and vice versa), which may also be related t...

  19. Light assisted drying (LAD) for protein stabilization: optical characterization of samples

    Science.gov (United States)

    Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

    2018-02-01

    Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

  20. Simultaneous isolation of mRNA and native protein from minute samples of cells

    DEFF Research Database (Denmark)

    Petersen, Tonny Studsgaard; Andersen, Claus Yding

    2014-01-01

    Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

  1. The Sorcerer II Global Ocean Sampling Expedition: Expanding theUniverse of Protein Families

    Energy Technology Data Exchange (ETDEWEB)

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B.; Halpern,Aaron L.; Williamson, Shannon J.; Remington, Karin; Eisen, Jonathan A.; Heidelberg, Karla B.; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S.; Li, Huiying; Mashiyama, Susan T.; Joachimiak, Marcin P.; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A.; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael,Benjamin J.; Bafna, Vineet; Friedman, Robert; Brenner, Steven E.; Godzik,Adam; Eisenberg, David; Dixon, Jack E.; Taylor, Susan S.; Strausberg,Robert L.; Frazier, Marvin; Venter, J.Craig

    2006-03-23

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  2. The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families.

    Science.gov (United States)

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J Craig

    2007-03-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  3. The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families.

    Directory of Open Access Journals (Sweden)

    Shibu Yooseph

    2007-03-01

    Full Text Available Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  4. Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid

    Directory of Open Access Journals (Sweden)

    Han Roelofsen

    2008-01-01

    Full Text Available Saliva is an easy accessible plasma ultra-filtrate. Therefore, saliva can be an attractive alternative to blood for measurement of diagnostic protein markers. Our aim was to determine stability and protein composition of saliva. Protein stability at room temperature was examined by incubating fresh whole saliva with and without inhibitors of proteases and bacterial metabolism followed by Surface Enhanced Laser Desorption/Ionization (SELDI analyses. Protein composition was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE fractionation of saliva proteins followed by digestion of excised bands and identification by liquid chromatography tandem mass spectrometry (LC-MS/MS. Results show that rapid protein degradation occurs within 30 minutes after sample collection. Degradation starts already during collection. Protease inhibitors partly prevented degradation while inhibition of bacterial metabolism did not affect degradation. Three stable degradation products of 2937 Da, 3370 Da and 4132 Da were discovered which can be used as markers to monitor sample quality. Saliva proteome analyses revealed 218 proteins of which 84 can also be found in blood plasma. Based on a comparison with seven other proteomics studies on whole saliva we identified 83 new saliva proteins. We conclude that saliva is a promising diagnostic fl uid when precautions are taken towards protein breakdown.

  5. Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

    DEFF Research Database (Denmark)

    Nesgaard, Lise W.; Hoffmann, Søren Vrønning; Andersen, Christian Beyschau

    2008-01-01

    Circular dichroism using synchrotron radiation (SRCD) can extend the spectral range down to approximately 130 nm for dry proteins, potentially providing new structural information. Using a selection of dried model proteins, including alpha-helical, beta-sheet, and mixed-structure proteins, we...... with previously published theoretical calculations related to pi-orbital transitions. We also show that drying does not lead to large changes in the secondary structure and does not induce orientational artifacts. In combination with principal component analysis, our SRCD data allow us to distinguish between two...... different types of protein fibrils, highlighting that bona fide fibrils formed by lysozyme are structurally more similar to the nonclassical fibrillar aggregates formed by the SerADan peptide than with the amyloid formed by alpha-synuclein. Thus, despite the lack of direct structural conclusions...

  6. Serial Sampling of Serum Protein Biomarkers for Monitoring Human Traumatic Brain Injury Dynamics: A Systematic Review.

    Science.gov (United States)

    Thelin, Eric Peter; Zeiler, Frederick Adam; Ercole, Ari; Mondello, Stefania; Büki, András; Bellander, Bo-Michael; Helmy, Adel; Menon, David K; Nelson, David W

    2017-01-01

    The proteins S100B, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and neurofilament light (NF-L) have been serially sampled in serum of patients suffering from traumatic brain injury (TBI) in order to assess injury severity and tissue fate. We review the current literature of serum level dynamics of these proteins following TBI and used the term "effective half-life" ( t 1/2 ) in order to describe the "fall" rate in serum. Through searches on EMBASE, Medline, and Scopus, we looked for articles where these proteins had been serially sampled in serum in human TBI. We excluded animal studies, studies with only one presented sample and studies without neuroradiological examinations. Following screening (10,389 papers), n  = 122 papers were included. The proteins S100B ( n  = 66) and NSE ( n  = 27) were the two most frequent biomarkers that were serially sampled. For S100B in severe TBI, a majority of studies indicate a t 1/2 of about 24 h, even if very early sampling in these patients reveals rapid decreases (1-2 h) though possibly of non-cerebral origin. In contrast, the t 1/2 for NSE is comparably longer, ranging from 48 to 72 h in severe TBI cases. The protein GFAP ( n  = 18) appears to have t 1/2 of about 24-48 h in severe TBI. The protein UCH-L1 ( n  = 9) presents a t 1/2 around 7 h in mild TBI and about 10 h in severe. Frequent sampling of these proteins revealed different trajectories with persisting high serum levels, or secondary peaks, in patients with unfavorable outcome or in patients developing secondary detrimental events. Finally, NF-L ( n  = 2) only increased in the few studies available, suggesting a serum availability of >10 days. To date, automated assays are available for S100B and NSE making them faster and more practical to use. Serial sampling of brain-specific proteins in serum reveals different temporal trajectories that should be

  7. Serial Sampling of Serum Protein Biomarkers for Monitoring Human Traumatic Brain Injury Dynamics: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Eric Peter Thelin

    2017-07-01

    Full Text Available BackgroundThe proteins S100B, neuron-specific enolase (NSE, glial fibrillary acidic protein (GFAP, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1, and neurofilament light (NF-L have been serially sampled in serum of patients suffering from traumatic brain injury (TBI in order to assess injury severity and tissue fate. We review the current literature of serum level dynamics of these proteins following TBI and used the term “effective half-life” (t1/2 in order to describe the “fall” rate in serum.Materials and methodsThrough searches on EMBASE, Medline, and Scopus, we looked for articles where these proteins had been serially sampled in serum in human TBI. We excluded animal studies, studies with only one presented sample and studies without neuroradiological examinations.ResultsFollowing screening (10,389 papers, n = 122 papers were included. The proteins S100B (n = 66 and NSE (n = 27 were the two most frequent biomarkers that were serially sampled. For S100B in severe TBI, a majority of studies indicate a t1/2 of about 24 h, even if very early sampling in these patients reveals rapid decreases (1–2 h though possibly of non-cerebral origin. In contrast, the t1/2 for NSE is comparably longer, ranging from 48 to 72 h in severe TBI cases. The protein GFAP (n = 18 appears to have t1/2 of about 24–48 h in severe TBI. The protein UCH-L1 (n = 9 presents a t1/2 around 7 h in mild TBI and about 10 h in severe. Frequent sampling of these proteins revealed different trajectories with persisting high serum levels, or secondary peaks, in patients with unfavorable outcome or in patients developing secondary detrimental events. Finally, NF-L (n = 2 only increased in the few studies available, suggesting a serum availability of >10 days. To date, automated assays are available for S100B and NSE making them faster and more practical to use.ConclusionSerial sampling of brain-specific proteins in serum reveals

  8. Modelling Protein Dynamics on the Microsecond Time Scale

    DEFF Research Database (Denmark)

    Siuda, Iwona Anna

    Recent years have shown an increase in coarse-grained (CG) molecular dynamics simulations, providing structural and dynamic details of large proteins and enabling studies of self-assembly of biological materials. It is not easy to acquire such data experimentally, and access is also still limited...... in atomistic simulations. During her PhD studies, Iwona Siuda used MARTINI CG models to study the dynamics of different globular and membrane proteins. In several cases, the MARTINI model was sufficient to study conformational changes of small, purely alpha-helical proteins. However, in studies of larger......ELNEDIN was therefore proposed as part of the work. Iwona Siuda’s results from the CG simulations had biological implications that provide insights into possible mechanisms of the periplasmic leucine-binding protein, the sarco(endo)plasmic reticulum calcium pump, and several proteins from the saposin-like proteins...

  9. Laser-Induced Breakdown Spectroscopy Based Protein Assay for Cereal Samples.

    Science.gov (United States)

    Sezer, Banu; Bilge, Gonca; Boyaci, Ismail Hakki

    2016-12-14

    Protein content is an important quality parameter in terms of price, nutritional value, and labeling of various cereal samples. However, conventional analysis methods, namely, Kjeldahl and Dumas, have major drawbacks such as long analysis time, titration mistakes, and carrier gas dependence with high purity. For this reason, there is an urgent need for rapid, reliable, and environmentally friendly technologies for protein analysis. The present study aims to develop a new method for protein analysis in wheat flour and whole meal by using laser-induced breakdown spectroscopy (LIBS), which is a multielemental, fast, and simple spectroscopic method. Unlike the Kjeldahl and Dumas methods, it has potential to analyze a high number of samples in considerably short time. In the study, nitrogen peaks in LIBS spectra of wheat flour and whole meal samples with different protein contents were correlated with results of the standard Dumas method with the aid of chemometric methods. A calibration graph showed good linearity with the protein content between 7.9 and 20.9% and a 0.992 coefficient of determination (R 2 ). The limit of detection was calculated as 0.26%. The results indicated that LIBS is a promising and reliable method with its high sensitivity for routine protein analysis in wheat flour and whole meal samples.

  10. Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

    International Nuclear Information System (INIS)

    Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

    2015-01-01

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii

  11. Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

    Energy Technology Data Exchange (ETDEWEB)

    Bittame, Amina [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Effantin, Grégory [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Schoehn, Guy; Weissenhorn, Winfried [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Cesbron-Delauw, Marie-France; Gagnon, Jean [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Mercier, Corinne, E-mail: corinne.mercier@ujf-grenoble.fr [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France)

    2015-03-27

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii.

  12. Evaluation of sampling plans to detect Cry9C protein in corn flour and meal.

    Science.gov (United States)

    Whitaker, Thomas B; Trucksess, Mary W; Giesbrecht, Francis G; Slate, Andrew B; Thomas, Francis S

    2004-01-01

    StarLink is a genetically modified corn that produces an insecticidal protein, Cry9C. Studies were conducted to determine the variability and Cry9C distribution among sample test results when Cry9C protein was estimated in a bulk lot of corn flour and meal. Emphasis was placed on measuring sampling and analytical variances associated with each step of the test procedure used to measure Cry9C in corn flour and meal. Two commercially available enzyme-linked immunosorbent assay kits were used: one for the determination of Cry9C protein concentration and the other for % StarLink seed. The sampling and analytical variances associated with each step of the Cry9C test procedures were determined for flour and meal. Variances were found to be functions of Cry9C concentration, and regression equations were developed to describe the relationships. Because of the larger particle size, sampling variability associated with cornmeal was about double that for corn flour. For cornmeal, the sampling variance accounted for 92.6% of the total testing variability. The observed sampling and analytical distributions were compared with the Normal distribution. In almost all comparisons, the null hypothesis that the Cry9C protein values were sampled from a Normal distribution could not be rejected at 95% confidence limits. The Normal distribution and the variance estimates were used to evaluate the performance of several Cry9C protein sampling plans for corn flour and meal. Operating characteristic curves were developed and used to demonstrate the effect of increasing sample size on reducing false positives (seller's risk) and false negatives (buyer's risk).

  13. Detection of secondary structure elements in proteins by hydrophobic cluster analysis.

    Science.gov (United States)

    Woodcock, S; Mornon, J P; Henrissat, B

    1992-10-01

    Hydrophobic cluster analysis (HCA) is a protein sequence comparison method based on alpha-helical representations of the sequences where the size, shape and orientation of the clusters of hydrophobic residues are primarily compared. The effectiveness of HCA has been suggested to originate from its potential ability to focus on the residues forming the hydrophobic core of globular proteins. We have addressed the robustness of the bidimensional representation used for HCA in its ability to detect the regular secondary structure elements of proteins. Various parameters have been studied such as those governing cluster size and limits, the hydrophobic residues constituting the clusters as well as the potential shift of the cluster positions with respect to the position of the regular secondary structure elements. The following results have been found to support the alpha-helical bidimensional representation used in HCA: (i) there is a positive correlation (clearly above background noise) between the hydrophobic clusters and the regular secondary structure elements in proteins; (ii) the hydrophobic clusters are centred on the regular secondary structure elements; (iii) the pitch of the helical representation which gives the best correspondence is that of an alpha-helix. The correspondence between hydrophobic clusters and regular secondary structure elements suggests a way to implement variable gap penalties during the automatic alignment of protein sequences.

  14. 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.

  15. On the accuracy of protein determination in large biological samples by prompt gamma neutron activation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kasviki, K. [Institute of Nuclear Technology and Radiation Protection, NCSR ' Demokritos' , Aghia Paraskevi, Attikis 15310 (Greece); Medical Physics Laboratory, Medical School, University of Ioannina, Ioannina 45110 (Greece); Stamatelatos, I.E. [Institute of Nuclear Technology and Radiation Protection, NCSR ' Demokritos' , Aghia Paraskevi, Attikis 15310 (Greece)], E-mail: ion@ipta.demokritos.gr; Yannakopoulou, E. [Institute of Physical Chemistry, NCSR ' Demokritos' , Aghia Paraskevi, Attikis 15310 (Greece); Papadopoulou, P. [Institute of Technology of Agricultural Products, NAGREF, Lycovrissi, Attikis 14123 (Greece); Kalef-Ezra, J. [Medical Physics Laboratory, Medical School, University of Ioannina, Ioannina 45110 (Greece)

    2007-10-15

    A prompt gamma neutron activation analysis (PGNAA) facility has been developed for the determination of nitrogen and thus total protein in large volume biological samples or the whole body of small animals. In the present work, the accuracy of nitrogen determination by PGNAA in phantoms of known composition as well as in four raw ground meat samples of about 1 kg mass was examined. Dumas combustion and Kjeldahl techniques were also used for the assessment of nitrogen concentration in the meat samples. No statistically significant differences were found between the concentrations assessed by the three techniques. The results of this work demonstrate the applicability of PGNAA for the assessment of total protein in biological samples of 0.25-1.5 kg mass, such as a meat sample or the body of small animal even in vivo with an equivalent radiation dose of about 40 mSv.

  16. On the accuracy of protein determination in large biological samples by prompt gamma neutron activation analysis

    International Nuclear Information System (INIS)

    Kasviki, K.; Stamatelatos, I.E.; Yannakopoulou, E.; Papadopoulou, P.; Kalef-Ezra, J.

    2007-01-01

    A prompt gamma neutron activation analysis (PGNAA) facility has been developed for the determination of nitrogen and thus total protein in large volume biological samples or the whole body of small animals. In the present work, the accuracy of nitrogen determination by PGNAA in phantoms of known composition as well as in four raw ground meat samples of about 1 kg mass was examined. Dumas combustion and Kjeldahl techniques were also used for the assessment of nitrogen concentration in the meat samples. No statistically significant differences were found between the concentrations assessed by the three techniques. The results of this work demonstrate the applicability of PGNAA for the assessment of total protein in biological samples of 0.25-1.5 kg mass, such as a meat sample or the body of small animal even in vivo with an equivalent radiation dose of about 40 mSv

  17. Why fibrous proteins are romantic.

    Science.gov (United States)

    Cohen, C

    1998-01-01

    Here I give a personal account of the great history of fibrous protein structure. I describe how Astbury first recognized the essential simplicity of fibrous proteins and their paradigmatic role in protein structure. The poor diffraction patterns yielded by these proteins were then deciphered by Pauling, Crick, Ramachandran and others (in part by model building) to reveal alpha-helical coiled coils, beta-sheets, and the collagen triple helical coiled coil-all characterized by different local sequence periodicities. Longer-range sequence periodicities (or "magic numbers") present in diverse fibrous proteins, such as collagen, tropomyosin, paramyosin, myosin, and were then shown to account for the characteristic axial repeats observed in filaments of these proteins. More recently, analysis of fibrous protein structure has been extended in many cases to atomic resolution, and some systems, such as "leucine zippers," are providing a deeper understanding of protein design than similar studies of globular proteins. In the last sections, I provide some dramatic examples of fibrous protein dynamics. One example is the so-called "spring-loaded" mechanism for viral fusion by the hemagglutinin protein of influenza. Another is the possible conformational changes in prion proteins, implicated in "mad cow disease," which may be related to similar transitions in a variety of globular and fibrous proteins. Copyright 1998 Academic Press.

  18. Sampling-based exploration of folded state of a protein under kinematic and geometric constraints

    KAUST Repository

    Yao, Peggy

    2011-10-04

    Flexibility is critical for a folded protein to bind to other molecules (ligands) and achieve its functions. The conformational selection theory suggests that a folded protein deforms continuously and its ligand selects the most favorable conformations to bind to. Therefore, one of the best options to study protein-ligand binding is to sample conformations broadly distributed over the protein-folded state. This article presents a new sampler, called kino-geometric sampler (KGS). This sampler encodes dominant energy terms implicitly by simple kinematic and geometric constraints. Two key technical contributions of KGS are (1) a robotics-inspired Jacobian-based method to simultaneously deform a large number of interdependent kinematic cycles without any significant break-up of the closure constraints, and (2) a diffusive strategy to generate conformation distributions that diffuse quickly throughout the protein folded state. Experiments on four very different test proteins demonstrate that KGS can efficiently compute distributions containing conformations close to target (e.g., functional) conformations. These targets are not given to KGS, hence are not used to bias the sampling process. In particular, for a lysine-binding protein, KGS was able to sample conformations in both the intermediate and functional states without the ligand, while previous work using molecular dynamics simulation had required the ligand to be taken into account in the potential function. Overall, KGS demonstrates that kino-geometric constraints characterize the folded subset of a protein conformation space and that this subset is small enough to be approximated by a relatively small distribution of conformations. © 2011 Wiley Periodicals, Inc.

  19. Conformational sampling in template-free protein loop structure modeling: an overview.

    Science.gov (United States)

    Li, Yaohang

    2013-01-01

    Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a "mini protein folding problem" under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

  20. CONFORMATIONAL SAMPLING IN TEMPLATE-FREE PROTEIN LOOP STRUCTURE MODELING: AN OVERVIEW

    Directory of Open Access Journals (Sweden)

    Yaohang Li

    2013-02-01

    Full Text Available Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

  1. Studies on voltammetric determination of cadmium in samples containing native and digested proteins

    Energy Technology Data Exchange (ETDEWEB)

    Drozd, Marcin; Pietrzak, Mariusz, E-mail: mariusz@ch.pw.edu.pl; Malinowska, Elżbieta

    2014-03-01

    Highlights: • Proteins exhibit diverse impact on the DPASV cadmium signals. • Proteins subjected to HNO{sub 3} introduce less interference, than the native ones. • Optimal amount of SDS depends on the kind of protein. • Presence of thiolated coating agents of QDs do not influence the analysis. - Abstract: This work focuses on determination of cadmium ions using anodic stripping voltammetry (ASV) on thin film mercury electrode in conditions corresponding to those obtained after digestion of cadmium-based quantum dots and their conjugates. It presents the impact of selected proteins, including potential receptors and surface blocking agents on the voltammetric determination of cadmium. Experiments regarding elimination of interferences related to proteins presence using sodium dodecyl sulfate (SDS) are also shown. Effect of SDS on selected analytical parameters and simplicity of analyses carried out was investigated in the framework of current studies. The significant differences of influence among tested proteins on ASV cadmium determination, as well as the variability in SDS effectiveness as the antifouling agent were observed and explained. This work is especially important for those, who design new bioassays and biosensors with a use of quantum dots as electrochemical labels, as it shows what problems may arise from presence of native and digested proteins in tested samples.

  2. Dynamically polarized samples for neutron protein crystallography at the Spallation Neutron Source

    International Nuclear Information System (INIS)

    Zhao, Jinkui; Pierce, Josh; Robertson, J. L.; Herwig, Kenneth W.; Myles, Dean; Cuneo, Matt; Li, Le; Meilleur, Flora; Standaert, Bob

    2016-01-01

    To prepare for the next generation neutron scattering instruments for the planned second target station at the Spallation Neutron Source (SNS) and to broaden the scientific impact of neutron protein crystallography at the Oak Ridge National Laboratory, we have recently ramped up our efforts to develop a dynamically polarized target for neutron protein crystallography at the SNS. Proteins contain a large amount of hydrogen which contributes to incoherent diffraction background and limits the sensitivity of neutron protein crystallography. This incoherent background can be suppressed by using polarized neutron diffraction, which in the same time also improves the coherent diffraction signal. Our plan is to develop a custom Dynamic Nuclear Polarization (DNP) setup tailored to neutron protein diffraction instruments. Protein crystals will be polarized at a magnetic field of 5 T and temperatures of below 1 K. After the dynamic polarization process, the sample will be brought to a frozen-spin mode in a 0.5 T holding field and at temperatures below 100 mK. In a parallel effort, we are also investigating various ways of incorporating polarization agents needed for DNP, such as site specific spin labels, into protein crystals. (paper)

  3. Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

    Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

  4. Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

    Science.gov (United States)

    Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

    2012-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. PMID:22520159

  5. 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

  6. Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs.

    Directory of Open Access Journals (Sweden)

    Dimitar V Pachov

    2015-07-01

    Full Text Available Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key

  7. Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

    Science.gov (United States)

    Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

    2008-09-01

    Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

  8. imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation.

    Science.gov (United States)

    Zhao, Qun; Fang, Fei; Wu, Ci; Wu, Qi; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-03-17

    An integrated sample preparation method, termed "imFASP", which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50-500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. CASP10-BCL::Fold efficiently samples topologies of large proteins.

    Science.gov (United States)

    Heinze, Sten; Putnam, Daniel K; Fischer, Axel W; Kohlmann, Tim; Weiner, Brian E; Meiler, Jens

    2015-03-01

    During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some β-strand proteins model refinement failed as β-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein. © 2015 Wiley Periodicals, Inc.

  10. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain

    DEFF Research Database (Denmark)

    Pedersen, LB; Birkelund, Svend; Holm, A

    1996-01-01

    The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may......-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel...

  11. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    International Nuclear Information System (INIS)

    Manno, D; Filippo, E; Fiore, R; Serra, A; Urso, E; Rizzello, A; Maffia, M

    2010-01-01

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP C . In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP C at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  12. Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.

    Science.gov (United States)

    Sinclair, John; Timms, John F

    2011-08-01

    Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Nanocapillaries for Open Tubular Chromatographic Separations of Proteins in Femtoliter to Picoliter Samples

    Science.gov (United States)

    Wang, Xiayan; Cheng, Chang; Wang, Shili; Zhao, Meiping; Dasgupta, Purnendu K.; Liu, Shaorong

    2009-01-01

    We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 106 theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 μm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto to picoliter (fL-pL) volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typ. 200 psi) as the pump and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit μL volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, α-lactalbumin, insulin, and α -2-macroglobulin. PMID:19663450

  14. Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples

    DEFF Research Database (Denmark)

    Poulsen, Nicklas N; Andersen, Nina Z; Østergaard, Jesper

    2015-01-01

    Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject...... to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system...... in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator...

  15. Quantifying Protein-Carbohydrate Interactions Using Liquid Sample Desorption Electrospray Ionization Mass Spectrometry

    Science.gov (United States)

    Yao, Yuyu; Shams-Ud-Doha, Km; Daneshfar, Rambod; Kitova, Elena N.; Klassen, John S.

    2015-01-01

    The application of liquid sample desorption electrospray ionization mass spectrometry (liquid sample DESI-MS) for quantifying protein-carbohydrate interactions in vitro is described. Association constants for the interactions between lysozyme and β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc and β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc, and between a single chain antibody and α-D-Galp-(1 → 2)-[α-D-Abep-(1 → 3)]-α-D-Manp-OCH3 and β-D-Glcp-(1 → 2)-[α-D-Abep-(1 → 3)]-α-D-Manp-OCH3 measured using liquid sample DESI-MS were found to be in good agreement with values measured by isothermal titration calorimetry and the direct ESI-MS assay. The reference protein method, which was originally developed to correct ESI mass spectra for the occurrence of nonspecific ligand-protein binding, was shown to reliably correct liquid sample DESI mass spectra for nonspecific binding. The suitability of liquid sample DESI-MS for quantitative binding measurements carried out using solutions containing high concentrations of the nonvolatile biological buffer phosphate buffered saline (PBS) was also explored. Binding of lysozyme to β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc in aqueous solutions containing up to 1× PBS was successfully monitored using liquid sample DESI-MS; with ESI-MS the binding measurements were limited to concentrations less than 0.02 X PBS.

  16. The challenging measurement of protein in complex biomass-derived samples

    DEFF Research Database (Denmark)

    Haven, M.O.; Jørgensen, H.

    2014-01-01

    and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay - alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant...... that the presence of cellulose, lignin and glucose (above 50 g/kg) could significantly affect the results of the assay. Comparison of analyses performed with the ninhydrin assay and with a CN analyser revealed that there was good agreement between these two analytical methods, but care has to be taken when applying...... the ninhydrin assay. If used correctly, the ninhydrin assay can be used as a fast method to evaluate the adsorption of cellulases to lignin....

  17. Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

    Science.gov (United States)

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

    Directory of Open Access Journals (Sweden)

    Christina Looße

    2015-06-01

    Full Text Available Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4 comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m. deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.

  19. Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

    KAUST Repository

    Schrittwieser, Stefan

    2017-06-30

    Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.

  20. Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

    KAUST Repository

    Schrittwieser, Stefan; Pelaz, Beatriz; Parak, Wolfgang J.; Lentijo Mozo, Sergio; Soulantica, Katerina; Dieckhoff, Jan; Ludwig, Frank; Schotter, Joerg

    2017-01-01

    Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.

  1. Sulfonate-terminated carbosilane dendron-coated nanotubes: a greener point of view in protein sample preparation.

    Science.gov (United States)

    González-García, Estefanía; Gutiérrez Ulloa, Carlos E; de la Mata, Francisco Javier; Marina, María Luisa; García, María Concepción

    2017-09-01

    Reduction or removal of solvents and reagents in protein sample preparation is a requirement. Dendrimers can strongly interact with proteins and have great potential as a greener alternative to conventional methods used in protein sample preparation. This work proposes the use of single-walled carbon nanotubes (SWCNTs) functionalized with carbosilane dendrons with sulfonate groups for protein sample preparation and shows the successful application of the proposed methodology to extract proteins from a complex matrix. SEM images of nanotubes and mixtures of nanotubes and proteins were taken. Moreover, intrinsic fluorescence intensity of proteins was monitored to observe the most significant interactions at increasing dendron generations under neutral and basic pHs. Different conditions for the disruption of interactions between proteins and nanotubes after protein extraction and different concentrations of the disrupting reagent and the nanotube were also tried. Compatibility of extraction and disrupting conditions with the enzymatic digestion of proteins for obtaining bioactive peptides was also studied. Finally, sulfonate-terminated carbosilane dendron-coated SWCNTs enabled the extraction of proteins from a complex sample without using non-environmentally friendly solvents that were required so far. Graphical Abstract Green protein extraction from a complex sample employing carbosilane dendron coated nanotubes.

  2. A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Jeremy Potriquet

    Full Text Available To facilitate high-throughput proteomic analyses we have developed a modified FASP protocol which improves the rate at which protein samples can be processed prior to mass spectrometry. Adapting the original FASP protocol to a 96-well format necessitates extended spin times for buffer exchange due to the low centrifugation speeds tolerated by these devices. However, by using 96-well plates with a more robust polyethersulfone molecular weight cutoff membrane, instead of the cellulose membranes typically used in these devices, we could use isopropanol as a wetting agent, decreasing spin times required for buffer exchange from an hour to 30 minutes. In a typical work flow used in our laboratory this equates to a reduction of 3 hours per plate, providing processing times similar to FASP for the processing of up to 96 samples per plate. To test whether our modified protocol produced similar results to FASP and other FASP-like protocols we compared the performance of our modified protocol to the original FASP and the more recently described eFASP and MStern-blot. We show that all FASP-like methods, including our modified protocol, display similar performance in terms of proteins identified and reproducibility. Our results show that our modified FASP protocol is an efficient method for the high-throughput processing of protein samples for mass spectral analysis.

  3. The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

    Energy Technology Data Exchange (ETDEWEB)

    Thompson,J.; Ryan, Z.; Salisbury, J.; Kumar, R.

    2006-01-01

    Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered {alpha}-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an {alpha}-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.

  4. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

    Science.gov (United States)

    Fischer, Roman; Kessler, Benedikt M

    2015-04-01

    We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. High-throughput simultaneous analysis of RNA, protein, and lipid biomarkers in heterogeneous tissue samples.

    Science.gov (United States)

    Reiser, Vladimír; Smith, Ryan C; Xue, Jiyan; Kurtz, Marc M; Liu, Rong; Legrand, Cheryl; He, Xuanmin; Yu, Xiang; Wong, Peggy; Hinchcliffe, John S; Tanen, Michael R; Lazar, Gloria; Zieba, Renata; Ichetovkin, Marina; Chen, Zhu; O'Neill, Edward A; Tanaka, Wesley K; Marton, Matthew J; Liao, Jason; Morris, Mark; Hailman, Eric; Tokiwa, George Y; Plump, Andrew S

    2011-11-01

    With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD). We used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured. The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting. The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.

  6. FRAGSION: ultra-fast protein fragment library generation by IOHMM sampling.

    Science.gov (United States)

    Bhattacharya, Debswapna; Adhikari, Badri; Li, Jilong; Cheng, Jianlin

    2016-07-01

    Speed, accuracy and robustness of building protein fragment library have important implications in de novo protein structure prediction since fragment-based methods are one of the most successful approaches in template-free modeling (FM). Majority of the existing fragment detection methods rely on database-driven search strategies to identify candidate fragments, which are inherently time-consuming and often hinder the possibility to locate longer fragments due to the limited sizes of databases. Also, it is difficult to alleviate the effect of noisy sequence-based predicted features such as secondary structures on the quality of fragment. Here, we present FRAGSION, a database-free method to efficiently generate protein fragment library by sampling from an Input-Output Hidden Markov Model. FRAGSION offers some unique features compared to existing approaches in that it (i) is lightning-fast, consuming only few seconds of CPU time to generate fragment library for a protein of typical length (300 residues); (ii) can generate dynamic-size fragments of any length (even for the whole protein sequence) and (iii) offers ways to handle noise in predicted secondary structure during fragment sampling. On a FM dataset from the most recent Critical Assessment of Structure Prediction, we demonstrate that FGRAGSION provides advantages over the state-of-the-art fragment picking protocol of ROSETTA suite by speeding up computation by several orders of magnitude while achieving comparable performance in fragment quality. Source code and executable versions of FRAGSION for Linux and MacOS is freely available to non-commercial users at http://sysbio.rnet.missouri.edu/FRAGSION/ It is bundled with a manual and example data. chengji@missouri.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Efficacy of independence sampling in replica exchange simulations of ordered and disordered proteins.

    Science.gov (United States)

    Lee, Kuo Hao; Chen, Jianhan

    2017-11-15

    Recasting temperature replica exchange (T-RE) as a special case of Gibbs sampling has led to a simple and efficient scheme for enhanced mixing (Chodera and Shirts, J. Chem. Phys., 2011, 135, 194110). To critically examine if T-RE with independence sampling (T-REis) improves conformational sampling, we performed T-RE and T-REis simulations of ordered and disordered proteins using coarse-grained and atomistic models. The results demonstrate that T-REis effectively increase the replica mobility in temperatures space with minimal computational overhead, especially for folded proteins. However, enhanced mixing does not translate well into improved conformational sampling. The convergences of thermodynamic properties interested are similar, with slight improvements for T-REis of ordered systems. The study re-affirms the efficiency of T-RE does not appear to be limited by temperature diffusion, but by the inherent rates of spontaneous large-scale conformational re-arrangements. Due to its simplicity and efficacy of enhanced mixing, T-REis is expected to be more effective when incorporated with various Hamiltonian-RE protocols. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Atomic resolution structure of cucurmosin, a novel type 1 ribosome-inactivating protein from the sarcocarp of Cucurbita moschata

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Xiaomin; Meehan, Edward J.; Xie, Jieming; Huang, Mingdong; Chen, Minghuang; Chen, Liqing (UAH); (Fujian); (Chinese Aca. Sci.)

    2008-10-27

    A novel type 1 ribosome-inactivating protein (RIP) designated cucurmosin was isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04 {angstrom}, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven {alpha}-helices and eight {beta}-strands, and a smaller C-terminal domain consisting of three {alpha}-helices and two {beta}-strands. The high resolution structure established a glycosylation pattern of GlcNAc{sub 2}Man3Xyl. Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. The structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed.

  9. Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation.

    Science.gov (United States)

    Iida, Shinji; Nakamura, Haruki; Higo, Junichi

    2016-06-15

    We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein-protein or protein-ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks. © 2016 The Author(s).

  10. Crude protein, fibre and phytic acid in vitro digestibility of selected legume and buckwheat samples

    Directory of Open Access Journals (Sweden)

    Petra Vojtíšková

    2013-01-01

    Full Text Available The aim of this study was to determine crude protein, fibre and phytic acid in vitro digestibility of selected legumes and buckwheat products. All analyses except the phytic acid contents were performed in the line with the Commission Regulation (EC No. 152/2009. A modified version of Holt’s Method was used for phytic acid (phytate determination. None of all samples contained more than 11% of moisture. Soybeans are rich in crude protein; they contain nearly 40% of this compound. The content of crude protein in buckwheat flours was about 14%. The highest amount of phytate was found in common beans and soybeans-about 2 g/100 g of dry matter. On the other hand, the lowest phytate content was observed in buckwheat pasta (F. esculentum groats was 1.9 g per 100 g of dry matter. In vitro digestibility was determined using an incubator Daisy and pepsin enzymes and the combination of pepsin and pancreatin. The highest coefficient of crude protein digestibility was discovered to be in peels and wholemeal flour. The greatest fibre digestibility coefficients were obtained for peels, which contain about 65% of fibre in their dry matter. When pepsin was used, a higher phytic acid digestibility coefficient for G. max, Ph. vulgaris, peels, flour, groats and broken groats was observed; while when the combination of pepsin and pancreatin was used, higher phytic acid digestibility coefficients for peas, lentil and wholemeal flour were observed.

  11. Sampling

    CERN Document Server

    Thompson, Steven K

    2012-01-01

    Praise for the Second Edition "This book has never had a competitor. It is the only book that takes a broad approach to sampling . . . any good personal statistics library should include a copy of this book." —Technometrics "Well-written . . . an excellent book on an important subject. Highly recommended." —Choice "An ideal reference for scientific researchers and other professionals who use sampling." —Zentralblatt Math Features new developments in the field combined with all aspects of obtaining, interpreting, and using sample data Sampling provides an up-to-date treat

  12. Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach.

    Science.gov (United States)

    Santa, Cátia; Anjo, Sandra I; Manadas, Bruno

    2016-07-01

    Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH-MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Probabilistic Equilibrium Sampling of Protein Structures from SAXS Data and a Coarse Grained Debye Formula

    DEFF Research Database (Denmark)

    Andreetta, Christian

    -likelihood estimators for the form factors employed in the Debye formula, a theoretical forward model for SAXS profiles. The resulting computation compares favorably with the state of the art tool in the field, the program CRYSOL in the suite ATSAS. A faster, parallel implementation on Graphical Processor Units (GPUs......The present work describes the design and the implementation of a protocol for arbitrary precision computation of Small Angle X-ray Scattering (SAXS) profiles, and its inclusion in a probabilistic framework for protein structure determination. This protocol identifies a set of maximum...... of protein structures all fitting the experimental data. For the first time, we describe in full atomic detail a set of different conformations attainable by flexible polypeptides in solution. This method is not limited by assumptions in shape or size of the samples. It allows therefore to investigate...

  14. Surface adsorption of lattice HP proteins: Thermodynamics and structural transitions using Wang-Landau sampling

    International Nuclear Information System (INIS)

    Li Yingwai; Landau, David P; Wüst, Thomas

    2012-01-01

    Wang-Landau sampling has been applied to investigate the thermodynamics and structural properties of a lattice hydrophobic-polar heteropolymer (the HP protein model) interacting with an attractive substrate. For simplicity, we consider a short HP sequence consisting of only 36 monomers interacting with a substrate which attracts all monomers in the sequence. The conformational “phase transitions” have been identified by a canonical analysis of the specific heat and suitable structural observables. Three major “transitions”, namely, adsorption, hydrophobic core formation and “flattening” of adsorbed structures, are observed. Depending on the surface attractive strength relative to the intra-protein attraction among the H monomers, these processes take place in different sequences upon cooling.

  15. Selection of peptides interfering with protein-protein interaction.

    Science.gov (United States)

    Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

    2009-01-01

    Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

  16. Assessing protein conformational sampling methods based on bivariate lag-distributions of backbone angles

    KAUST Repository

    Maadooliat, Mehdi; Gao, Xin; Huang, Jianhua Z.

    2012-01-01

    Despite considerable progress in the past decades, protein structure prediction remains one of the major unsolved problems in computational biology. Angular-sampling-based methods have been extensively studied recently due to their ability to capture the continuous conformational space of protein structures. The literature has focused on using a variety of parametric models of the sequential dependencies between angle pairs along the protein chains. In this article, we present a thorough review of angular-sampling-based methods by assessing three main questions: What is the best distribution type to model the protein angles? What is a reasonable number of components in a mixture model that should be considered to accurately parameterize the joint distribution of the angles? and What is the order of the local sequence-structure dependency that should be considered by a prediction method? We assess the model fits for different methods using bivariate lag-distributions of the dihedral/planar angles. Moreover, the main information across the lags can be extracted using a technique called Lag singular value decomposition (LagSVD), which considers the joint distribution of the dihedral/planar angles over different lags using a nonparametric approach and monitors the behavior of the lag-distribution of the angles using singular value decomposition. As a result, we developed graphical tools and numerical measurements to compare and evaluate the performance of different model fits. Furthermore, we developed a web-tool (http://www.stat.tamu. edu/~madoliat/LagSVD) that can be used to produce informative animations. © The Author 2012. Published by Oxford University Press.

  17. Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

    Science.gov (United States)

    Hong, Xinguo; Hao, Quan

    2009-01-01

    In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan (2D mode), with an interval as small as 1.5 times the beam size, at low temperature (e.g., 4 °C). A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio (better than the reported data on the same protein using a flow cell). The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

  18. Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

    International Nuclear Information System (INIS)

    Hong Xinguo; Hao Quan

    2009-01-01

    In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan (2D mode), with an interval as small as 1.5 times the beam size, at low temperature (e.g., 4 deg. C). A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio (better than the reported data on the same protein using a flow cell). The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

  19. Assessing protein conformational sampling methods based on bivariate lag-distributions of backbone angles

    KAUST Repository

    Maadooliat, Mehdi

    2012-08-27

    Despite considerable progress in the past decades, protein structure prediction remains one of the major unsolved problems in computational biology. Angular-sampling-based methods have been extensively studied recently due to their ability to capture the continuous conformational space of protein structures. The literature has focused on using a variety of parametric models of the sequential dependencies between angle pairs along the protein chains. In this article, we present a thorough review of angular-sampling-based methods by assessing three main questions: What is the best distribution type to model the protein angles? What is a reasonable number of components in a mixture model that should be considered to accurately parameterize the joint distribution of the angles? and What is the order of the local sequence-structure dependency that should be considered by a prediction method? We assess the model fits for different methods using bivariate lag-distributions of the dihedral/planar angles. Moreover, the main information across the lags can be extracted using a technique called Lag singular value decomposition (LagSVD), which considers the joint distribution of the dihedral/planar angles over different lags using a nonparametric approach and monitors the behavior of the lag-distribution of the angles using singular value decomposition. As a result, we developed graphical tools and numerical measurements to compare and evaluate the performance of different model fits. Furthermore, we developed a web-tool (http://www.stat.tamu. edu/~madoliat/LagSVD) that can be used to produce informative animations. © The Author 2012. Published by Oxford University Press.

  20. Near-native protein loop sampling using nonparametric density estimation accommodating sparcity.

    Science.gov (United States)

    Joo, Hyun; Chavan, Archana G; Day, Ryan; Lennox, Kristin P; Sukhanov, Paul; Dahl, David B; Vannucci, Marina; Tsai, Jerry

    2011-10-01

    Unlike the core structural elements of a protein like regular secondary structure, template based modeling (TBM) has difficulty with loop regions due to their variability in sequence and structure as well as the sparse sampling from a limited number of homologous templates. We present a novel, knowledge-based method for loop sampling that leverages homologous torsion angle information to estimate a continuous joint backbone dihedral angle density at each loop position. The φ,ψ distributions are estimated via a Dirichlet process mixture of hidden Markov models (DPM-HMM). Models are quickly generated based on samples from these distributions and were enriched using an end-to-end distance filter. The performance of the DPM-HMM method was evaluated against a diverse test set in a leave-one-out approach. Candidates as low as 0.45 Å RMSD and with a worst case of 3.66 Å were produced. For the canonical loops like the immunoglobulin complementarity-determining regions (mean RMSD 7.0 Å), this sampling method produces a population of loop structures to around 3.66 Å for loops up to 17 residues. In a direct test of sampling to the Loopy algorithm, our method demonstrates the ability to sample nearer native structures for both the canonical CDRH1 and non-canonical CDRH3 loops. Lastly, in the realistic test conditions of the CASP9 experiment, successful application of DPM-HMM for 90 loops from 45 TBM targets shows the general applicability of our sampling method in loop modeling problem. These results demonstrate that our DPM-HMM produces an advantage by consistently sampling near native loop structure. The software used in this analysis is available for download at http://www.stat.tamu.edu/~dahl/software/cortorgles/.

  1. Near-native protein loop sampling using nonparametric density estimation accommodating sparcity.

    Directory of Open Access Journals (Sweden)

    Hyun Joo

    2011-10-01

    Full Text Available Unlike the core structural elements of a protein like regular secondary structure, template based modeling (TBM has difficulty with loop regions due to their variability in sequence and structure as well as the sparse sampling from a limited number of homologous templates. We present a novel, knowledge-based method for loop sampling that leverages homologous torsion angle information to estimate a continuous joint backbone dihedral angle density at each loop position. The φ,ψ distributions are estimated via a Dirichlet process mixture of hidden Markov models (DPM-HMM. Models are quickly generated based on samples from these distributions and were enriched using an end-to-end distance filter. The performance of the DPM-HMM method was evaluated against a diverse test set in a leave-one-out approach. Candidates as low as 0.45 Å RMSD and with a worst case of 3.66 Å were produced. For the canonical loops like the immunoglobulin complementarity-determining regions (mean RMSD 7.0 Å, this sampling method produces a population of loop structures to around 3.66 Å for loops up to 17 residues. In a direct test of sampling to the Loopy algorithm, our method demonstrates the ability to sample nearer native structures for both the canonical CDRH1 and non-canonical CDRH3 loops. Lastly, in the realistic test conditions of the CASP9 experiment, successful application of DPM-HMM for 90 loops from 45 TBM targets shows the general applicability of our sampling method in loop modeling problem. These results demonstrate that our DPM-HMM produces an advantage by consistently sampling near native loop structure. The software used in this analysis is available for download at http://www.stat.tamu.edu/~dahl/software/cortorgles/.

  2. Near-Native Protein Loop Sampling Using Nonparametric Density Estimation Accommodating Sparcity

    Science.gov (United States)

    Day, Ryan; Lennox, Kristin P.; Sukhanov, Paul; Dahl, David B.; Vannucci, Marina; Tsai, Jerry

    2011-01-01

    Unlike the core structural elements of a protein like regular secondary structure, template based modeling (TBM) has difficulty with loop regions due to their variability in sequence and structure as well as the sparse sampling from a limited number of homologous templates. We present a novel, knowledge-based method for loop sampling that leverages homologous torsion angle information to estimate a continuous joint backbone dihedral angle density at each loop position. The φ,ψ distributions are estimated via a Dirichlet process mixture of hidden Markov models (DPM-HMM). Models are quickly generated based on samples from these distributions and were enriched using an end-to-end distance filter. The performance of the DPM-HMM method was evaluated against a diverse test set in a leave-one-out approach. Candidates as low as 0.45 Å RMSD and with a worst case of 3.66 Å were produced. For the canonical loops like the immunoglobulin complementarity-determining regions (mean RMSD 7.0 Å), this sampling method produces a population of loop structures to around 3.66 Å for loops up to 17 residues. In a direct test of sampling to the Loopy algorithm, our method demonstrates the ability to sample nearer native structures for both the canonical CDRH1 and non-canonical CDRH3 loops. Lastly, in the realistic test conditions of the CASP9 experiment, successful application of DPM-HMM for 90 loops from 45 TBM targets shows the general applicability of our sampling method in loop modeling problem. These results demonstrate that our DPM-HMM produces an advantage by consistently sampling near native loop structure. The software used in this analysis is available for download at http://www.stat.tamu.edu/~dahl/software/cortorgles/. PMID:22028638

  3. Toward structural dynamics: protein motions viewed by chemical shift modulations and direct detection of C'N multiple-quantum relaxation.

    Science.gov (United States)

    Mori, Mirko; Kateb, Fatiha; Bodenhausen, Geoffrey; Piccioli, Mario; Abergel, Daniel

    2010-03-17

    Multiple quantum relaxation in proteins reveals unexpected relationships between correlated or anti-correlated conformational backbone dynamics in alpha-helices or beta-sheets. The contributions of conformational exchange to the relaxation rates of C'N coherences (i.e., double- and zero-quantum coherences involving backbone carbonyl (13)C' and neighboring amide (15)N nuclei) depend on the kinetics of slow exchange processes, as well as on the populations of the conformations and chemical shift differences of (13)C' and (15)N nuclei. The relaxation rates of C'N coherences, which reflect concerted fluctuations due to slow chemical shift modulations (CSMs), were determined by direct (13)C detection in diamagnetic and paramagnetic proteins. In well-folded proteins such as lanthanide-substituted calbindin (CaLnCb), copper,zinc superoxide dismutase (Cu,Zn SOD), and matrix metalloproteinase (MMP12), slow conformational exchange occurs along the entire backbone. Our observations demonstrate that relaxation rates of C'N coherences arising from slow backbone dynamics have positive signs (characteristic of correlated fluctuations) in beta-sheets and negative signs (characteristic of anti-correlated fluctuations) in alpha-helices. This extends the prospects of structure-dynamics relationships to slow time scales that are relevant for protein function and enzymatic activity.

  4. Calculation of absolute protein-ligand binding free energy using distributed replica sampling.

    Science.gov (United States)

    Rodinger, Tomas; Howell, P Lynne; Pomès, Régis

    2008-10-21

    Distributed replica sampling [T. Rodinger et al., J. Chem. Theory Comput. 2, 725 (2006)] is a simple and general scheme for Boltzmann sampling of conformational space by computer simulation in which multiple replicas of the system undergo a random walk in reaction coordinate or temperature space. Individual replicas are linked through a generalized Hamiltonian containing an extra potential energy term or bias which depends on the distribution of all replicas, thus enforcing the desired sampling distribution along the coordinate or parameter of interest regardless of free energy barriers. In contrast to replica exchange methods, efficient implementation of the algorithm does not require synchronicity of the individual simulations. The algorithm is inherently suited for large-scale simulations using shared or heterogeneous computing platforms such as a distributed network. In this work, we build on our original algorithm by introducing Boltzmann-weighted jumping, which allows moves of a larger magnitude and thus enhances sampling efficiency along the reaction coordinate. The approach is demonstrated using a realistic and biologically relevant application; we calculate the standard binding free energy of benzene to the L99A mutant of T4 lysozyme. Distributed replica sampling is used in conjunction with thermodynamic integration to compute the potential of mean force for extracting the ligand from protein and solvent along a nonphysical spatial coordinate. Dynamic treatment of the reaction coordinate leads to faster statistical convergence of the potential of mean force than a conventional static coordinate, which suffers from slow transitions on a rugged potential energy surface.

  5. Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure.

    Science.gov (United States)

    Cobb, Nathan J; Sönnichsen, Frank D; McHaourab, Hassane; Surewicz, Witold K

    2007-11-27

    Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and alpha-helical prion protein, PrP(C), to the beta-sheet-rich PrP(Sc). This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP(C) monomer, structures of the pathogenic PrP(Sc) or synthetic PrP(Sc)-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP(C). Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrP(C) involves major refolding of the C-terminal alpha-helical region. The core of the amyloid maps to C-terminal residues from approximately 160-220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of beta-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to beta-structure.

  6. Design of a minimal protein oligomerization domain by a structural approach.

    Science.gov (United States)

    Burkhard, P; Meier, M; Lustig, A

    2000-12-01

    Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.

  7. Structural and thermodynamic studies of the tobacco calmodulin-like rgs-CaM protein.

    Science.gov (United States)

    Makiyama, Rodrigo K; Fernandes, Carlos A H; Dreyer, Thiago R; Moda, Bruno S; Matioli, Fabio F; Fontes, Marcos R M; Maia, Ivan G

    2016-11-01

    The tobacco calmodulin-like protein rgs-CaM is involved in host defense against virus and is reported to possess an associated RNA silencing suppressor activity. Rgs-CaM is also believed to act as an antiviral factor by interacting and targeting viral silencing suppressors for autophagic degradation. Despite these functional data, calcium interplay in the modulation of rgs-CaM is still poorly understood. Here we show that rgs-CaM displays a prevalent alpha-helical conformation and possesses three functional Ca 2+ -binding sites. Using computational modeling and molecular dynamics simulation, we demonstrate that Ca 2+ binding to rgs-CaM triggers expansion of its tertiary structure with reorientation of alpha-helices within the EF-hands. This conformational change leads to the exposure of a large negatively charged region that may be implicated in the electrostatic interactions between rgs-CaM and viral suppressors. Moreover, the k d values obtained for Ca 2+ binding to the three functional sites are not within the affinity range of a typical Ca 2+ sensor. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    Science.gov (United States)

    Sheen, Hyukho

    2016-04-01

    Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

    Science.gov (United States)

    Mullins, Christina S; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

    2016-01-01

    A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

  10. Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

    Directory of Open Access Journals (Sweden)

    Christina S Mullins

    Full Text Available A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC.The expression of HERV-H Gag proteins (Gag-H was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium.Taken together, the Gag-H antibody clone(s present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

  11. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    Energy Technology Data Exchange (ETDEWEB)

    Manno, D; Filippo, E; Fiore, R; Serra, A [Dipartimento di Scienza dei Materiali, Universita del Salento, Lecce (Italy); Urso, E; Rizzello, A; Maffia, M [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita del Salento, Lecce (Italy)

    2010-04-23

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP{sup C}. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP{sup C} at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  12. Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples

    Science.gov (United States)

    Mullins, Christina S.; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

    2016-01-01

    Introduction A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). Results The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Conclusion Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut. PMID:27119520

  13. Adaptive enhanced sampling with a path-variable for the simulation of protein folding and aggregation

    Science.gov (United States)

    Peter, Emanuel K.

    2017-12-01

    In this article, we present a novel adaptive enhanced sampling molecular dynamics (MD) method for the accelerated simulation of protein folding and aggregation. We introduce a path-variable L based on the un-biased momenta p and displacements dq for the definition of the bias s applied to the system and derive 3 algorithms: general adaptive bias MD, adaptive path-sampling, and a hybrid method which combines the first 2 methodologies. Through the analysis of the correlations between the bias and the un-biased gradient in the system, we find that the hybrid methodology leads to an improved force correlation and acceleration in the sampling of the phase space. We apply our method on SPC/E water, where we find a conservation of the average water structure. We then use our method to sample dialanine and the folding of TrpCage, where we find a good agreement with simulation data reported in the literature. Finally, we apply our methodologies on the initial stages of aggregation of a hexamer of Alzheimer's amyloid β fragment 25-35 (Aβ 25-35) and find that transitions within the hexameric aggregate are dominated by entropic barriers, while we speculate that especially the conformation entropy plays a major role in the formation of the fibril as a rate limiting factor.

  14. {sup 137}Cs Absorption factors (Afs) some vegetable and protein samples in cooking

    Energy Technology Data Exchange (ETDEWEB)

    Malek, M A [Bangladesh Atomic Energy Commission, Dhaka (Bangladesh); Nakahara, M [National Institute of Radiological Sciences, Hitachinaka City (Japan)

    2004-07-01

    Full text: The human race uses fresh water for cooking, drinking and washing purposes. The source of fresh water may be radioactively contaminated by various sources of contaminants. With the increased use of radioisotopes, nuclear testing, possible nuclear warfare and terror activities, the apprehension of widespread contamination of the surface and ground water is increasing day by day. In the case of widespread contamination of freshwater sources, people may be compelled to use the contaminated water. The radionuclides in fresh water may enter the human body through two major routes: drinking and cooking food with the water. During cooking, the radionuclide present in the water may be transferred to the various ingredients of the cooked dish. The degree of contamination in the ingredients during the cooking depends on the absorption power of the individual ingredients and on the level of radionuclide present in the water. The ratio of the concentration of the radionuclide absorbed in the ingredients to the concentration in the water can be designated as the 'Absorption factor' (Af). A consumer always has the choice of eating either the whole dish or a part of the dish. The Af of every consumed ingredient can be used to predict the radionuclide absorbed by the individual ingredients cooked with contaminated water, and as such to predict the dose to the consumer. The factor can also be used to assess the dispersion of radionuclide from the water used in cooking to the ingredients in the cooked dish. A better understanding of variables in the cooking that affect the Af in various ingredients is central to deriving the contamination level of the ingredients. To the best of our knowledge, no investigation on this topic has been conducted before. In order to assess this topic and for obtaining base line data, a research project was undertaken to determine the Afs of some vegetable and protein samples, and to investigate the effects of salinity and cooling on Af

  15. A sampling framework for incorporating quantitative mass spectrometry data in protein interaction analysis.

    Science.gov (United States)

    Tucker, George; Loh, Po-Ru; Berger, Bonnie

    2013-10-04

    Comprehensive protein-protein interaction (PPI) maps are a powerful resource for uncovering the molecular basis of genetic interactions and providing mechanistic insights. Over the past decade, high-throughput experimental techniques have been developed to generate PPI maps at proteome scale, first using yeast two-hybrid approaches and more recently via affinity purification combined with mass spectrometry (AP-MS). Unfortunately, data from both protocols are prone to both high false positive and false negative rates. To address these issues, many methods have been developed to post-process raw PPI data. However, with few exceptions, these methods only analyze binary experimental data (in which each potential interaction tested is deemed either observed or unobserved), neglecting quantitative information available from AP-MS such as spectral counts. We propose a novel method for incorporating quantitative information from AP-MS data into existing PPI inference methods that analyze binary interaction data. Our approach introduces a probabilistic framework that models the statistical noise inherent in observations of co-purifications. Using a sampling-based approach, we model the uncertainty of interactions with low spectral counts by generating an ensemble of possible alternative experimental outcomes. We then apply the existing method of choice to each alternative outcome and aggregate results over the ensemble. We validate our approach on three recent AP-MS data sets and demonstrate performance comparable to or better than state-of-the-art methods. Additionally, we provide an in-depth discussion comparing the theoretical bases of existing approaches and identify common aspects that may be key to their performance. Our sampling framework extends the existing body of work on PPI analysis using binary interaction data to apply to the richer quantitative data now commonly available through AP-MS assays. This framework is quite general, and many enhancements are likely

  16. Effective sampling range of a synthetic protein-based attractant for Ceratitis capitata (Diptera: Tephritidae).

    Science.gov (United States)

    Epsky, Nancy D; Espinoza, Hernán R; Kendra, Paul E; Abernathy, Robert; Midgarden, David; Heath, Robert R

    2010-10-01

    Studies were conducted in Honduras to determine effective sampling range of a female-targeted protein-based synthetic attractant for the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Multilure traps were baited with ammonium acetate, putrescine, and trimethylamine lures (three-component attractant) and sampled over eight consecutive weeks. Field design consisted of 38 traps (over 0.5 ha) placed in a combination of standard and high-density grids to facilitate geostatistical analysis, and tests were conducted in coffee (Coffea arabica L.),mango (Mangifera indica L.),and orthanique (Citrus sinensis X Citrus reticulata). Effective sampling range, as determined from the range parameter obtained from experimental variograms that fit a spherical model, was approximately 30 m for flies captured in tests in coffee or mango and approximately 40 m for flies captured in orthanique. For comparison, a release-recapture study was conducted in mango using wild (field-collected) mixed sex C. capitata and an array of 20 baited traps spaced 10-50 m from the release point. Contour analysis was used to document spatial distribution of fly recaptures and to estimate effective sampling range, defined by the area that encompassed 90% of the recaptures. With this approach, effective range of the three-component attractant was estimated to be approximately 28 m, similar to results obtained from variogram analysis. Contour maps indicated that wind direction had a strong influence on sampling range, which was approximately 15 m greater upwind compared with downwind from the release point. Geostatistical analysis of field-captured insects in appropriately designed trapping grids may provide a supplement or alternative to release-recapture studies to estimate sampling ranges for semiochemical-based trapping systems.

  17. Structure of an odorant-binding protein from the mosquito Aedes aegypti suggests a binding pocket covered by a pH-sensitive "Lid".

    Directory of Open Access Journals (Sweden)

    Ney Ribeiro Leite

    Full Text Available BACKGROUND: The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. METHODOLOGY: Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 A resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. CONCLUSION: The structure of AaegOBP1 ( = AaegOBP39 shares the common fold of insect OBPs with six alpha-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors. A C-terminal loop covers the binding cavity and this "lid" may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

  18. Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique.

    Science.gov (United States)

    Gama, Fernanda Gomes Velasque; Santana, Aureo Evangelista; Filho, Eugênio de Campos; Nogueira, Cláudia Aparecida da Silva

    2007-03-01

    Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.

  19. Bottom–up protein identifications from microliter quantities of individual human tear samples. Important steps towards clinical relevance.

    Directory of Open Access Journals (Sweden)

    Peter Raus

    2015-12-01

    With 375 confidently identified proteins in the healthy adult tear, the obtained results are comprehensive and in large agreement with previously published observations on pooled samples of multiple patients. We conclude that, to a limited extent, bottom–up tear protein identifications from individual patients may have clinical relevance.

  20. 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

  1. Adaptive local learning in sampling based motion planning for protein folding.

    Science.gov (United States)

    Ekenna, Chinwe; Thomas, Shawna; Amato, Nancy M

    2016-08-01

    Simulating protein folding motions is an important problem in computational biology. Motion planning algorithms, such as Probabilistic Roadmap Methods, have been successful in modeling the folding landscape. Probabilistic Roadmap Methods and variants contain several phases (i.e., sampling, connection, and path extraction). Most of the time is spent in the connection phase and selecting which variant to employ is a difficult task. Global machine learning has been applied to the connection phase but is inefficient in situations with varying topology, such as those typical of folding landscapes. We develop a local learning algorithm that exploits the past performance of methods within the neighborhood of the current connection attempts as a basis for learning. It is sensitive not only to different types of landscapes but also to differing regions in the landscape itself, removing the need to explicitly partition the landscape. We perform experiments on 23 proteins of varying secondary structure makeup with 52-114 residues. We compare the success rate when using our methods and other methods. We demonstrate a clear need for learning (i.e., only learning methods were able to validate against all available experimental data) and show that local learning is superior to global learning producing, in many cases, significantly higher quality results than the other methods. We present an algorithm that uses local learning to select appropriate connection methods in the context of roadmap construction for protein folding. Our method removes the burden of deciding which method to use, leverages the strengths of the individual input methods, and it is extendable to include other future connection methods.

  2. 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.

  3. Identification of proteins from cambium tissues of the chinese white poplar (populus tomentosa) sampled during the growing season

    International Nuclear Information System (INIS)

    Xie, J.; Liu, S.; Qi, Q.; Hou, Y.

    2014-01-01

    Various protein extraction methods have been used to investigate Chinese white poplar (Populus tomentosa) proteomics. However, extracting and characterizing proteins from woody plants remains a challenge. Two-dimensional gel electrophoresis is a powerful, widely used method for the analysis of complex protein mixtures extracted from biological samples. The technique separates mixtures of proteins along two dimensions, by isoelectric point and molecular weight, and can resolve thousands of different proteins. Here, we report a new application of two-dimensional gel electrophoresis to investigate the proteomics of P. tomentosa cambium tissues over the course of a growing season. Of three protein extraction methods that we compared (the Tris-phenol method, trichloroacetic acid-acetone method, and trichloroacetic acid-acetone-phenol method), trichloroacetic acid-acetone was the most efficient approach for protein extraction from cambium tissues of P. tomentosa. After extraction, the proteins were separated using two-dimensional gel electrophoresis. The protein quantities of six spots changed over the course of the growing season from February to July. Five spots were identified using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry, and the sixth spot was identified by liquid chromatography-mass spectrometry. The proteins included enolase, class Ia chitinase, and four unnamed proteins. Our results show the best approach to proteomics in P. tomentosa and reveal trends in protein activities during a growing season in this tree species. (author)

  4. Amino acid code of protein secondary structure.

    Science.gov (United States)

    Shestopalov, B V

    2003-01-01

    The calculation of protein three-dimensional structure from the amino acid sequence is a fundamental problem to be solved. This paper presents principles of the code theory of protein secondary structure, and their consequence--the amino acid code of protein secondary structure. The doublet code model of protein secondary structure, developed earlier by the author (Shestopalov, 1990), is part of this theory. The theory basis are: 1) the name secondary structure is assigned to the conformation, stabilized only by the nearest (intraresidual) and middle-range (at a distance no more than that between residues i and i + 5) interactions; 2) the secondary structure consists of regular (alpha-helical and beta-structural) and irregular (coil) segments; 3) the alpha-helices, beta-strands and coil segments are encoded, respectively, by residue pairs (i, i + 4), (i, i + 2), (i, i = 1), according to the numbers of residues per period, 3.6, 2, 1; 4) all such pairs in the amino acid sequence are codons for elementary structural elements, or structurons; 5) the codons are divided into 21 types depending on their strength, i.e. their encoding capability; 6) overlappings of structurons of one and the same structure generate the longer segments of this structure; 7) overlapping of structurons of different structures is forbidden, and therefore selection of codons is required, the codon selection is hierarchic; 8) the code theory of protein secondary structure generates six variants of the amino acid code of protein secondary structure. There are two possible kinds of model construction based on the theory: the physical one using physical properties of amino acid residues, and the statistical one using results of statistical analysis of a great body of structural data. Some evident consequences of the theory are: a) the theory can be used for calculating the secondary structure from the amino acid sequence as a partial solution of the problem of calculation of protein three

  5. A method for analysing small samples of floral pollen for free and protein-bound amino acids.

    Science.gov (United States)

    Stabler, Daniel; Power, Eileen F; Borland, Anne M; Barnes, Jeremy D; Wright, Geraldine A

    2018-02-01

    Pollen provides floral visitors with essential nutrients including proteins, lipids, vitamins and minerals. As an important nutrient resource for pollinators, including honeybees and bumblebees, pollen quality is of growing interest in assessing available nutrition to foraging bees. To date, quantifying the protein-bound amino acids in pollen has been difficult and methods rely on large amounts of pollen, typically more than 1 g. More usual is to estimate a crude protein value based on the nitrogen content of pollen, however, such methods provide no information on the distribution of essential and non-essential amino acids constituting the proteins.Here, we describe a method of microwave-assisted acid hydrolysis using low amounts of pollen that allows exploration of amino acid composition, quantified using ultra high performance liquid chromatography (UHPLC), and a back calculation to estimate the crude protein content of pollen.Reliable analysis of protein-bound and free amino acids as well as an estimation of crude protein concentration was obtained from pollen samples as low as 1 mg. Greater variation in both protein-bound and free amino acids was found in pollen sample sizes amino acids in smaller sample sizes, we suggest a correction factor to apply to specific sample sizes of pollen in order to estimate total crude protein content.The method described in this paper will allow researchers to explore the composition of amino acids in pollen and will aid research assessing the available nutrition to pollinating animals. This method will be particularly useful in assaying the pollen of wild plants, from which it is difficult to obtain large sample weights.

  6. Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

    DEFF Research Database (Denmark)

    Sasaki, T; Brakebusch, C; Engel, J

    1998-01-01

    Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

  7. Sample processing, protocol, and statistical analysis of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of protein, cell, and tissue samples.

    Science.gov (United States)

    Barreto, Goncalo; Soininen, Antti; Sillat, Tarvo; Konttinen, Yrjö T; Kaivosoja, Emilia

    2014-01-01

    Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is increasingly being used in analysis of biological samples. For example, it has been applied to distinguish healthy and osteoarthritic human cartilage. This chapter discusses ToF-SIMS principle and instrumentation including the three modes of analysis in ToF-SIMS. ToF-SIMS sets certain requirements for the samples to be analyzed; for example, the samples have to be vacuum compatible. Accordingly, sample processing steps for different biological samples, i.e., proteins, cells, frozen and paraffin-embedded tissues and extracellular matrix for the ToF-SIMS are presented. Multivariate analysis of the ToF-SIMS data and the necessary data preprocessing steps (peak selection, data normalization, mean-centering, and scaling and transformation) are discussed in this chapter.

  8. What befalls the proteins and water in a living cell when the cell dies?

    Science.gov (United States)

    Ling, Gilbert N; Fu, Ya-zhen

    2005-01-01

    The solvency of solutes of varying molecular size in the intracellular water of freshly-killed Ehrlich carcinoma cells fits the same theoretical curve that describes the solvency of similar solutes in a 36% solution of native bovine hemoglobin--a protein found only in red blood cells and making up 97.3% of the red cell's total intracellular proteins. The merging of the two sets of data confirms the prediction of the AI Hypothesis that key intracellular protein(s) in dying cells undergo(es) a transition from: (1) one in which the polypeptide NHCO groups assume a fully-extended conformation with relatively strong power of polarizing and orienting the bulk-phase water in multilayers; to (2) one in which most of the polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformations (see below for definition) with much weaker power in polarizing-orienting multilayers of bulk-phase water. This concordance of the two sets of data also shows that what we now call native hemoglobin--supposedly denoting hemoglobin found in its natural state in living red blood cells--, in fact, more closely resembles the water-polarizing, and -orienting intracellular proteins in dead cells. Although in the dead Ehrlich carcinoma cells as well as in the 36% solution of native hemoglobin, much of the protein's polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformation (Perutz 1969; Weissbluth 1974), both systems produce a weak but nonetheless pervasive and "long-range" water polarization and orientation. It is suggested that in both the dead Ehrlich carcinoma ascites cells and in the 36% native bovine hemoglobin solution, enough polypeptide NHCO groups assume the fully-extended conformation to produce the weak but far-reaching multilayer water polarization and orientation observed.

  9. Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper.

    Science.gov (United States)

    Jiang, Weidong; Mao, Ying Qing; Huang, Ruochun; Duan, Chaohui; Xi, Yun; Yang, Kai; Huang, Ruo-Pan

    2014-01-31

    Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Sampling-based exploration of folded state of a protein under kinematic and geometric constraints

    KAUST Repository

    Yao, Peggy; Zhang, Liangjun; Latombe, Jean-Claude

    2011-01-01

    Flexibility is critical for a folded protein to bind to other molecules (ligands) and achieve its functions. The conformational selection theory suggests that a folded protein deforms continuously and its ligand selects the most favorable

  11. GIS: a comprehensive source for protein structure similarities.

    Science.gov (United States)

    Guerler, Aysam; Knapp, Ernst-Walter

    2010-07-01

    A web service for analysis of protein structures that are sequentially or non-sequentially similar was generated. Recently, the non-sequential structure alignment algorithm GANGSTA+ was introduced. GANGSTA+ can detect non-sequential structural analogs for proteins stated to possess novel folds. Since GANGSTA+ ignores the polypeptide chain connectivity of secondary structure elements (i.e. alpha-helices and beta-strands), it is able to detect structural similarities also between proteins whose sequences were reshuffled during evolution. GANGSTA+ was applied in an all-against-all comparison on the ASTRAL40 database (SCOP version 1.75), which consists of >10,000 protein domains yielding about 55 x 10(6) possible protein structure alignments. Here, we provide the resulting protein structure alignments as a public web-based service, named GANGSTA+ Internet Services (GIS). We also allow to browse the ASTRAL40 database of protein structures with GANGSTA+ relative to an externally given protein structure using different constraints to select specific results. GIS allows us to analyze protein structure families according to the SCOP classification scheme. Additionally, users can upload their own protein structures for pairwise protein structure comparison, alignment against all protein structures of the ASTRAL40 database (SCOP version 1.75) or symmetry analysis. GIS is publicly available at http://agknapp.chemie.fu-berlin.de/gplus.

  12. On plate graphite supported sample processing for simultaneous lipid and protein identification by matrix assisted laser desorption ionization mass spectrometry.

    Science.gov (United States)

    Calvano, Cosima Damiana; van der Werf, Inez Dorothé; Sabbatini, Luigia; Palmisano, Francesco

    2015-05-01

    The simultaneous identification of lipids and proteins by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) after direct on-plate processing of micro-samples supported on colloidal graphite is demonstrated. Taking advantages of large surface area and thermal conductivity, graphite provided an ideal substrate for on-plate proteolysis and lipid extraction. Indeed proteins could be efficiently digested on-plate within 15 min, providing sequence coverages comparable to those obtained by conventional in-solution overnight digestion. Interestingly, detection of hydrophilic phosphorylated peptides could be easily achieved without any further enrichment step. Furthermore, lipids could be simultaneously extracted/identified without any additional treatment/processing step as demonstrated for model complex samples such as milk and egg. The present approach is simple, efficient, of large applicability and offers great promise for protein and lipid identification in very small samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Mass spectrometric identification of diagnostic markers for chronic prostatitis in seminal plasma by analysis of seminal plasma protein clinical samples.

    Science.gov (United States)

    Rokka, A; Mehik, A; Tonttila, P; Vaarala, M

    2017-08-15

    There are few specific diagnostic markers for chronic prostatitis. Therefore, we used mass spectrometry to evaluate differences in seminal plasma protein expression among patients with prostatitis and young and middle-aged healthy controls. We analysed pooled seminal plasma protein samples from four prostatitis patients (two pools), three young controls (one pool), and three middle-aged controls (one pool). The samples were analysed by liquid chromatography-tandem mass spectrometry. Of the 349 proteins identified, 16 were differentially expressed between the two control pools. Five proteins were up- or down-regulated in both of the prostatitis pools compared to middle-aged controls but not between young and middle-aged pools. Progestagen-associated endometrial protein (PAEP) was over-expressed in prostatitis samples compared to young and middle-aged controls. Our findings and those of previous studies indicate that PAEP is a potential seminal plasma marker for chronic prostatitis. In conclusion, we found age-related changes in seminal plasma protein expression. PAEP expression in seminal plasma should be investigated further to evaluate its potential as a diagnostic marker for chronic prostatitis.

  14. Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

    OpenAIRE

    Li, Yaohang

    2013-01-01

    Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increas...

  15. StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

    Science.gov (United States)

    Xu, Yongtao; Zhou, Xu; Huang, Meilan

    2015-01-01

    Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

  16. Elemental analysis of samples of biological origin relative to their protein content by means of charged particle bombardment

    International Nuclear Information System (INIS)

    Szoekefalvi-Nagy, Z.; Demeter, I.; Varga, L.; Hollos-Nagy, K.; Keszthelyi, L.

    1981-04-01

    The particle excited X-ray emission (PIXE) and the 14 N(d,p) 15 N nuclear reaction is combined for simultaneous elemental composition and nitrogen content determination in biological samples. Using the correlation between nitrogen and proton content the elemental composition is related to the protein content of the sample. The principles and main characteristics of the method are described and illustrative applications are also given. (author)

  17. A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns.

    Science.gov (United States)

    Azzam, Sausan; Broadwater, Laurie; Li, Shuo; Freeman, Ernest J; McDonough, Jennifer; Gregory, Roger B

    2013-05-01

    Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 k

  18. Serum Protein Profile Study of Clinical Samples Using High Performance Liquid Chromatography-Laser Induced Fluorescence

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya

    2009-01-01

    The serum protein profiles of normal subjects, patients diagnosed with cervical cancer, and oral cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence detection (HPLC-LIF). Serum protein profiles of the above three classes were tested for estab...

  19. Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

    International Nuclear Information System (INIS)

    Liang Liang; Dou Peng; Dong Mingming; Ke Xiaokang; Bian Ningsheng; Liu Zhen

    2009-01-01

    Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 ± 0.83 x 10 -3 s -1 . The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

  20. Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

    NARCIS (Netherlands)

    Lu, J.; Boeren, J.A.; Vries, de S.C.; Valenberg, van H.J.F.; Vervoort, J.J.M.; Hettinga, K.A.

    2011-01-01

    Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as

  1. Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample

    Science.gov (United States)

    Guo, Shifeng; Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; He, Tao; Teo, Serena Lay Ming; Vancso, G. Julius

    2016-09-01

    Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the ‘footprint’ of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

  2. Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius

    Directory of Open Access Journals (Sweden)

    Zheng Xiaojuan

    2009-10-01

    Full Text Available Abstract Background Two-dimensional gel electrophoresis (2-DE is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF, a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS. Results An extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v 3-[(3-cholamidopropyl-dimethylammonio]-1-propanesulfonate (CHAPS, 50 mM dithiothreitol (DTT, 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF, 20 U/ml Deoxyribonuclease I (DNase I, and 0.25 mg/ml Ribonuclease A (RNase A, combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF. When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found. Conclusion These results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

  3. Combination of atomic force microscopy and mass spectrometry for the detection of target protein in the serum samples of children with autism spectrum disorders

    Science.gov (United States)

    Kaysheva, A. L.; Pleshakova, T. O.; Kopylov, A. T.; Shumov, I. D.; Iourov, I. Y.; Vorsanova, S. G.; Yurov, Y. B.; Ziborov, V. S.; Archakov, A. I.; Ivanov, Y. D.

    2017-10-01

    Possibility of detection of target proteins associated with development of autistic disorders in children with use of combined atomic force microscopy and mass spectrometry (AFM/MS) method is demonstrated. The proposed method is based on the combination of affine enrichment of proteins from biological samples and visualization of these proteins by AFM and MS analysis with quantitative detection of target proteins.

  4. PDB2CD visualises dynamics within protein structures.

    Science.gov (United States)

    Janes, Robert W

    2017-10-01

    Proteins tend to have defined conformations, a key factor in enabling their function. Atomic resolution structures of proteins are predominantly obtained by either solution nuclear magnetic resonance (NMR) or crystal structure methods. However, when considering a protein whose structure has been determined by both these approaches, on many occasions, the resultant conformations are subtly different, as illustrated by the examples in this study. The solution NMR approach invariably results in a cluster of structures whose conformations satisfy the distance boundaries imposed by the data collected; it might be argued that this is evidence of the dynamics of proteins when in solution. In crystal structures, the proteins are often in an energy minimum state which can result in an increase in the extent of regular secondary structure present relative to the solution state depicted by NMR, because the more dynamic ends of alpha helices and beta strands can become ordered at the lower temperatures. This study examines a novel way to display the differences in conformations within an NMR ensemble and between these and a crystal structure of a protein. Circular dichroism (CD) spectroscopy can be used to characterise protein structures in solution. Using the new bioinformatics tool, PDB2CD, which generates CD spectra from atomic resolution protein structures, the differences between, and possible dynamic range of, conformations adopted by a protein can be visualised.

  5. Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    Science.gov (United States)

    Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

    2013-08-01

    Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

  6. Determination of soy proteins in food samples by dispersive solid-phase immunoextraction and dynamic long-wavelength fluorometry

    International Nuclear Information System (INIS)

    Molina-Delgado, María Ángeles; Aguilar-Caballos, María Paz; Gomez-Hens, Agustina

    2013-01-01

    We report on a method for the determination of soy proteins in food samples via dispersive solid-phase immunoextraction using gold-coated magnetic nanoparticles (NPs) as a support. Soy proteins were first extracted using anti-soy protein antibodies immobilized on the NPs, and then quantified by measuring the increase in fluorescence of the long-wavelength fluorophore cresyl violet in the presence of the anionic surfactant sodium dodecyl sulfate at neutral pH in a flow system. The method involves the use of two standard or sample aliquots. The fluorescence intensity of one aliquot is directly measured whereas that of the other aliquot is measured after immunoextraction. The difference between the peak heights of both aliquots serves as the analytical information that is directly proportional to the protein concentration. The limit of detection is 0.35 mg L −1 , the linear range is from 1 to 15 mg L −1 , and the relative standard deviation is < 5 %. Proteins such as bovine serum albumin and globulins do not interfere at the same concentration level. The method was applied to the analysis of soy-based beverages and gave recoveries in the range between 80.0 and 107.3 %. (author)

  7. Antibody reactivity against Helicobacter pylori proteins in a sample of the Spanish adult population in 2008-2013.

    Science.gov (United States)

    Fernández-de-Larrea, Nerea; Michel, Angelika; Romero, Beatriz; Butt, Julia; Pawlita, Michael; Pérez-Gómez, Beatriz; Castaño-Vinyals, Gemma; Moreno, Victor; Martín, Vicente; Amiano, Pilar; Castilla, Jesús; Fernández-Tardón, Guillermo; Dierssen-Sotos, Trinidad; Clofent, Juan; Alguacil, Juan; Huerta, José María; Jiménez-Moleón, José Juan; Barricarte, Aurelio; Molinuevo, Amaia; Fernández-Villa, Tania; Casabonne, Delphine; Sierra, Ángeles; Kogevinas, Manolis; de Sanjosé, Silvia; Pollán, Marina; Del Campo, Rosa; Waterboer, Tim; Aragonés, Nuria

    2017-10-01

    Differences in Helicobacter pylori protein expression have been related to the risk of severe gastric diseases. In Spain, a marked geographic pattern in gastric cancer mortality has long been reported. To characterize antibody reactivity patterns against 16 H. pylori proteins, by age, sex, and region of birth, in a large sample of the Spanish adult population. Antibody reactivity was quantified by H. pylori multiplex serology in a sample from the control group of the multicase-control study MCC-Spain. For this analysis, 2555 population-based controls were included. Each participant was classified as seropositive or seronegative for each protein according to specific cutoffs. Overall H. pylori seroprevalence was defined as positivity against ≥4 proteins. Descriptive analyses by age, sex, and region of birth were performed for both seroprevalence and seroreactivity (continuous measure). Differences among groups were tested by logistic and linear regression models. Overall H. pylori seroprevalence increased with age in both sexes. For ages 55-74, seroprevalence was lower in women than in men (84% vs 92%, Ppylori seropositive subjects, proteins with the highest seroprevalence were GroEL, NapA, HP231, and Omp. Seropositivity for most of the proteins increased or remained stable with age, rising mainly for CagA, GroEL, and HyuA in women. A clear cohort effect was not observed. This is the first study to describe the antibody patterns against 16 H. pylori proteins in the Spanish population. We found variability in the H. pylori antibody profiles according to both individual factors such as age and sex, and environmental factors such as the region of birth. The slightness of the reduction in seropositivity with decreasing age highlights the ongoing importance of this infection. © 2017 John Wiley & Sons Ltd.

  8. Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.

    Science.gov (United States)

    Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per; Holmbjerg, Anne Fich; Grimstrup, Marie; Mørtz, Ejvind; Kofoed, Thomas; Højrup, Peter

    2018-07-01

    Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein.

    Science.gov (United States)

    Yuan, Ping; Leser, George P; Demeler, Borries; Lamb, Robert A; Jardetzky, Theodore S

    2008-09-01

    The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high alpha-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

  10. Multivariate data analysis of two-dimensional gel electrophoresis protein patterns from few samples

    DEFF Research Database (Denmark)

    Jensen, Kristina Nedenskov; Jessen, Flemming; Jørgensen, Bo

    2008-01-01

    One application of 2D gel electrophoresis is to reveal differences in protein pattern between two or more groups of individuals, attributable to their group membership. Multivariate data analytical methods are useful in pinpointing the spots relevant for discrimination by focusing not only...... on single spot differences, but on the covariance structure between proteins. However, their outcome is dependent on data scaling, and they may fail in producing valid multivariate models due to the much higher number of "irrelevant" spots present in the gels. The case where only few gels are available...... and where the aim is to find as many as possible of the group-dependent proteins seems particularly difficult to handle. The present paper investigates such a case regarding the effect of scaling and of prefiltering by univariate nonparametric statistics on the selection of spots. Besides, a modified...

  11. An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

    Science.gov (United States)

    Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

    2017-09-22

    Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

  12. Proteome-wide identification of WRN-interacting proteins in untreated and nuclease-treated samples

    DEFF Research Database (Denmark)

    Lachapelle, Sophie; Gagné, Jean-Philippe; Garand, Chantal

    2011-01-01

    Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, telomere maintenance, and transcription. Here, we present the results of a large-scale prot...

  13. Increasing the sampling efficiency of protein conformational transition using velocity-scaling optimized hybrid explicit/implicit solvent REMD simulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yuqi; Wang, Jinan; Shao, Qiang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn; Zhu, Weiliang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn [ACS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203 (China); Shi, Jiye, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn [UCB Pharma, 216 Bath Road, Slough SL1 4EN (United Kingdom)

    2015-03-28

    The application of temperature replica exchange molecular dynamics (REMD) simulation on protein motion is limited by its huge requirement of computational resource, particularly when explicit solvent model is implemented. In the previous study, we developed a velocity-scaling optimized hybrid explicit/implicit solvent REMD method with the hope to reduce the temperature (replica) number on the premise of maintaining high sampling efficiency. In this study, we utilized this method to characterize and energetically identify the conformational transition pathway of a protein model, the N-terminal domain of calmodulin. In comparison to the standard explicit solvent REMD simulation, the hybrid REMD is much less computationally expensive but, meanwhile, gives accurate evaluation of the structural and thermodynamic properties of the conformational transition which are in well agreement with the standard REMD simulation. Therefore, the hybrid REMD could highly increase the computational efficiency and thus expand the application of REMD simulation to larger-size protein systems.

  14. Predicting protein-ATP binding sites from primary sequence through fusing bi-profile sampling of multi-view features

    Directory of Open Access Journals (Sweden)

    Zhang Ya-Nan

    2012-05-01

    Full Text Available Abstract Background Adenosine-5′-triphosphate (ATP is one of multifunctional nucleotides and plays an important role in cell biology as a coenzyme interacting with proteins. Revealing the binding sites between protein and ATP is significantly important to understand the functionality of the proteins and the mechanisms of protein-ATP complex. Results In this paper, we propose a novel framework for predicting the proteins’ functional residues, through which they can bind with ATP molecules. The new prediction protocol is achieved by combination of sequence evolutional information and bi-profile sampling of multi-view sequential features and the sequence derived structural features. The hypothesis for this strategy is single-view feature can only represent partial target’s knowledge and multiple sources of descriptors can be complementary. Conclusions Prediction performances evaluated by both 5-fold and leave-one-out jackknife cross-validation tests on two benchmark datasets consisting of 168 and 227 non-homologous ATP binding proteins respectively demonstrate the efficacy of the proposed protocol. Our experimental results also reveal that the residue structural characteristics of real protein-ATP binding sites are significant different from those normal ones, for example the binding residues do not show high solvent accessibility propensities, and the bindings prefer to occur at the conjoint points between different secondary structure segments. Furthermore, results also show that performance is affected by the imbalanced training datasets by testing multiple ratios between positive and negative samples in the experiments. Increasing the dataset scale is also demonstrated useful for improving the prediction performances.

  15. Using the multi-objective optimization replica exchange Monte Carlo enhanced sampling method for protein-small molecule docking.

    Science.gov (United States)

    Wang, Hongrui; Liu, Hongwei; Cai, Leixin; Wang, Caixia; Lv, Qiang

    2017-07-10

    In this study, we extended the replica exchange Monte Carlo (REMC) sampling method to protein-small molecule docking conformational prediction using RosettaLigand. In contrast to the traditional Monte Carlo (MC) and REMC sampling methods, these methods use multi-objective optimization Pareto front information to facilitate the selection of replicas for exchange. The Pareto front information generated to select lower energy conformations as representative conformation structure replicas can facilitate the convergence of the available conformational space, including available near-native structures. Furthermore, our approach directly provides min-min scenario Pareto optimal solutions, as well as a hybrid of the min-min and max-min scenario Pareto optimal solutions with lower energy conformations for use as structure templates in the REMC sampling method. These methods were validated based on a thorough analysis of a benchmark data set containing 16 benchmark test cases. An in-depth comparison between MC, REMC, multi-objective optimization-REMC (MO-REMC), and hybrid MO-REMC (HMO-REMC) sampling methods was performed to illustrate the differences between the four conformational search strategies. Our findings demonstrate that the MO-REMC and HMO-REMC conformational sampling methods are powerful approaches for obtaining protein-small molecule docking conformational predictions based on the binding energy of complexes in RosettaLigand.

  16. Domain organizations of modular extracellular matrix proteins and their evolution.

    Science.gov (United States)

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  17. Divergent flow isoelectric focusing: fast and efficient method for protein sample preparation for mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Mazanec, Karel; Bobálová, Janette; Šlais, Karel

    2009-01-01

    Roč. 393, 6-7 (2009), s. 1769-1778 ISSN 1618-2642 R&D Projects: GA MŠk 1M0570; GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : Isoelectric focusing * proteins * MALDI-TOF/TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.480, year: 2009

  18. Direct analysis of site-specific N-glycopeptides of serological proteins in dried blood spot samples.

    Science.gov (United States)

    Choi, Na Young; Hwang, Heeyoun; Ji, Eun Sun; Park, Gun Wook; Lee, Ju Yeon; Lee, Hyun Kyoung; Kim, Jin Young; Yoo, Jong Shin

    2017-08-01

    Dried blood spot (DBS) samples have a number of advantages, especially with respect to ease of collection, transportation, and storage and to reduce biohazard risk. N-glycosylation is a major post-translational modification of proteins in human blood that is related to a variety of biological functions, including metastasis, cell-cell interactions, inflammation, and immunization. Here, we directly analyzed tryptic N-glycopeptides from glycoproteins in DBS samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without centrifugation of blood samples, depletion of major proteins, desalting of tryptic peptides, and enrichment of N-glycopeptides. Using this simple method, we identified a total of 41 site-specific N-glycopeptides from 16 glycoproteins in the DBS samples, from immunoglobulin gamma 1 (IgG-1, 10 mg/mL) down to complement component C7 (50 μg/mL). Of these, 32 N-glycopeptides from 14 glycoproteins were consistently quantified over 180 days stored at room temperature. The major abundant glycoproteins in the DBS samples were IgG-1 and IgG-2, which contain nine asialo-fucosylated complex types of 16 different N-glycopeptide isoforms. Sialo-non-fucosylated complex types were primarily detected in the other glycoproteins such as alpha-1-acid glycoprotein 1, 2, alpha-1-antitypsin, alpha-2-macroglobulin, haptoglobin, hemopexin, Ig alpha 1, 2 chain C region, kininogen-1, prothrombin, and serotransferrin. We first report the characterization of site-specific N-glycoproteins in DBS samples by LC-MS/MS with minimal sample preparation.

  19. Exploring structural variability in X-ray crystallographic models using protein local optimization by torsion-angle sampling

    International Nuclear Information System (INIS)

    Knight, Jennifer L.; Zhou, Zhiyong; Gallicchio, Emilio; Himmel, Daniel M.; Friesner, Richard A.; Arnold, Eddy; Levy, Ronald M.

    2008-01-01

    Torsion-angle sampling, as implemented in the Protein Local Optimization Program (PLOP), is used to generate multiple structurally variable single-conformer models which are in good agreement with X-ray data. An ensemble-refinement approach to differentiate between positional uncertainty and conformational heterogeneity is proposed. Modeling structural variability is critical for understanding protein function and for modeling reliable targets for in silico docking experiments. Because of the time-intensive nature of manual X-ray crystallographic refinement, automated refinement methods that thoroughly explore conformational space are essential for the systematic construction of structurally variable models. Using five proteins spanning resolutions of 1.0–2.8 Å, it is demonstrated how torsion-angle sampling of backbone and side-chain libraries with filtering against both the chemical energy, using a modern effective potential, and the electron density, coupled with minimization of a reciprocal-space X-ray target function, can generate multiple structurally variable models which fit the X-ray data well. Torsion-angle sampling as implemented in the Protein Local Optimization Program (PLOP) has been used in this work. Models with the lowest R free values are obtained when electrostatic and implicit solvation terms are included in the effective potential. HIV-1 protease, calmodulin and SUMO-conjugating enzyme illustrate how variability in the ensemble of structures captures structural variability that is observed across multiple crystal structures and is linked to functional flexibility at hinge regions and binding interfaces. An ensemble-refinement procedure is proposed to differentiate between variability that is a consequence of physical conformational heterogeneity and that which reflects uncertainty in the atomic coordinates

  20. Monte Carlo Methods Development and Applications in Conformational Sampling of Proteins

    DEFF Research Database (Denmark)

    Tian, Pengfei

    quantitative insights into their thermodynamic and mechanistic properties that are difficult to probe in laboratory experiments. However, despite the rapid progress in the development of molecular simulation, there are still two limiting factors, (1), the current molecular mechanics force fields alone...... sampling methods to address these two problems. First of all, a novel technique has been developed for reliably estimating diffusion coefficients for use in the enhanced sampling of molecular simulations. A broad applicability of this method is illustrated by studying various simulation problems...

  1. Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

    Science.gov (United States)

    Kemenesi, Gábor; Kurucz, Kornélia; Zana, Brigitta; Földes, Fanni; Urbán, Péter; Vlaschenko, Anton; Kravchenko, Kseniia; Budinski, Ivana; Szodoray-Parádi, Farkas; Bücs, Szilárd; Jére, Csaba; Csősz, István; Szodoray-Parádi, Abigél; Estók, Péter; Görföl, Tamás; Boldogh, Sándor; Jakab, Ferenc

    2018-03-01

    Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

  2. Collective estimation of multiple bivariate density functions with application to angular-sampling-based protein loop modeling

    KAUST Repository

    Maadooliat, Mehdi

    2015-10-21

    This paper develops a method for simultaneous estimation of density functions for a collection of populations of protein backbone angle pairs using a data-driven, shared basis that is constructed by bivariate spline functions defined on a triangulation of the bivariate domain. The circular nature of angular data is taken into account by imposing appropriate smoothness constraints across boundaries of the triangles. Maximum penalized likelihood is used to fit the model and an alternating blockwise Newton-type algorithm is developed for computation. A simulation study shows that the collective estimation approach is statistically more efficient than estimating the densities individually. The proposed method was used to estimate neighbor-dependent distributions of protein backbone dihedral angles (i.e., Ramachandran distributions). The estimated distributions were applied to protein loop modeling, one of the most challenging open problems in protein structure prediction, by feeding them into an angular-sampling-based loop structure prediction framework. Our estimated distributions compared favorably to the Ramachandran distributions estimated by fitting a hierarchical Dirichlet process model; and in particular, our distributions showed significant improvements on the hard cases where existing methods do not work well.

  3. Collective estimation of multiple bivariate density functions with application to angular-sampling-based protein loop modeling

    KAUST Repository

    Maadooliat, Mehdi; Zhou, Lan; Najibi, Seyed Morteza; Gao, Xin; Huang, Jianhua Z.

    2015-01-01

    This paper develops a method for simultaneous estimation of density functions for a collection of populations of protein backbone angle pairs using a data-driven, shared basis that is constructed by bivariate spline functions defined on a triangulation of the bivariate domain. The circular nature of angular data is taken into account by imposing appropriate smoothness constraints across boundaries of the triangles. Maximum penalized likelihood is used to fit the model and an alternating blockwise Newton-type algorithm is developed for computation. A simulation study shows that the collective estimation approach is statistically more efficient than estimating the densities individually. The proposed method was used to estimate neighbor-dependent distributions of protein backbone dihedral angles (i.e., Ramachandran distributions). The estimated distributions were applied to protein loop modeling, one of the most challenging open problems in protein structure prediction, by feeding them into an angular-sampling-based loop structure prediction framework. Our estimated distributions compared favorably to the Ramachandran distributions estimated by fitting a hierarchical Dirichlet process model; and in particular, our distributions showed significant improvements on the hard cases where existing methods do not work well.

  4. 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.

  5. Digital ELISA for the quantification of attomolar concentrations of Alzheimer's disease biomarker protein Tau in biological samples.

    Science.gov (United States)

    Pérez-Ruiz, Elena; Decrop, Deborah; Ven, Karen; Tripodi, Lisa; Leirs, Karen; Rosseels, Joelle; van de Wouwer, Marlies; Geukens, Nick; De Vos, Ann; Vanmechelen, Eugeen; Winderickx, Joris; Lammertyn, Jeroen; Spasic, Dragana

    2018-07-26

    The close correlation between Tau pathology and Alzheimer's disease (AD) progression makes this protein a suitable biomarker for diagnosis and monitoring of the disorder evolution. However, the use of Tau in diagnostics has been hampered, as it currently requires collection of cerebrospinal fluid (CSF), which is an invasive clinical procedure. Although measuring Tau-levels in blood plasma would be favorable, the concentrations are below the detection limit of a conventional ELISA. In this work, we developed a digital ELISA for the quantification of attomolar protein Tau concentrations in both buffer and biological samples. Individual Tau molecules were first captured on the surface of magnetic particles using in-house developed antibodies and subsequently isolated into the femtoliter-sized wells of a 2 × 2 mm 2 microwell array. Combination of high-affinity antibodies, optimal assay conditions and a digital quantification approach resulted in a 24 ± 7 aM limit of detection (LOD) in buffer samples. Additionally, a dynamic range of 6 orders of magnitude was achieved by combining the digital readout with an analogue approach, allowing quantification from attomolar to picomolar levels of Tau using the same platform. This proves the compatibility of the presented assay with the wide range of Tau concentrations encountered in different biological samples. Next, the developed digital assay was applied to detect total Tau levels in spiked blood plasma. A similar LOD (55 ± 29 aM) was obtained compared to the buffer samples, which was 5000-fold more sensitive than commercially available ELISAs and even outperformed previously reported digital assays with 10-fold increase in sensitivity. Finally, the performance of the developed digital ELISA was assessed by quantifying protein Tau in three clinical CSF samples. Here, a high correlation (i.e. Pearson coefficient of 0.99) was found between the measured percentage of active particles and the reference protein Tau

  6. Surface plasmon resonance biosensor for detection of pregnancy associated plasma protein A2 in clinical samples

    Czech Academy of Sciences Publication Activity Database

    Bocková, Markéta; Chadtová Song, Xue; Gedeonová, Erika; Levová, K.; Kalousová, M.; Zima, T.; Homola, Jiří

    2016-01-01

    Roč. 408, č. 26 (2016), s. 7265-7269 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GBP205/12/G118 Grant - others:AV ČR(CZ) AP1101 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:67985882 Keywords : Nanoparticles * Blood sample * Surface plasmon resonance Subject RIV: BO - Biophysics Impact factor: 3.431, year: 2016

  7. A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample.

    Science.gov (United States)

    Salem, Mohamed; Bernach, Michal; Bajdzienko, Krzysztof; Giavalisco, Patrick

    2017-06-01

    Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

  8. Immunoassay of C-reactive protein by hot electron induced electrochemiluminescence using integrated electrodes with hydrophobic sample confinement

    Energy Technology Data Exchange (ETDEWEB)

    Ylinen-Hinkka, T., E-mail: tiina.ylinen-hinkka@aalto.fi [Laboratory of Analytical Chemistry, Aalto University School of Chemical Technology, P.O. Box 16100, FI-00076 Aalto (Finland); Niskanen, A.J.; Franssila, S. [Department of Materials Science and Engineering, Aalto University School of Chemical Technology, P.O. Box 16200, FI-00076 Aalto (Finland); Kulmala, S. [Laboratory of Analytical Chemistry, Aalto University School of Chemical Technology, P.O. Box 16100, FI-00076 Aalto (Finland)

    2011-09-19

    Highlights: {center_dot} C-reactive protein has been determined in the concentration range 0.01-10 mg L{sup -1} using an electrochemiluminescence microchip which employs integrated electrodes with hydrophobic sample confinement. {center_dot} This arrangement enables very simple and fast CRP analysis amenable to point-of-care applications. - Abstract: C-reactive protein (CRP) was determined in the concentration range 0.01-10 mg L{sup -1} using hot electron induced electrochemiluminescence (HECL) with devices combining both working and counter electrodes and sample confinement on a single chip. The sample area on the electrodes was defined by a hydrophobic ring, which enabled dispensing the reagents and the analyte directly on the electrode. Immunoassay of CRP by HECL using integrated electrodes is a good candidate for a high-sensitivity point-of-care CRP-test, because the concentration range is suitable, miniaturisation of the measurement system has been demonstrated and the assay method with integrated electrodes is easy to use. High-sensitivity CRP tests can be used to monitor the current state of cardiovascular disease and also to predict future cardiovascular problems in apparently healthy people.

  9. Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins.

    Science.gov (United States)

    Schaffer, Miroslava; Mahamid, Julia; Engel, Benjamin D; Laugks, Tim; Baumeister, Wolfgang; Plitzko, Jürgen M

    2017-02-01

    While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. 137Cs absorption factors (AFs) from contaminated cooking water to some vegetable and protein samples

    International Nuclear Information System (INIS)

    Malek, M.A.

    2006-01-01

    The radionuclide in contaminated freshwater may directly gain access to the human body through two major routes: drinking and cooking food with fresh water. During cooking, the radionuclide present in the water may be transferred to the various ingredients of the cooked food. The degree of contamination of food during cooking depends both on absorption power of the individual ingredients and the level of radionuclide present in the water. The ratio of the concentration of the radionuclide absorbed in the individual ingredients to the concentration in the cooking water can be designated as 'Absorption factor' (AF). AF can be used to predict the radionuclide absorbed by the ingredients cooked with contaminated water, to assess the internal radiation dose to the consumer and radionuclide transfer from the cooking water to the ingredients. A better understanding of the variables that affect the AF in various ingredients during cooking is central to deriving the contamination level of the ingredients. 10 kinds of greens and vegetable and 3 kinds of animal protein were boiled with 37 Cs contaminated freshwater and corresponding AFs were determined in both hot and cooled condition

  11. Current status and future prospects of an automated sample exchange system PAM for protein crystallography

    Science.gov (United States)

    Hiraki, M.; Yamada, Y.; Chavas, L. M. G.; Matsugaki, N.; Igarashi, N.; Wakatsuki, S.

    2013-03-01

    To achieve fully-automated and/or remote data collection in high-throughput X-ray experiments, the Structural Biology Research Centre at the Photon Factory (PF) has installed PF automated mounting system (PAM) for sample exchange robots at PF macromolecular crystallography beamlines BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. We are upgrading the experimental systems, including the PAM for stable and efficient operation. To prevent human error in automated data collection, we installed a two-dimensional barcode reader for identification of the cassettes and sample pins. Because no liquid nitrogen pipeline in the PF experimental hutch is installed, the users commonly add liquid nitrogen using a small Dewar. To address this issue, an automated liquid nitrogen filling system that links a 100-liter tank to the robot Dewar has been installed on the PF macromolecular beamline. Here we describe this new implementation, as well as future prospects.

  12. Advancements in mass spectrometry for biological samples: Protein chemical cross-linking and metabolite analysis of plant tissues

    Energy Technology Data Exchange (ETDEWEB)

    Klein, Adam [Iowa State Univ., Ames, IA (United States)

    2015-01-01

    This thesis presents work on advancements and applications of methodology for the analysis of biological samples using mass spectrometry. Included in this work are improvements to chemical cross-linking mass spectrometry (CXMS) for the study of protein structures and mass spectrometry imaging and quantitative analysis to study plant metabolites. Applications include using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to further explore metabolic heterogeneity in plant tissues and chemical interactions at the interface between plants and pests. Additional work was focused on developing liquid chromatography-mass spectrometry (LC-MS) methods to investigate metabolites associated with plant-pest interactions.

  13. Label-Free Quantitative Analysis of Mitochondrial Proteomes Using the Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP) and "Total Protein Approach".

    Science.gov (United States)

    Wiśniewski, Jacek R

    2017-01-01

    Determination of proteome composition and measuring of changes in protein titers provide important information with a substantial value for studying mitochondria.This chapter describes a workflow for the quantitative analysis of mitochondrial proteome with a focus on sample preparation and quantitative analysis of the data. The workflow involves the multienzyme digestion-filter aided sample preparation (MED-FASP) protocol enabling efficient extraction of proteins and high rate of protein-to-peptide conversion. Consecutive protein digestion with Lys C and trypsin enables generation of peptide fractions with minimal overlap, largely increases the number of identified proteins, and extends their sequence coverage. Abundances of proteins identified by multiple peptides can be assessed by the "Total Protein Approach."

  14. [Interaction of protein with charged colloidal particles].

    Science.gov (United States)

    Durdenko, E V; Kuznetsova, S M; Basova, L V; Tikhonenko, S A; Saburova, E A

    2011-01-01

    The functional state of three proteins of different molecular weight (urease, lactate dehydrogenase, and hemoglobin) in the presence of the linear polyelectrolytes poly(allylamine hydrochloride) (PAA) and sodium poly(styrenesulfonate) (PSS) in the dissolved state and of the same polyelectrolytes bound to the surface of microspheres has been investigated. Microspheres were prepared by consecutive absorption of oppositely charged polyelectrolytes so that the outer layer of the shell was PAA for the acidic protein urease, and PSS for the alkaline proteins LDH and hemoglobin. It was shown that the dissolved polyelectrolyte completely inactivates all three proteins within one minute with a slight difference in the time constant. (By Hb inactivation are conventionally meant changes in the heme environment observed from the spectrum in the Soret band.) In the presence of microspheres, the proteins were adsorbed on their surface; in this case, more than 95% of the activity was retained within two hours. The proportion of the protein adsorbed on microspheres accounted for about 98% for urease, 72% for Hb, and 35% for LDH, as determined from the tryptophan fluorescence data. The interaction of hemoglobin with another type of charged colloidal particles, phospholipid vesicles, leads to the destruction of the tertiary structure of the protein, which made itself evident in the optical absorption spectra in the Soret band, as well as the spectra of tryptophan fluorescence and circular dichroism. In this case, according to circular dichroism, the percentage of alpha-helical structure of Hb was maintained. The differences in the physical and chemical mechanisms of interaction of proteins with these two types of charged colloidal particles that leads to differences in the degree of denaturing effects are discussed.

  15. Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

    Science.gov (United States)

    Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

    1998-08-28

    The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

  16. All-atom simulation study of protein PTH(1-34) by using the Wang-Landau sampling method

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seung-Yeon [Korea National University of Transportation, Chungju (Korea, Republic of); Kwak, Woo-Seop [Chosun University, Gwangju (Korea, Republic of)

    2014-12-15

    We perform simulations of the N-terminal 34-residue protein fragment PTH(1-34), consisting of 581 atoms, of the 84-residue human parathyroid hormone by using the all-atom ECEPP/3 force field and the Wang-Landau sampling method. Through a massive high-performance computation, the density of states and the partition function Z(T), as a continuous function of T, are obtained for PTH(1-34). From the continuous partition function Z(T), the partition function zeros of PTH(1-34) are evaluated for the first time. From both the specific heat and the partition function zeros, two characteristic transition temperatures are obtained for the all-atom protein PTH(1-34). The higher transition temperature T{sub 1} and the lower transition temperature T{sub 2} of PTH(1-34) can be interpreted as the collapse temperature T{sub θ} and the folding temperature T{sub f} , respectively.

  17. Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS

    Directory of Open Access Journals (Sweden)

    Dritan Siliqi

    2018-02-01

    Full Text Available The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N- and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

  18. Modeling of allergen proteins found in sea food products

    Directory of Open Access Journals (Sweden)

    Nataly Galán-Freyle

    2012-06-01

    Full Text Available Shellfish are a source of food allergens, and their consumption is the cause of severe allergic reactions in humans. Tropomyosins, a family of muscle proteins, have been identified as the major allergens in shellfish and mollusks species. Nevertheless, few experimentally determined three-dimensional structures are available in the Protein Data Base (PDB. In this study, 3D models of several homologous of tropomyosins present in marine shellfish and mollusk species (Chaf 1, Met e1, Hom a1, Per v1, and Pen a1 were constructed, validated, and their immunoglobulin E binding epitopes were identified using bioinformatics tools. All protein models for these allergens consisted of long alpha-helices. Chaf 1, Met e1, and Hom a1 had six conserved regions with sequence similarities to known epitopes, whereas Per v1 and Pen a1 contained only one. Lipophilic potentials of identified epitopes revealed a high propensity of hydrophobic amino acids in the immunoglobulin E binding site. This information could be useful to design tropomyosin-specific immunotherapy for sea food allergies.

  19. Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-01

    The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.

  20. Pico-litre Sample Introduction and Acoustic Levitation Systems for Time Resolved Protein Crystallography Experiments at XFELS

    Directory of Open Access Journals (Sweden)

    Peter Docker

    2017-07-01

    Full Text Available The system described in this work is a variant from traditional acoustic levitation first described by, Marzo et al. It uses multiple transducers eliminating the requirement for a mirror surface, allowing for an open geometry as the sound from multiple transducers combines to generate the acoustic trap which is configured to catch pico litres of crystal slurries. These acoustic traps also have the significant benefit of eliminating potential beam attenuation due to support structures or microfluidic devices. Additionally they meet the need to eliminate sample environments when experiments are carried out using an X-ray Free Electron Lasers (XFEL such as the Linac Coherent Light Source (LCLS as any sample environment would not survive the exposure to the X-Ray beam. XFELs generate Light a billion times brighter than the sun. The application for this system will be to examine turn over in Beta lactamase proteins which is responsible for bacteria developing antibiotic resistance and therefore of significant importance to future world health. The system will allow for diffraction data to be collected before and after turnover allowing for a better understanding of the underling processes. The authors first described this work at Nanotech 2017.

  1. Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

    Science.gov (United States)

    Wellenreuther, G.; Fittschen, U. E. A.; Achard, M. E. S.; Faust, A.; Kreplin, X.; Meyer-Klaucke, W.

    2008-12-01

    Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence (μXRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

  2. Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

    Energy Technology Data Exchange (ETDEWEB)

    Wellenreuther, G. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany); Fittschen, U.E.A. [Department of Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg (Germany); Achard, M.E.S.; Faust, A.; Kreplin, X. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany); Meyer-Klaucke, W. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany)], E-mail: Wolfram@embl-hamburg.de

    2008-12-15

    Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence ({mu}XRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

  3. Validation of an semi-automated multi component method using protein precipitation LC-MS-MS for the analysis of whole blood samples

    DEFF Research Database (Denmark)

    Slots, Tina

    BACKGROUND: Solid phase extraction (SPE) are one of many multi-component methods, but can be very time-consuming and labour-intensive. Protein precipitation is, on the other hand, a much simpler and faster sample pre-treatment than SPE, and protein precipitation also has the ability to cover a wi......-mortem whole blood sample preparation for toxicological analysis; from the primary sample tube to a 96-deepwell plate ready for injection on the liquid chromatography mass spectrometry (LC-MS/MS)....

  4. Structure, Function, Self-Assembly and Origin of Simple Membrane Proteins

    Science.gov (United States)

    Pohorille, Andrew

    2003-01-01

    Integral membrane proteins perform such essential cellular functions as transport of ions, nutrients and waste products across cell walls, transduction of environmental signals, regulation of cell fusion, recognition of other cells, energy capture and its conversion into high-energy compounds. In fact, 30-40% of genes in modem organisms codes for membrane proteins. Although contemporary membrane proteins or their functional assemblies can be quite complex, their transmembrane fragments are usually remarkably simple. The most common structural motif for these fragments is a bundle of alpha-helices, but occasionally it could be a beta-barrel. In a series of molecular dynamics computer simulations we investigated self-organizing properties of simple membrane proteins based on these structural motifs. Specifically, we studied folding and insertion into membranes of short, nonpolar or amphiphatic peptides. We also investigated glycophorin A, a peptide that forms sequence-specific dimers, and a transmembrane aggregate of four identical alpha-helices that forms an efficient and selective voltage-gated proton channel was investigated. Many peptides are attracted to water-membrane interfaces. Once at the interface, nonpolar peptides spontaneously fold to a-helices. Whenever the sequence permits, peptides that contain both polar and nonpolar amino also adopt helical structures, in which polar and nonpolar amino acid side chains are immersed in water and membrane, respectively. Specific identity of side chains is less important. Helical peptides at the interface could insert into the membrane and adopt a transmembrane conformation. However, insertion of a single helix is unfavorable because polar groups in the peptide become completely dehydrated upon insertion. The unfavorable free energy of insertion can be regained by spontaneous association of peptides in the membrane. The first step in this process is the formation of dimers, although the most common are aggregates of 4

  5. Investigating effects of sample pretreatment on protein stability using size-exclusion chromatography and high-resolution continuum source atomic absorption spectrometry.

    Science.gov (United States)

    Rakow, Tobias; El Deeb, Sami; Hahne, Thomas; El-Hady, Deia Abd; AlBishri, Hassan M; Wätzig, Hermann

    2014-09-01

    In this study, size-exclusion chromatography and high-resolution atomic absorption spectrometry methods have been developed and evaluated to test the stability of proteins during sample pretreatment. This especially includes different storage conditions but also adsorption before or even during the chromatographic process. For the development of the size exclusion method, a Biosep S3000 5 μm column was used for investigating a series of representative model proteins, namely bovine serum albumin, ovalbumin, monoclonal immunoglobulin G antibody, and myoglobin. Ambient temperature storage was found to be harmful to all model proteins, whereas short-term storage up to 14 days could be done in an ordinary refrigerator. Freezing the protein solutions was always complicated and had to be evaluated for each protein in the corresponding solvent. To keep the proteins in their native state a gentle freezing temperature should be chosen, hence liquid nitrogen should be avoided. Furthermore, a high-resolution continuum source atomic absorption spectrometry method was developed to observe the adsorption of proteins on container material and chromatographic columns. Adsorption to any container led to a sample loss and lowered the recovery rates. During the pretreatment and high-performance size-exclusion chromatography, adsorption caused sample losses of up to 33%. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

    Science.gov (United States)

    Morozov, Sergey Y; Milyutina, Irina A; Bobrova, Vera K; Ryazantsev, Dmitry Y; Erokhina, Tatiana N; Zavriev, Sergey K; Agranovsky, Alexey A; Solovyev, Andrey G; Troitsky, Alexey V

    2015-12-01

    The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  7. Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins.

    Science.gov (United States)

    Wiśniewski, Jacek R; Duś, Kamila; Mann, Matthias

    2013-04-01

    Archival formalin-fixed and paraffin-embedded clinical samples represent a very diverse source of material for proteomic investigation of diseases, often with follow-up patient information. Here, we describe an analytical workflow for analysis of laser-capture microdissected formalin-fixed and paraffin-embedded samples that allows studying proteomes to a depth of 10 000 proteins per sample. The workflow involves lysis of tissue in SDS-containing buffer, detergent removal, and consecutive digestion of the proteins with two enzymes by the multienzyme digestion filter-aided sample preparation method. Resulting peptides are fractionated by pipette-tip based strong anion exchange into six fractions and analyzed by LC-MS/MS on a bench top quadrupole Orbitrap mass spectrometer. Analysis of the data using the MaxQuant software resulted in the identification of 9502 ± 28 protein groups per a 110 nL sample of microdissected cells from human colonic adenoma. This depth of proteome analysis enables systemic insights into the organization of the adenoma cells and an estimation of the abundances of known biomarkers. It also allows the identification of proteins expressed from tumor suppressors, oncogenes, and other key players in the development and progression of the colorectal cancer. Our proteomic platform can be used for quantitative comparisons between samples representing different stages of diseases and thus can be applied to the discovery of biomarkers or drug targets. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development.

    Science.gov (United States)

    Hoane, Jessica S; Carruthers, Vernon B; Striepen, Boris; Morrison, David P; Entzeroth, Rolf; Howe, Daniel K

    2003-07-01

    Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the

  9. Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen.

    Science.gov (United States)

    Al-Abdul-Wahid, M Sameer; Verardi, Raffaello; Veglia, Gianluigi; Prosser, R Scott

    2011-09-01

    In studies of membrane proteins, knowledge of protein topology can provide useful insight into both structure and function. In this work, we present a solution NMR method for the measurement the tilt angle and average immersion depth of alpha helices in membrane proteins, from analysis of the paramagnetic relaxation rate enhancements arising from dissolved oxygen. No modification to the micelle or protein is necessary, and the topology of both transmembrane and amphipathic helices are readily determined. We apply this method to the measure the topology of a monomeric mutant of phospholamban (AFA-PLN), a 52-residue membrane protein containing both an amphipathic and a transmembrane alpha helix. In dodecylphosphocholine micelles, the amphipathic helix of AFA-PLN was found to have a tilt angle of 87° ± 1° and an average immersion depth of 13.2 Å. The transmembrane helix was found to have an average immersion depth of 5.4 Å, indicating residues 41 and 42 are closest to the micelle centre. The resolution of paramagnetic relaxation rate enhancements from dissolved oxygen compares favourably to those from Ni (II), a hydrophilic paramagnetic species.

  10. Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen

    International Nuclear Information System (INIS)

    Al-Abdul-Wahid, M. Sameer; Verardi, Raffaello; Veglia, Gianluigi; Prosser, R. Scott

    2011-01-01

    In studies of membrane proteins, knowledge of protein topology can provide useful insight into both structure and function. In this work, we present a solution NMR method for the measurement the tilt angle and average immersion depth of alpha helices in membrane proteins, from analysis of the paramagnetic relaxation rate enhancements arising from dissolved oxygen. No modification to the micelle or protein is necessary, and the topology of both transmembrane and amphipathic helices are readily determined. We apply this method to the measure the topology of a monomeric mutant of phospholamban (AFA-PLN), a 52-residue membrane protein containing both an amphipathic and a transmembrane alpha helix. In dodecylphosphocholine micelles, the amphipathic helix of AFA-PLN was found to have a tilt angle of 87° ± 1° and an average immersion depth of 13.2 Å. The transmembrane helix was found to have an average immersion depth of 5.4 Å, indicating residues 41 and 42 are closest to the micelle centre. The resolution of paramagnetic relaxation rate enhancements from dissolved oxygen compares favourably to those from Ni (II), a hydrophilic paramagnetic species.

  11. Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface

    DEFF Research Database (Denmark)

    Stensballe, A; Jensen, Ole Nørregaard

    2001-01-01

    /ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used as the first protein screening method in many laboratories because of its inherent simplicity, mass accuracy, sensitivity and relatively high sample throughput. We present a simplified sample preparation method for MALDI-MS that enables in-gel digestion...... for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation...... protocol while being less labour intensive and more cost-effective due to minimal consumption of reagents, enzymes and consumables. Preliminary data obtained on a MALDI quadrupole-TOF tandem mass spectrometer demonstrated the utility of the on-probe digestion protocol for peptide mass mapping and peptide...

  12. Advanced fluidic handling and use of two-phase flow for high throughput structural investigation of proteins on a microfluidic sample preparation platform

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Møller, M.

    2010-01-01

    Research on the structure of proteins can bring forth a wealth of information about biological function and can be used to better understand the processes in living cells. This paper reports a new microfluidic sample preparation system for the structural investigation of proteins by Small Angle X......-ray Scattering (SAXS). The system includes hardware and software features for precise fluidic control, synchrotron beamline control, UV absorbance measurements and automated data analysis. The precise fluidic handling capabilities are used to transport and precisely position samples as small as 500 n...

  13. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Nielsen, Søren Skou

    2011-01-01

    A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure...... control, UV absorbance measurements and automated data analysis. As little as 15 l of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis...

  14. Development of a Univariate Membrane-Based Mid-Infrared Method for Protein Quantitation and Total Lipid Content Analysis of Biological Samples

    Directory of Open Access Journals (Sweden)

    Ivona Strug

    2014-01-01

    Full Text Available Biological samples present a range of complexities from homogeneous purified protein to multicomponent mixtures. Accurate qualification of such samples is paramount to downstream applications. We describe the development of an MIR spectroscopy-based analytical method offering simultaneous protein quantitation (0.25–5 mg/mL and analysis of total lipid or detergent species, as well as the identification of other biomolecules present in biological samples. The method utilizes a hydrophilic PTFE membrane engineered for presentation of aqueous samples in a dried format compatible with fast infrared analysis. Unlike classical quantification techniques, the reported method is amino acid sequence independent and thus applicable to complex samples of unknown composition. By comparison to existing platforms, this MIR-based method enables direct quantification using minimal sample volume (2 µL; it is well-suited where repeat access and limited sample size are critical parameters. Further, accurate results can be derived without specialized training or knowledge of IR spectroscopy. Overall, the simplified application and analysis system provides a more cost-effective alternative to high-throughput IR systems for research laboratories with minimal throughput demands. In summary, the MIR-based system provides a viable alternative to current protein quantitation methods; it also uniquely offers simultaneous qualification of other components, notably lipids and detergents.

  15. [Detection of serum proteins in the patients of lung adenocarcinoma by the method of magnetic bead based sample fractionation and MALDI-TOF-MS].

    Science.gov (United States)

    Liu, Dan; Liu, Lun-Xu; Yuan, Quan; Li, Xiao-Liang; Huang, Na; Yang, Xiao-Dong

    2010-05-01

    To screen the serum proteins related to human lung adenocarcinoma using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) technology. The blood samples were collected from 10 patients of lung adenocarcinoma before and one week after the surgery, while 10 healthy subjects were used as control. The differential protein expression between the two groups and the change of those proteins after surgery were studied by ClinProt magnetic bead enrichment and MALDI-TOF-MS. Six protein peaks were identified, 2 of them were highly expressed protein biomarkers with relative molecular weights of 2661, 2991, and increased after the surgery, 4 of them were lowly expressed protein biomarkers with relative molecular weights of 4091, 4210, 4644, 5336, which continuously decreased after the surgery. ClinProt magnetic bead enrichment and MALDI-TOF-MS is a quick, easy and sensitive method of proteomics. The differential expressed proteins may be the latent tumor marker of lung adenocarcinoma. The alteration of those proteins after surgery might be helpful to assess the therapeutic effect and prognosis.

  16. Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2008-02-01

    Full Text Available Abstract Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G, were established for Thogoto virus (THOV GP and Autographa california multiple NPV (AcMNPV GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes.

  17. Compositional and conformational analysis of yam proteins by near infrared fourier transform Raman spectroscopy.

    Science.gov (United States)

    Liao, Yu-Hsiu; Wang, Chih-Hsien; Tseng, Chin-Yin; Chen, Hsiu-Ling; Lin, Long-Liu; Chen, Wenlung

    2004-12-29

    Fourier transform (FT)-Raman spectroscopy was employed to study the molecular structure of yam proteins isolated from three commonly consumed yam species including Dioscorea alata L., D. alata L. var. purpurea, and Dioscorea japonica. Although D. alata L. and D. alata L. var. purpurea consisted of similar amino acid residues, they still exhibited significant differences in conformational arrangement. The secondary structure of D. alata L. was mainly an alpha-helix, while D. alata L. var. purpurea was mostly in antiparallel beta-sheets. In contrast, D. japonica, which belongs to a different species, exhibited explicit differences in amino acid compositions and molecular structures of which the conformation was a mixed form of alpha-helices and antiparallel beta-sheets. FT-Raman directly proved the existence of S-S in yam proteins, implying that oligomer formation in yam proteins might be due to disulfide linking of dioscorin (32 kDa). The microenvironment of aromatic amino acids and the state of S-S in yam proteins were also discussed.

  18. Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

    International Nuclear Information System (INIS)

    Jhun, E.; Jhun, B.H.; Jones, L.R.; Jung, C.Y.

    1991-01-01

    The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100 degree C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein

  19. Rapid, large-scale purification and characterization of Ada protein (O sup 6 methylguanine-DNA methyltransferase) of E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharyya, D.; Tano, K.; Bunick, G.J.; Uberbacher, E.C.; Mitra, S. (Oak Ridge National Laboratory, TN (USA)); Behnke, W.D. (Univ. of Cincinnati College of Medicine, OH (USA))

    1988-07-25

    The E. coli Ada protein (O{sup 6}-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E{sup 280nm}{sub 1%}) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O{sup 6}-methylguanine in DNA. Its reaction with O{sup 6}-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 {times} 10{sup 9} M{sup {minus}1} min{sup {minus}1} at 0{degree}C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low {alpha}-helical content and the radius of gyration of 23 {angstrom} indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.

  20. Ion-Exchange Sample Displacement Chromatography as a Method for Fast and Simple Isolation of Low- and High-Abundance Proteins from Complex Biological Mixtures

    Directory of Open Access Journals (Sweden)

    Martina Srajer Gajdosik

    2014-01-01

    Full Text Available Sample displacement chromatography (SDC in reversed phase and ion-exchange modes was introduced at the end of 1980s. This chromatographic method was first used for preparative purification of synthetic peptides, and subsequently adapted for protein fractionation, mainly in anion-exchange mode. In the past few years, SDC has been successfully used for enrichment of low- and medium-abundance proteins from complex biological fluids on both monolithic and bulk chromatographic supports. If aqueous mobile phase is used with the application of mild chromatographic conditions, isolated proteins are not denatured and can also keep their biological activity. In this paper, the use of SDC in anion-exchange mode on a high-capacity chromatographic resin for separation of proteins from complex biological mixtures such as human plasma is demonstrated. By use of three and more columns coupled in series during sample application, and subsequent parallel elution of detached columns, additional separation of bound proteins was achieved. Highly enriched human serum albumin fraction and a number of physiologically active medium- and low-abundance proteins could be fractionated and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS and matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS. The use of the aforementioned columns that can be sanitized with 1 M sodium hydroxide for further application of SDC in biotechnology and food technology was discussed.

  1. Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

    Science.gov (United States)

    Wang, Wei-Qing; Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-04-01

    Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (Pseeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. © 2015 American Society of Plant Biologists. All Rights Reserved.

  2. How proteases from Enterococcus faecalis contribute to its resistance to short alpha-helical antimicrobial peptides

    Czech Academy of Sciences Publication Activity Database

    Nešuta, Ondřej; Buděšínský, Miloš; Hadravová, Romana; Monincová, Lenka; Humpolíčková, Jana; Čeřovský, Václav

    2017-01-01

    Roč. 75, č. 7 (2017), č. článku ftx091. ISSN 2049-632X R&D Projects: GA TA ČR(CZ) TA04010638; GA MZd(CZ) NV16-27726A Institutional support: RVO:61388963 Keywords : antimicrobial peptides * biofilm * C-terminal deamidation * gelatinase * protease Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 2.335, year: 2016

  3. Molecular dynamics study of the solvation of an alpha-helical transmembrane peptide by DMSO

    NARCIS (Netherlands)

    Duarte, A.M.; Mierlo, van C.P.M.; Hemminga, M.A.

    2008-01-01

    10-ns molecular dynamics study of the solvation of a hydrophobic transmembrane helical peptide in dimethyl sulfoxide (DMSO) is presented. The objective is to analyze how this aprotic polar solvent is able to solvate three groups of amino acid residues (i.e., polar, apolar, and charged) that are

  4. Double-brush Langmuir-Blodgett monolayers of alpha-helical diblock copolypeptides

    NARCIS (Netherlands)

    Nguyen, Le-Thu T.; Vorenkamp, Eltjo J.; Daumont, Christophe J. M.; ten Brinke, Gerrit; Schouten, Arend J.; Vukovic, Ivana; Loos, Katja

    2010-01-01

    The synthesis of amphiphilic diblock copolypeptides consisting of poly(alpha-L-glutamic acid) (PLGA) and poly(gamma-methyl-L-glutamate-ran-gamma-stearyl-L-glutamate) with 30 mol % of stearyl substituents (PMLGSLG) and their monolayer behavior at the air-water interface have been studied.

  5. Rational Design of Alpha-Helical Antimicrobial Peptides: Do's and Don'ts

    DEFF Research Database (Denmark)

    Uggerhøj, Lars Erik; Poulsen, Tanja Juul; Munk, Jens Kristian

    2015-01-01

    Antimicrobial peptides (AMPs) are promising candidates for battling multiresistant bacteria. Despite extensive research, structure–activity relationships of AMPs are not fully understood, and there is a lack of structural data relating to AMPs in lipids. Here we present the NMR structure of anoplin...... (GLLKRIKTLL-NH2) in a micellar environment. A vast library of substitutions was designed and tested for antimicrobial and hemolytic activity, as well as for changes in structure and lipid interactions. This showed that improvement of antimicrobial activity without concomitant introduction of strong hemolytic...

  6. MZDASoft: a software architecture that enables large-scale comparison of protein expression levels over multiple samples based on liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    Ghanat Bari, Mehrab; Ramirez, Nelson; Wang, Zhiwei; Zhang, Jianqiu Michelle

    2015-10-15

    Without accurate peak linking/alignment, only the expression levels of a small percentage of proteins can be compared across multiple samples in Liquid Chromatography/Mass Spectrometry/Tandem Mass Spectrometry (LC/MS/MS) due to the selective nature of tandem MS peptide identification. This greatly hampers biomedical research that aims at finding biomarkers for disease diagnosis, treatment, and the understanding of disease mechanisms. A recent algorithm, PeakLink, has allowed the accurate linking of LC/MS peaks without tandem MS identifications to their corresponding ones with identifications across multiple samples collected from different instruments, tissues and labs, which greatly enhanced the ability of comparing proteins. However, PeakLink cannot be implemented practically for large numbers of samples based on existing software architectures, because it requires access to peak elution profiles from multiple LC/MS/MS samples simultaneously. We propose a new architecture based on parallel processing, which extracts LC/MS peak features, and saves them in database files to enable the implementation of PeakLink for multiple samples. The software has been deployed in High-Performance Computing (HPC) environments. The core part of the software, MZDASoft Parallel Peak Extractor (PPE), can be downloaded with a user and developer's guide, and it can be run on HPC centers directly. The quantification applications, MZDASoft TandemQuant and MZDASoft PeakLink, are written in Matlab, which are compiled with a Matlab runtime compiler. A sample script that incorporates all necessary processing steps of MZDASoft for LC/MS/MS quantification in a parallel processing environment is available. The project webpage is http://compgenomics.utsa.edu/zgroup/MZDASoft. The proposed architecture enables the implementation of PeakLink for multiple samples. Significantly more (100%-500%) proteins can be compared over multiple samples with better quantification accuracy in test cases. MZDASoft

  7. Metabolic fingerprinting of high-fat plasma samples processed by centrifugation- and filtration-based protein precipitation delineates significant differences in metabolite information coverage.

    Science.gov (United States)

    Barri, Thaer; Holmer-Jensen, Jens; Hermansen, Kjeld; Dragsted, Lars O

    2012-03-09

    Metabolomics and metabolic fingerprinting are being extensively employed for improved understanding of biological changes induced by endogenous or exogenous factors. Blood serum or plasma samples are often employed for metabolomics studies. Plasma protein precipitation (PPP) is currently performed in most laboratories before LC-MS analysis. However, the impact of fat content in plasma samples on metabolite coverage has not previously been investigated. Here, we have studied whether PPP procedures influence coverage of plasma metabolites from high-fat plasma samples. An optimized UPLC-QTOF/MS metabolic fingerprinting approach and multivariate modeling (PCA and OPLS-DA) were utilized for finding characteristic metabolite changes induced by two PPP procedures; centrifugation and filtration. We used 12-h fasting samples and postprandial samples collected at 2h after a standardized high-fat protein-rich meal in obese non-diabetic subjects recruited in a dietary intervention. The two PPP procedures as well as external and internal standards (ISs) were used to track errors in response normalization and quantification. Remarkably and sometimes uniquely, the fPPP, but not the cPPP approach, recovered not only high molecular weight (HMW) lipophilic metabolites, but also small molecular weight (SMW) relatively polar metabolites. Characteristic SMW markers of postprandial samples were aromatic and branched-chain amino acids that were elevated (p<0.001) as a consequence of the protein challenge. In contrast, some HMW lipophilic species, e.g. acylcarnitines, were moderately lower (p<0.001) in postprandial samples. LysoPCs were largely unaffected. In conclusion, the fPPP procedure is recommended for processing high-fat plasma samples in metabolomics studies. While method improvements presented here were clear, use of several ISs revealed substantial challenges to untargeted metabolomics due to large and variable matrix effects. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min-Sung; Saunders, Adam M.; Hamaoka, Brent Y.; Beachy, Philip A.; Leahy, Daniel J. (Stanford-MED); (JHU)

    2011-09-20

    Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-{angstrom} crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, {alpha}-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

  9. Identification of a key structural element for protein folding within beta-hairpin turns.

    Science.gov (United States)

    Kim, Jaewon; Brych, Stephen R; Lee, Jihun; Logan, Timothy M; Blaber, Michael

    2003-05-09

    Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.

  10. Insights into alternative prion protein topologies induced under high hydrostatic pressure

    Energy Technology Data Exchange (ETDEWEB)

    Torrent, Joan [INSERM U128, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France); Alvarez-Martinez, Maria Teresa [INSERM U431, IFR 122, Place Eugene Bataillon, F-34095 Montpellier cedex 5 (France); Heitz, Frederic [CRBM, CNRS-UPR 1086, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France); Liautard, Jean-Pierre [INSERM U431, IFR 122, Place Eugene Bataillon, F-34095 Montpellier cedex 5 (France); Balny, Claude [INSERM U128, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France); Lange, Reinhard [INSERM U128, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France)

    2004-04-14

    The critical step in the pathogenesis of transmissible spongiform encephalopathies (TSEs) appears to be a conformational transition of a normal prion protein (PrP{sup C}) into a misfolded isoform (PrP{sup Sc}). To gain insight into the structural conversion of the prion protein we have exploited the use of high hydrostatic pressure combined with various spectroscopic techniques. In vitro transitions of the recombinant PrP to a scrapie-like form have never resulted in an infectious structure. It is our hypothesis that the acquisition of the disease-causing conformation depends on folding pathways which are difficult to attain. We attempt to favour, via specific reaction conditions at high pressure, alternative routes of misfolding leading to a stable infectious amyloidogenic conformer. Our results have demonstrated the potential of high pressure to reveal various prion structural changes, which are inaccessible by conventional methods. Especially, we have characterized a pressure-induced conformer in which the normal {alpha}-helical structure is changed into a highly aggregated {beta}-sheet conformation showing markedly increased resistance to proteolysis (key markers of potential infectious agents). Our work may have important implications, not only for ultimately proving the protein-only hypothesis and for understanding the basic mechanism of the disease, but also for developing preventative and therapeutic measures.

  11. A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy.

    Science.gov (United States)

    Gaupels, Frank; Knauer, Torsten; van Bel, Aart J E

    2008-01-01

    This study investigated advantages and drawbacks of two sieve-tube sap sampling methods for comparison of phloem proteins in powdery mildew-infested vs. non-infested Hordeum vulgare plants. In one approach, sieve tube sap was collected by stylectomy. Aphid stylets were cut and immediately covered with silicon oil to prevent any contamination or modification of exudates. In this way, a maximum of 1muL pure phloem sap could be obtained per hour. Interestingly, after pathogen infection exudation from microcauterized stylets was reduced to less than 40% of control plants, suggesting that powdery mildew induced sieve tube-occlusion mechanisms. In contrast to the laborious stylectomy, facilitated exudation using EDTA to prevent calcium-mediated callose formation is quick and easy with a large volume yield. After two-dimensional (2D) electrophoresis, a digital overlay of the protein sets extracted from EDTA solutions and stylet exudates showed that some major spots were the same with both sampling techniques. However, EDTA exudates also contained large amounts of contaminative proteins of unknown origin. A combinatory approach may be most favourable for studies in which the protein composition of phloem sap is compared between control and pathogen-infected plants. Facilitated exudation may be applied for subtractive identification of differentially expressed proteins by 2D/mass spectrometry, which requires large amounts of protein. A reference gel loaded with pure phloem sap from stylectomy may be useful for confirmation of phloem origin of candidate spots by digital overlay. The method provides a novel opportunity to study differential expression of phloem proteins in monocotyledonous plant species.

  12. UPF201 Archaeal Specific Family Members Reveals Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.N.; Swaminathan, S.; Burley, S. K.

    2008-12-11

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  13. Analyzing the Effects of Climate Factors on Soybean Protein, Oil Contents, and Composition by Extensive and High-Density Sampling in China.

    Science.gov (United States)

    Song, Wenwen; Yang, Ruping; Wu, Tingting; Wu, Cunxiang; Sun, Shi; Zhang, Shouwei; Jiang, Bingjun; Tian, Shiyan; Liu, Xiaobing; Han, Tianfu

    2016-05-25

    From 2010 to 2013, 763 soybean samples were collected from an extensive area of China. The correlations between seed compositions and climate data were analyzed. The contents of crude protein and water-soluble protein, total amount of protein plus oil, and most of the amino acids were positively correlated with an accumulated temperature ≥15 °C (AT15) and the mean daily temperature (MDT) but were negatively correlated with hours of sunshine (HS) and diurnal temperature range (DTR). The correlations of crude oil and most fatty acids with climate factors were opposite to those of crude protein. Crude oil content had a quadratic regression relationship with MDT, and a positive correlation between oil content and MDT was found when the daily temperature was soybean protein and oil contents. The study illustrated the effects of climate factors on soybean protein and oil contents and proposed agronomic practices for improving soybean quality in different regions of China. The results provide a foundation for the regionalization of high-quality soybean production in China and similar regions in the world.

  14. A novel one-class SVM based negative data sampling method for reconstructing proteome-wide HTLV-human protein interaction networks.

    Science.gov (United States)

    Mei, Suyu; Zhu, Hao

    2015-01-26

    Protein-protein interaction (PPI) prediction is generally treated as a problem of binary classification wherein negative data sampling is still an open problem to be addressed. The commonly used random sampling is prone to yield less representative negative data with considerable false negatives. Meanwhile rational constraints are seldom exerted on model selection to reduce the risk of false positive predictions for most of the existing computational methods. In this work, we propose a novel negative data sampling method based on one-class SVM (support vector machine, SVM) to predict proteome-wide protein interactions between HTLV retrovirus and Homo sapiens, wherein one-class SVM is used to choose reliable and representative negative data, and two-class SVM is used to yield proteome-wide outcomes as predictive feedback for rational model selection. Computational results suggest that one-class SVM is more suited to be used as negative data sampling method than two-class PPI predictor, and the predictive feedback constrained model selection helps to yield a rational predictive model that reduces the risk of false positive predictions. Some predictions have been validated by the recent literature. Lastly, gene ontology based clustering of the predicted PPI networks is conducted to provide valuable cues for the pathogenesis of HTLV retrovirus.

  15. 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,

  16. Search for Breast Cancer Biomarkers in Fractionated Serum Samples by Protein Profiling With SELDI-TOF MS

    NARCIS (Netherlands)

    Opstal - van Winden, A.W.J.; Beijnen, J.H.; de Loof, A.; van Heerde, W.L.; Vermeulen, R.; Peeters, P.H.M.; van Gils, C.H.

    2012-01-01

    BackgroundMany high-abundant acute phase reactants have been previously detected as potential breast cancer biomar-kers. However, they are unlikely to be specific for breast cancer. Cancer-specific biomarkers are thought to be among the lower abundant proteins.MethodsWe aimed to detect lower

  17. Proteomics computational analyses suggest that the bornavirus glycoprotein is a class III viral fusion protein (γ penetrene

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2009-09-01

    Full Text Available Abstract Background Borna disease virus (BDV is the type member of the Bornaviridae, a family of viruses that induce often fatal neurological diseases in horses, sheep and other animals, and have been proposed to have roles in certain psychiatric diseases of humans. The BDV glycoprotein (G is an extensively glycosylated protein that migrates with an apparent molecular mass of 84,000 to 94,000 kilodaltons (kDa. BDV G is post-translationally cleaved by the cellular subtilisin-like protease furin into two subunits, a 41 kDa amino terminal protein GP1 and a 43 kDa carboxyl terminal protein GP2. Results Class III viral fusion proteins (VFP encoded by members of the Rhabdoviridae, Herpesviridae and Baculoviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Proteomics computational analyses suggest that the structural/functional motifs that characterize class III VFP are located collinearly in BDV G. Structural models were established for BDV G based on the post-fusion structure of a prototypic class III VFP, vesicular stomatitis virus glycoprotein (VSV G. Conclusion These results suggest that G encoded by members of the Bornavirdae are class III VFPs (gamma-penetrenes.

  18. Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

    Science.gov (United States)

    Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

    2009-10-01

    Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

  19. Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation1

    Science.gov (United States)

    Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-01-01

    Germination and thermoinhibition in lettuce (Lactuca sativa ‘Jianyexianfeng No. 1’) seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. PMID:25736209

  20. Estimasi Sintesis Protein Mikrobia Rumen Menggunakan Ekskresi Derivat Purin dalam Urin dengan Teknik Spot Sampling pada Kambing Bligon dan Kambing Kejobong

    Directory of Open Access Journals (Sweden)

    Dianestu Putra

    2016-11-01

    Full Text Available This study were aimed to determine the correlation between concentration of purine derivatives (PD in spot sample with PD total excretion in Bligon and Kejobong goats and determine the appropriate sampling time, in order to predicting microbial protein synthesis in both breeds. Six male Bligon goats and six male Kejobong goats with age range from 8 to 14 months and body weight from 16 to 21 kg were placed in metabolism cages. Peanut straw and water were given to both groups of goats through ad libitum feeding and drinking. The study was done in 14 days for adaptation, 3 days for collection. Sample of feeds, feed residues, and feces were collected and then analyzed to determine dry matter and organic matter content. Spot urine and the total daily urine samples were also collected. Samples collection of spot sampling technique was run by taking the urine periodically with 3 hours intervals at 24 hours. Urine samples were analyzed for the content of creatinine and PD which includes allantoin, uric acid, xanthine, and hypoxanthine. Data were tested for the correlation between concentration of PD spot urine sample with total PD daily excretion. It is known that the concentration of PD and creatinine (µmol/L for Bligon were 1,418.40 and 202.85 respectively, while for Kejobong were 1,547.40 and 219.68 respectively. Total excretion of PD, allantoin, uric acid, xanthyne and hypoxanthine and creatinine (µmol/W0,75/day for Bligon were 114.14, 95.86, 17.31, 0.97, and 16.40 respectively, with microbial protein synthesis efficiency was 4.61 g N/kg degraded of organic matter in rumen (DOMR. Total excretion of PD allantoin, uric acid, xanthyne and hypoxanthine and creatinine (µmol/W0,75/day for Kejobong were 180.18, 158.17, 20.60, 1.40, and 24.87 respectively, with microbial protein synthesis efficiency was 6.90 g N/kg DOMR. Based on this study also known that the best time for spot sampling to determine the total excretion of PD in Bligon was in the range

  1. Structural Basis for dsRNA Recognition by NS1 Protein of Influenza A Virus

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, A.; Wong, S; Yuan, Y

    2009-01-01

    Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel alpha-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

  2. The Plasmodium PHIST and RESA-Like Protein Families of Human and Rodent Malaria Parasites

    Science.gov (United States)

    Moreira, Cristina K.; Naissant, Bernina; Coppi, Alida; Bennett, Brandy L.; Aime, Elena; Franke-Fayard, Blandine; Janse, Chris J.; Coppens, Isabelle; Sinnis, Photini; Templeton, Thomas J.

    2016-01-01

    The phist gene family has members identified across the Plasmodium genus, defined by the presence of a domain of roughly 150 amino acids having conserved aromatic residues and an all alpha-helical structure. The family is highly amplified in P. falciparum, with 65 predicted genes in the genome of the 3D7 isolate. In contrast, in the rodent malaria parasite P. berghei 3 genes are identified, one of which is an apparent pseudogene. Transcripts of the P. berghei phist genes are predominant in schizonts, whereas in P. falciparum transcript profiles span different asexual blood stages and gametocytes. We pursued targeted disruption of P. berghei phist genes in order to characterize a simplistic model for the expanded phist gene repertoire in P. falciparum. Unsuccessful attempts to disrupt P. berghei PBANKA_114540 suggest that this phist gene is essential, while knockout of phist PBANKA_122900 shows an apparent normal progression and non-essential function throughout the life cycle. Epitope-tagging of P. falciparum and P. berghei phist genes confirmed protein export to the erythrocyte cytoplasm and localization with a punctate pattern. Three P. berghei PEXEL/HT-positive exported proteins exhibit at least partial co-localization, in support of a common vesicular compartment in the cytoplasm of erythrocytes infected with rodent malaria parasites. PMID:27022937

  3. Secondary Structure Preferences of Mn2+ Binding Sites in Bacterial Proteins

    Directory of Open Access Journals (Sweden)

    Tatyana Aleksandrovna Khrustaleva

    2014-01-01

    Full Text Available 3D structures of proteins with coordinated Mn2+ ions from bacteria with low, average, and high genomic GC-content have been analyzed (149 PDB files were used. Major Mn2+ binders are aspartic acid (6.82% of Asp residues, histidine (14.76% of His residues, and glutamic acid (3.51% of Glu residues. We found out that the motif of secondary structure “beta strand-major binder-random coil” is overrepresented around all the three major Mn2+ binders. That motif may be followed by either alpha helix or beta strand. Beta strands near Mn2+ binding residues should be stable because they are enriched by such beta formers as valine and isoleucine, as well as by specific combinations of hydrophobic and hydrophilic amino acid residues characteristic to beta sheet. In the group of proteins from GC-rich bacteria glutamic acid residues situated in alpha helices frequently coordinate Mn2+ ions, probably, because of the decrease of Lys usage under the influence of mutational GC-pressure. On the other hand, the percentage of Mn2+ sites with at least one amino acid in the “beta strand-major binder-random coil” motif of secondary structure (77.88% does not depend on genomic GC-content.

  4. Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

    Directory of Open Access Journals (Sweden)

    Caroline Kulangara

    Full Text Available In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1. In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.

  5. Tissue and serum samples of patients with papillary thyroid cancer with and without benign background demonstrate different altered expression of proteins

    Directory of Open Access Journals (Sweden)

    Mardiaty Iryani Abdullah

    2016-09-01

    Full Text Available Background Papillary thyroid cancer (PTC is mainly diagnosed using fine-needle aspiration biopsy. This most common form of well-differentiated thyroid cancer occurs with or without a background of benign thyroid goiter (BTG. Methods In the present study, a gel-based proteomics analysis was performed to analyse the expression of proteins in tissue and serum samples of PTC patients with (PTCb; n = 6 and without a history of BTG (PTCa; n = 8 relative to patients with BTG (n = 20. This was followed by confirmation of the levels of proteins which showed significant altered abundances of more than two-fold difference (p < 0.01 in the tissue and serum samples of the same subjects using ELISA. Results The data of our study showed that PTCa and PTCb distinguish themselves from BTG in the types of tissue and serum proteins of altered abundance. While higher levels of alpha-1 antitrypsin (A1AT and heat shock 70 kDa protein were associated with PTCa, lower levels of A1AT, protein disulfide isomerase and ubiquitin-conjugating enzyme E2 N seemed apparent in the PTCb. In case of the serum proteins, higher abundances of A1AT and alpha 1-beta glycoprotein were detected in PTCa, while PTCb was associated with enhanced apolipoprotein A-IV and alpha 2-HS glycoprotein (AHSG. The different altered expression of tissue and serum A1AT as well as serum AHSG between PTCa and PTCb patients were also validated by ELISA. Discussion The distinctive altered abundances of the tissue and serum proteins form preliminary indications that PTCa and PTCb are two distinct cancers of the thyroid that are etiologically and mechanistically different although it is currently not possible to rule out that they may also be due other reasons such as the different stages of the malignant disease. These proteins stand to have a potential use as tissue or serum biomarkers to discriminate the three different thyroid neoplasms although this requires further validation in clinically

  6. Effect of blood contamination on results of dipstick evaluation and urine protein-to-urine creatinine ratio for urine samples from dogs and cats.

    Science.gov (United States)

    Vientós-Plotts, Aida I; Behrend, Ellen N; Welles, Elizabeth G; Chew, Dennis J; Gaillard, Philippe R; Busler, Jessica N; Lee, Hollie P

    2018-05-01

    OBJECTIVE To evaluate effects of blood contamination on dipstick results, specific gravity (SG), and urine protein-to-urine creatinine ratio (UPCR) for urine samples from dogs and cats. SAMPLE Urine samples collected from 279 dogs and 120 cats. PROCEDURES Urine pools were made for each species (dogs [n = 60] and cats [30]). Blood was added to an aliquot of a pool, and serial dilutions were prepared with the remaining urine. Color and dipstick variables were recorded, and SG and UPCR were measured. For cats, 1 set of pools was used; for dogs, 2 sets were used. Comparisons were made between undiluted urine and spiked urine samples for individual colors. Repeated-measures ANOVA on ranks was used to compare dipstick scores and UPCR results; χ 2 tests were used to compare proteinuria categorizations (nonproteinuric, borderline, or proteinuric). RESULTS Any blood in the urine resulted in significantly increased dipstick scores for blood. In both species, scores for bilirubin and ketones, pH, and SG were affected by visible blood contamination. No significant difference for the dipstick protein reagent results was evident until a sample was visibly hematuric. The UPCR was significantly increased in dark yellow samples of both species. Proteinuria categorizations differed significantly between undiluted urine and urine of all colors, except light yellow. CONCLUSIONS AND CLINICAL RELEVANCE Any degree of blood contamination affected results of dipstick analysis. Effects depended on urine color and the variable measured. Microscopic blood contamination may affect the UPCR; thus, blood contamination may be a differential diagnosis for proteinuria in yellow urine samples.

  7. Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

    Science.gov (United States)

    Ikeda, Mayumi; Ishima, Yu; Shibata, Akitomo; Chuang, Victor T G; Sawa, Tomohiro; Ihara, Hideshi; Watanabe, Hiroshi; Xian, Ming; Ouchi, Yuya; Shimizu, Taro; Ando, Hidenori; Ukawa, Masami; Ishida, Tatsuhiro; Akaike, Takaaki; Otagiri, Masaki; Maruyama, Toru

    2017-05-29

    Hydrogen sulfide (H 2 S) signaling involves polysulfide (RSS n SR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α 1 -anti-trypsin, α 1 -acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. β-trace protein as a diagnostic marker for perilymphatic fluid fistula: a prospective controlled pilot study to test a sample collection technique.

    Science.gov (United States)

    Bachmann-Harildstad, Gregor; Stenklev, Niels Christian; Myrvoll, Elin; Jablonski, Greg; Klingenberg, Olav

    2011-01-01

    The diagnosis of perilymphatic fluid (PLF) fistula is still challenging. Perilymphatic fluid fistula is one possible complication after stapedotomy or cochlear implant surgery. We have performed a prospective diagnostic pilot study to further investigate β-trace protein (β-TP) as a marker for PLF fistula. In this pilot study, we tested the sensitivity of the β-TP marker using a simple method for sample collection from the tympanic cavity. Prospective controlled diagnostic study. Two-center tertiary referral hospitals. A total of 35 adult patients undergoing ear surgery were included. Subjects were divided into 2 groups: 1) 19 patients undergoing stapedotomy were investigated for PLF fistula in samples obtained from the tympanic cavity and 2) 16 patients undergoing myringoplasty were investigated for PLF fistula in samples from the tympanic cavity. This group served as the control. Mean age +/- SD at surgery was 49.9 +/- 8.0 years in the study group and 39.69 +/- 15.47 years in the control group. β-Trace protein (prostaglandin D synthase) in tympanic cavity samples and serum samples was analyzed. The samples were collected by gradually filling the tympanic cavity with 100 to 200 μl sodium chloride and by immediately collecting a volume of 60 to 100 μl in a mucus specimen set container. The concentration of β-TP was quantified using laser nephelometry. The median β-TP in the study group was 0.8 mg/L (range, 0.05-4.5 mg/L). In the control group, the median β-TP value was 0.16 mg/L (range, 0.01-0.36 mg/L). Thirty-five percent of the values in the study group were below the highest value in the negative control group. The β-TP values of the tympanic cavity samples were significantly higher in the study group than in controls (p = 0.0001). The serum values were 0.55 +/- 0.18 and 0.53 +/- 0.11 mg/L, respectively. It may be feasible to test for PLF fistula using β-TP in samples from the tympanic cavity. Our results, however, suggest a relative low diagnostic

  9. An evaluation of multiplex bead-based analysis of cytokines and soluble proteins in archived lithium heparin plasma, EDTA plasma and serum samples

    DEFF Research Database (Denmark)

    Brøndum, Line; Sørensen, Brita Singers; Eriksen, Jesper Grau

    2016-01-01

    -13, and VEGF, LH plasma levels vs. EDTA plasma: IL-2 and IL-4. CONCLUSION: Stored serum, LH plasma, and EDTA plasma from clinical trials can be used for analysis of circulating cytokines and proteins. Variations in measurements occur, but are within reasonable ranges. The optimal type of media...... in plasma and serum from 86 head and neck cancer patients and 33 controls were evaluated: EGFR, leptin, OPN, VEGFR-1, VEGFR-2, IL-2, IL-13, PDGF-bb, TNF, PAI-1, SDF-1a, IL-4, IL-6, IL-8, eotaxin, G-CSF, VEGF, GRO-a, and HGF. RESULTS: The correlation between measurements of the same samples analyzed...... on different dates was reasonable. However, samples run on different dates could exhibit different absolute values. The 75th percentile of the fold differences for samples run on different dates was 2.2. No significant difference was found between one and four freeze-thaw cycles (except for HGF...

  10. Estimation of Daily Proteinuria in Patients with Amyloidosis by Using the Protein-To-Creatinine ratio in Random Urine Samples.

    Science.gov (United States)

    Talamo, Giampaolo; Mir Muhammad, A; Pandey, Manoj K; Zhu, Junjia; Creer, Michael H; Malysz, Jozef

    2015-02-11

    Measurement of daily proteinuria in patients with amyloidosis is recommended at the time of diagnosis for assessing renal involvement, and for monitoring disease activity. Renal involvement is usually defined by proteinuria >500 mg/day. We evaluated the accuracy of the random urine protein-to-creatinine ratio (Pr/Cr) in predicting 24 hour proteinuria in patient with amyloidosis. We compared results of random urine Pr/Cr ratio and concomitant 24-hour urine collections in 44 patients with amyloidosis. We found a strong correlation (Spearman's ρ=0.874) between the Pr/Cr ratio and the 24 hour urine protein excretion. For predicting renal involvement, the optimal cut-off point of the Pr/Cr ratio was 715 mg/g. The sensitivity and specificity for this point were 91.8% and 95.5%, respectively, and the area under the curve value was 97.4%. We conclude that the random urine Pr/Cr ratio could be useful in the screening of renal involvement in patients with amyloidosis. If validated in a prospective study, the random urine Pr/Cr ratio could replace the 24 hour urine collection for the assessment of daily proteinuria and presence of nephrotic syndrome in patients with amyloidosis.

  11. Estimation of daily proteinuria in patients with amyloidosis by using the protein-to-creatinine ratio in random urine sample

    Directory of Open Access Journals (Sweden)

    Giampaolo Talamo

    2015-02-01

    Full Text Available Measurement of daily proteinuria in patients with amyloidosis is recommended at the time of diagnosis for assessing renal involvement, and for monitoring disease activity. Renal involvement is usually defined by proteinuria >500 mg/day. We evaluated the accuracy of the random urine protein-to-creatinine ratio (Pr/Cr in predicting 24 hour proteinuria in patient with amyloidosis. We com- pared results of random urine Pr/Cr ratio and concomitant 24-hour urine collections in 44 patients with amyloidosis. We found a strong correlation (Spearman’s ρ=0.874 between the Pr/Cr ratio and the 24 hour urine protein excretion. For predicting renal involvement, the optimal cut-off point of the Pr/Cr ratio was 715 mg/g. The sensitivity and specificity for this point were 91.8% and 95.5%, respectively, and the area under the curve value was 97.4%. We conclude that the random urine Pr/Cr ratio could be useful in the screening of renal involvement in patients with amyloidosis. If validated in a prospective study, the random urine Pr/Cr ratio could replace the 24 hour urine collection for the assessment of daily proteinuria and presence of nephrotic syndrome in patients with amyloidosis.

  12. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  13. Density functional calculations of backbone 15N shielding tensors in beta-sheet and turn residues of protein G

    International Nuclear Information System (INIS)

    Cai Ling; Kosov, Daniel S.; Fushman, David

    2011-01-01

    We performed density functional calculations of backbone 15 N shielding tensors in the regions of beta-sheet and turns of protein G. The calculations were carried out for all twenty-four beta-sheet residues and eight beta-turn residues in the protein GB3 and the results were compared with the available experimental data from solid-state and solution NMR measurements. Together with the alpha-helix data, our calculations cover 39 out of the 55 residues (or 71%) in GB3. The applicability of several computational models developed previously (Cai et al. in J Biomol NMR 45:245–253, 2009) to compute 15 N shielding tensors of alpha-helical residues is assessed. We show that the proposed quantum chemical computational model is capable of predicting isotropic 15 N chemical shifts for an entire protein that are in good correlation with experimental data. However, the individual components of the predicted 15 N shielding tensor agree with experiment less well: the computed values show much larger spread than the experimental data, and there is a profound difference in the behavior of the tensor components for alpha-helix/turns and beta-sheet residues. We discuss possible reasons for this.

  14. Visual detection of copper(II) ions in blood samples by controlling the leaching of protein-capped gold nanoparticles.

    Science.gov (United States)

    Lee, Yen-Fei; Deng, Ting-Wei; Chiu, Wei-Jane; Wei, Tsao-Yen; Roy, Prathik; Huang, Chih-Ching

    2012-04-21

    We have developed a simple, low-cost, paper-based probe for the selective colorimetric detection of copper ions (Cu(2+)) in aqueous solutions. The bovine serum albumin (BSA)-modified 13.3-nm Au nanoparticle (BSA-Au NP) probe was designed to detect Cu(2+) ions using lead ions (Pb(2+)) and 2-mercaptoethanol (2-ME) as leaching agents in a glycine-NaOH (pH 12.0) solution. In addition, a nitrocellulose membrane (NCM) was used to trap the BSA-Au NPs, leading to the preparation of a nanocomposite film consisting of a BSA-Au NP-decorated membrane (BSA-Au NPs/NCM). The BSA-Au NPs probe operates on the principle that Cu deposition on the surface of the BSA-Au NPs inhibits their leaching ability, which is accelerated by Pb(2+) ions in the presence of 2-ME. Under optimal solution conditions (5 mM glycine-NaOH (pH 12.0), Pb(2+) (50 μM), and 2-ME (1.0 M)), the Pb(2+)/2-ME-BSA-Au NPs/NCM enabled the detection of Cu(2+) at nanomolar concentrations in aqueous solutions by the naked eye with high selectivity (at least 100-fold over other metal ions). In addition, this cost-effective probe allowed for the rapid and simple determination of Cu(2+) ions in not only natural water samples but also in a complex biological sample (in this case, blood sample).

  15. 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.

  16. The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

    Science.gov (United States)

    Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

    2015-10-01

    Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

  17. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    Energy Technology Data Exchange (ETDEWEB)

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  18. Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

    Directory of Open Access Journals (Sweden)

    Yolanda Bel

    2017-04-01

    Full Text Available Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w. If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices.

  19. Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins

    DEFF Research Database (Denmark)

    Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per

    2018-01-01

    in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining...... the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced...... accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data....

  20. Flow injection on-line oxidizing fluorometry coupled to dialysis sampling for the study of carbamazepine-protein binding

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Zhiqi [School of Chemistry and Materials Science, Shaanxi Normal University, Xi' an 710062 (China)]. E-mail: zqzhang@snnu.edu.cn; Liang Guoxi [School of Chemistry and Materials Science, Shaanxi Normal University, Xi' an 710062 (China)

    2005-04-22

    The mechanism of binding of carbamazepine (CBZ) with bovine serum albumin (BSA) has been investigated in vitro based on a new flow injection fluorometry coupled to the technique of dialysis sampling. The CBZ and BSA were mixed in different molar ratios in 0.050 mol L{sup -1} phosphate buffer (containing 0.9% NaCl), pH 7.4, and incubated at 37 {+-} 0.5 deg. C in a water bath. The dialysis sampler was utilized to sample free CBZ from mixed solution with a relative dialytic efficiency of 7.6%. Then the CBZ in dialysis solution was injected into carrier and on-line oxidized by lead dioxide solid-phase reactor into fluorescent product with a maximum excitation wavelength of 355 nm and a maximum emission wavelength of 478 nm. The fluorescence intensity measured was linear proportional with the concentration of free CBZ in mixed solution over the range of 1 x 10{sup -5} to 2 x 10{sup -4} mol L{sup -1} with the detection limit of 6 x 10{sup -6} mol L{sup -1}. According to the fluorescence measurement results from mixed solution, the association constant (K) estimated for CBZ-BSA binding and the number of the binding site (n) with Scatchard analysis were 1.08 x 10{sup 4} L mol{sup -1} and 0.94, respectively. Stern-Volmer plots indicated the presence of dynamic component in the quenching mechanism. The acting force was suggested to be mainly hydrophobic and the distance between the acceptor and donor was 3.12 nm. The estimated binding parameters agreed well with literature values.

  1. Evaluation of the use of purine derivatives:creatinine ratio in spot urine samples as an index of microbial protein supply in Yerli Kara crossbred cattle

    International Nuclear Information System (INIS)

    Cetinkaya, N.; Ozdemir, H.; Gucus, A.I.; Ozcan, H.; Sogut, A.; Yaman, S.

    2004-01-01

    In Experiment I, response of daily purine derivatives (PD) excretion to feed intake in Yerli Kara crossbred (YK-C) cattle on state farms was measured. Animals were fed a mixed diet containing 30% wheat straw and 70% compounded feed. Crude protein and organic matter contents of the diet were 12.4% and 95%, respectively. In Experiment II, spot urine sampling technique was applied at state farm. Four Yerli Kara crossbred bulls with a mean live weight of 211.0 ± 41.3 kg were used. The experimental design, feeding and diet were the same as in Experiment I. The treatments were allocated according to a 4 x 4 Latin Square design. In Experiment III, spot urine sampling technique was applied at smallholder farms. Two to three kg of compound feed (crude protein 12%) containing 65% barley, 25% bran, 6% sunflower seed meal, 3% marmer dust and 1% mineral and vitamin mixture was offered in two parts, one in the morning (0730 h) and the other in the afternoon (1700 h). The ingredients in the compound feed were similar for all animals, but animals in Groups I, II and III received 1 to 2 kg/d of straw (crude protein 3%), grass hay (crude protein 7%), or both straw and grass hay respectively. In Experiment I, a significant correlation (R 2 =0.99) between PD excretion (Y, mmol/d) and digestible organic matter intake, DOMI (X, kg/d) for YK-C cattle was observed (Y = 12.5 + 19.7 X). Moreover, daily PD excretion (mmol/d) was correlated with the PDC index, which was defined as [PD molar concentration] / [Creatinine molar concentration] x kgW 0.75 . In Experiment II, the PDC index increased with level of intake. The coefficient of variation due to time of sampling for uric acid, allantoin, PD, creatinine, total-N, the PDC Index in spot urine samples were less than 5%. In Experiment III, the PDC index were 49.95 ± 13.5, 45.6 ± 13.0, 48.95 ± 15.3 for the three groups respectively. These values were similar to those for 60% intake level in Experiment I. Using the equation DOMI = 344 + 48

  2. Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture.

    Science.gov (United States)

    Eswaran, Jeyanthy; Bernad, Antonio; Ligos, Jose M; Guinea, Barbara; Debreczeni, Judit E; Sobott, Frank; Parker, Sirlester A; Najmanovich, Rafael; Turk, Benjamin E; Knapp, Stefan

    2008-01-01

    The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

  3. Distribution and evolution of stable single α-helices (SAH domains in myosin motor proteins.

    Directory of Open Access Journals (Sweden)

    Dominic Simm

    Full Text Available Stable single-alpha helices (SAHs are versatile structural elements in many prokaryotic and eukaryotic proteins acting as semi-flexible linkers and constant force springs. This way SAH-domains function as part of the lever of many different myosins. Canonical myosin levers consist of one or several IQ-motifs to which light chains such as calmodulin bind. SAH-domains provide flexibility in length and stiffness to the myosin levers, and may be particularly suited for myosins working in crowded cellular environments. Although the function of the SAH-domains in human class-6 and class-10 myosins has well been characterised, the distribution of the SAH-domain in all myosin subfamilies and across the eukaryotic tree of life remained elusive. Here, we analysed the largest available myosin sequence dataset consisting of 7919 manually annotated myosin sequences from 938 species representing all major eukaryotic branches using the SAH-prediction algorithm of Waggawagga, a recently developed tool for the identification of SAH-domains. With this approach we identified SAH-domains in more than one third of the supposed 79 myosin subfamilies. Depending on the myosin class, the presence of SAH-domains can range from a few to almost all class members indicating complex patterns of independent and taxon-specific SAH-domain gain and loss.

  4. Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

    Science.gov (United States)

    Zhou, Lijun; Lv, Zhiqiang; Shao, Jing; Xu, Ying; Luo, Xiaohong; Zhang, Yuming; Hu, Yang; Zhang, Wenji; Luo, Shuhong; Fang, Jianmin; Wang, Ying; Duan, Chaohui; Huang, Ruopan

    2016-09-01

    The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas. © 2015 Wiley Periodicals, Inc.

  5. Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

    Science.gov (United States)

    Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

    2003-04-01

    Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

  6. Interpreting meta-analysis according to the adequacy of sample size. An example using isoniazid chemoprophylaxis for tuberculosis in purified protein derivative negative HIV-infected individuals

    Directory of Open Access Journals (Sweden)

    Kristian Thorlund

    2010-04-01

    Full Text Available Kristian Thorlund1,2, Aranka Anema3, Edward Mills41Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, Canada; 2The Copenhagen Trial Unit, Centre for Clinical Intervention Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark; 3British Columbia Centre for Excellence in HIV/AIDS, University of British Columbia, Vancouver, British Columbia, Canada; 4Faculty of Health Sciences, University of Ottawa, Ottawa, Ontario, CanadaObjective: To illustrate the utility of statistical monitoring boundaries in meta-analysis, and provide a framework in which meta-analysis can be interpreted according to the adequacy of sample size. To propose a simple method for determining how many patients need to be randomized in a future trial before a meta-analysis can be deemed conclusive.Study design and setting: Prospective meta-analysis of randomized clinical trials (RCTs that evaluated the effectiveness of isoniazid chemoprophylaxis versus placebo for preventing the incidence of tuberculosis disease among human immunodeficiency virus (HIV-positive individuals testing purified protein derivative negative. Assessment of meta-analysis precision using trial sequential analysis (TSA with LanDeMets monitoring boundaries. Sample size determination for a future trials to make the meta-analysis conclusive according to the thresholds set by the monitoring boundaries.Results: The meta-analysis included nine trials comprising 2,911 trial participants and yielded a relative risk of 0.74 (95% CI, 0.53–1.04, P = 0.082, I2 = 0%. To deem the meta-analysis conclusive according to the thresholds set by the monitoring boundaries, a future RCT would need to randomize 3,800 participants.Conclusion: Statistical monitoring boundaries provide a framework for interpreting meta-analysis according to the adequacy of sample size and project the required sample size for a future RCT to make a meta-analysis conclusive

  7. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

    LENUS (Irish Health Repository)

    Mee, Blanaid C.

    2011-12-29

    The Saint James\\'s Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep® (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy® and QIAamp® (both Qiagen), and (4) to examine the effectiveness of Allprotect® (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software®). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A260\\/280 values using QIAamp were 33.2 ng\\/μL and 1.86, respectively, and using AllPrep were 23.2 ng\\/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng\\/μL and 8.16, respectively, and with AllPrep were 74.8 ng\\/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.

  8. Laser-cut paper-based device for the detection of dengue non-structural NS1 protein and specific IgM in human samples.

    Science.gov (United States)

    Theillet, G; Rubens, A; Foucault, F; Dalbon, P; Rozand, C; Leparc-Goffart, I; Bedin, F

    2018-03-10

    The incidence of flavivirus infections has increased dramatically in recent decades in tropical and sub-tropical areas worldwide, affecting hundreds of millions of people each year. Dengue viruses are typically transmitted by mosquitoes and can cause a wide range of symptoms from flu-like fever to organ impairment and death. Although conventional diagnostic tests can provide early diagnosis of acute dengue infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop affordable, simple, rapid, and robust diagnostic tools that can be used at 'Point of Care' settings. Early diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, a novel laser-cut device made of glass-fiber paper was designed and tested for the detection of the dengue Non Structural 1 (NS1) viral protein and specific IgM in blood and plasma. The device, called PAD, was able to detect around 25 ng/mL of NS1 protein in various sample types in 8 minutes, following a few simple steps. The PAD was also able to detect specific IgM in human plasmas in less than 10 minutes. The PAD appears to have all the potential to assist health workers in early diagnosis of dengue fever or other tropical fevers caused by flaviviruses.

  9. Focused conformational sampling in proteins

    Czech Academy of Sciences Publication Activity Database

    Bacci, M.; Langini, C.; Vymětal, Jiří; Caflisch, A.; Vitalis, A.

    2017-01-01

    Roč. 147, č. 19 (2017), č. článku 195102. ISSN 0021-9606 Institutional support: RVO:61388963 Keywords : molecular dynamics simulations * histone recognition * fibril elongation Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 2.965, year: 2016

  10. Self-assembling triblock proteins for biofunctional surface modification

    Science.gov (United States)

    Fischer, Stephen E.

    Despite the tremendous promise of cell/tissue engineering, significant challenges remain in engineering functional scaffolds to precisely regulate the complex processes of tissue growth and development. As the point of contact between the cells and the scaffold, the scaffold surface plays a major role in mediating cellular behaviors. In this dissertation, the development and utility of self-assembling, artificial protein hydrogels as biofunctional surface modifiers is described. The design of these recombinant proteins is based on a telechelic triblock motif, in which a disordered polyelectrolyte central domain containing embedded bioactive ligands is flanked by two leucine zipper domains. Under moderate conditions of temperature and pH, the leucine zipper end domains form amphiphilic alpha-helices that reversibly associate into homo-trimeric aggregates, driving hydrogel formation. Moreover, the amphiphilic nature of these helical domains enables surface adsorption to a variety of scaffold materials to form biofunctional protein coatings. The nature and stability of these coatings in various solution conditions, and their interaction with mammalian cells is the primary focus of this dissertation. In particular, triblock protein coatings functionalized with cell recognition sequences are shown to produce well-defined surfaces with precise control over ligand density. The impact of this is demonstrated in multiple cell types through ligand density-dependent cell-substrate interactions. To improve the stability of these physically self-assembled coatings, two covalent crosslinking strategies are described---one in which a zero-length chemical crosslinker (EDC) is utilized and a second in which disulfide bonds are engineered into the recombinant proteins. These targeted crosslinking approaches are shown to increase the stability of surface adsorbed protein layers with minimal effect on the presentation of many bioactive ligands. Finally, to demonstrate the versatility

  11. Solution Structure of Pfu RPP21, a Component of the Archaeal RNase P Holoenzyme, and Interactions with its RPP29 Protein Partner

    Science.gov (United States)

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2009-01-01

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5′-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentration, four proteins subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30 and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with Pfu RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step towards understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  12. Amide or amine: determining the origin of the 3300 cm(-1) NH mode in protein SFG spectra using 15N isotope labels.

    Science.gov (United States)

    Weidner, Tobias; Breen, Nicholas F; Drobny, Gary P; Castner, David G

    2009-11-26

    Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases, a strong NH mode near 3300 cm(-1) is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain, since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode, we studied (15)N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an alpha-helical secondary structure (LKalpha14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. (15)N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm(-1) on SiO(2) and 13 cm(-1) on CaF(2). This clearly shows the 3300 cm(-1) NH feature is associated with side chain NH stretches and not with backbone amide modes.

  13. Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization.

    Science.gov (United States)

    Beuming, Thijs; Che, Ye; Abel, Robert; Kim, Byungchan; Shanmugasundaram, Veerabahu; Sherman, Woody

    2012-03-01

    Water plays an essential role in determining the structure and function of all biological systems. Recent methodological advances allow for an accurate and efficient estimation of the thermodynamic properties of water molecules at the surface of proteins. In this work, we characterize these thermodynamic properties and relate them to various structural and functional characteristics of the protein. We find that high-energy hydration sites often exist near protein motifs typically characterized as hydrophilic, such as backbone amide groups. We also find that waters around alpha helices and beta sheets tend to be less stable than waters around loops. Furthermore, we find no significant correlation between the hydration site-free energy and the solvent accessible surface area of the site. In addition, we find that the distribution of high-energy hydration sites on the protein surface can be used to identify the location of binding sites and that binding sites of druggable targets tend to have a greater density of thermodynamically unstable hydration sites. Using this information, we characterize the FKBP12 protein and show good agreement between fragment screening hit rates from NMR spectroscopy and hydration site energetics. Finally, we show that water molecules observed in crystal structures are less stable on average than bulk water as a consequence of the high degree of spatial localization, thereby resulting in a significant loss in entropy. These findings should help to better understand the characteristics of waters at the surface of proteins and are expected to lead to insights that can guide structure-based drug design efforts. Copyright © 2011 Wiley Periodicals, Inc.

  14. Immunohistochemical quantification of expression of a tight junction protein, claudin-7, in human lung cancer samples using digital image analysis method.

    Science.gov (United States)

    Lu, Zhe; Liu, Yi; Xu, Junfeng; Yin, Hongping; Yuan, Haiying; Gu, Jinjing; Chen, Yan-Hua; Shi, Liyun; Chen, Dan; Xie, Bin

    2018-03-01

    Tight junction proteins are correlated with cancer development. As the pivotal proteins in epithelial cells, altered expression and distribution of different claudins have been reported in a wide variety of human malignancies. We have previously reported that claudin-7 was strongly expressed in benign bronchial epithelial cells at the cell-cell junction while expression of claudin-7 was either altered with discontinued weak expression or completely absent in lung cancers. Based on these results, we continued working on the expression pattern of claudin-7 and its relationship with lung cancer development. We herein proposed a new Digital Image Classification, Fragmentation index, Morphological analysis (DICFM) method for differentiating the normal lung tissues and lung cancer tissues based on the claudin-7 immunohistochemical staining. Seventy-seven lung cancer samples were obtained from the Second Affiliated Hospital of Zhejiang University and claudin-7 immunohistochemical staining was performed. Based on C++ and Open Source Computer Vision Library (OpenCV, version 2.4.4), the DICFM processing module was developed. Intensity and fragmentation of claudin-7 expression, as well as the morphological parameters of nuclei were calculated. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis. Agreement between these computational results and the results obtained by two pathologists was demonstrated. The intensity of claudin-7 expression was significantly decreased while the fragmentation was significantly increased in the lung cancer tissues compared to the normal lung tissues and the intensity was strongly positively associated with the differentiation of lung cancer cells. Moreover, the perimeters of the nuclei of lung cancer cells were significantly greater than that of the normal lung cells, while the parameters of area and circularity revealed no statistical significance. Taken together, our DICFM approach may be applied as an

  15. LEAping to conclusions: a computational reanalysis of late embryogenesis abundant proteins and their possible roles.

    Science.gov (United States)

    Wise, Michael J

    2003-10-29

    The late embryogenesis abundant (LEA) proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties. Previous research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins. There is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones, via a role in DNA-binding. In general, non-globular and

  16. LEAping to conclusions: A computational reanalysis of late embryogenesis abundant proteins and their possible roles

    Directory of Open Access Journals (Sweden)

    Wise Michael J

    2003-10-01

    Full Text Available Abstract Background The late embryogenesis abundant (LEA proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties. Results Previous research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins. Conclusions There is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones

  17. Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins.

    Science.gov (United States)

    Palmieri, Ferdinando; Agrimi, Gennaro; Blanco, Emanuela; Castegna, Alessandra; Di Noia, Maria A; Iacobazzi, Vito; Lasorsa, Francesco M; Marobbio, Carlo M T; Palmieri, Luigi; Scarcia, Pasquale; Todisco, Simona; Vozza, Angelo; Walker, John

    2006-01-01

    The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

  18. Solution structure of human intestinal fatty acid binding protein: Implications for ligand entry and exit

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Fengli [Boston University School of Medicine, Department of Biophysics (United States); Luecke, Christian [Johann Wolfgang Goethe-Universitaet (Germany); Baier, Leslie J. [NIDDK, NIH, Phoenix Epidemiology and Clinical Research Branch (United States); Sacchettini, James C. [Texas A and M University, Department of Biochemistry and Biophysics (United States); Hamilton, James A. [Boston University School of Medicine, Department of Biophysics (United States)

    1997-04-15

    The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy.Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra(NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel {beta}-strands which form two nearly orthogonal {beta}-sheets of five strands each, and two short {alpha}-helices that connect the {beta}-strands A and B. The interior of the protein consists of a water-filled cavity between the two {beta}-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP.The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.

  19. Simple protein precipitation extraction technique followed by validated chromatographic method for linezolid analysis in real human plasma samples to study its pharmacokinetics.

    Science.gov (United States)

    Mohammed, Samah A; Eissa, Maya S; Ahmed, Hytham M

    2017-02-01

    Fast and sensitive HPLC method was developed, optimized and validated for quantification of linezolid (LNZ) in human plasma using guaifenesin as an internal standard (IS). Analyte and IS were extracted from plasma by simple protein precipitation extraction technique using methanol as the precipitating solvent. The pretreated samples were injected in a mobile phase formed of acetonitrile:water:methanol (20:70:10v/v/v) in an isocratic mode at a flow rate of 1.5mL/min with UV detection at 251nm. Separation was done using Aglient ODS C 18 . The method showed linearity in the range of 0.75-50μg/mL with correlation coefficients equals to 0.9991. Precision and accuracy were in conformity with the criteria normally accepted in bio-analytical method validation. The RSDs for intra- and inter-day assays were <3.56 and 4.63%, respectively. The intra- and inter-day accuracies were 94.67-98.28% and 91.25-96.18%, respectively. The mean absolute recoveries ranged from 92.56±1.78 to 95.24±2.84. According to stability results, LNZ was stable in human plasma during the storage and analysis. LNZ a pharmacokinetic behavior was studied by applying the proposed analytical method. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Transcription Factor Activating Protein-2β (TFAP-2β) genotype and symptoms of attention deficit hyperactivity disorder in relation to symptoms of depression in two independent samples.

    Science.gov (United States)

    Nilsson, Kent W; Sonnby, Karin; Nordquist, Niklas; Comasco, Erika; Leppert, Jerzy; Oreland, Lars; Sjöberg, Rickard L

    2014-04-01

    The Transcription Factor Activating Protein-2β (TFAP-2β) gene has been shown to influence monoaminergic neurotransmission, and several genes important for monoaminergic function have binding sites for TFAP-2β. Familial studies of attention deficit hyperactivity disorder (ADHD) suggest a hereditary-determined subtype of ADHD with comorbid depression. We examined a functional variation of the TFAP-2β gene in the context of co-occurring symptoms of ADHD and depression in two independent population-based samples of adolescents (Group A, n = 175 and Group B, n = 1,506) from Sweden. Results indicated 6.1 to 7.8% of adolescents screened positively for ADHD and depression symptoms. Symptoms of depression were more common among girls who screened positively for ADHD and did not carry the nine-repeat allele of the TFAP-2β intron 1 Variable Number Tandem Repeat (VNTR) polymorphism. The presence of the nine-repeat variant of the TFAP-2β intron 1 VNTR appears to protect girls with ADHD symptoms from the co-expression of symptoms of depression.

  1. Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Arun K Upadhyay

    Full Text Available The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

  2. Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

    Science.gov (United States)

    Upadhyay, Arun K.; Murmu, Aruna; Singh, Anupam; Panda, Amulya K.

    2012-01-01

    The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies. PMID:22479486

  3. Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

    Science.gov (United States)

    Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

    2016-01-05

    A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

  4. Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques.

    Science.gov (United States)

    de Jesus, Jemmyson Romário; da Silva Fernandes, Rafael; de Souza Pessôa, Gustavo; Raimundo, Ivo Milton; Arruda, Marco Aurélio Zezzi

    2017-08-01

    The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL -1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL -1 . Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells.

    Science.gov (United States)

    Dennison, Sarah Rachel; Harris, Frederick; Brandenburg, Klaus; Phoenix, David Andrew

    2007-11-01

    The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic alpha-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be alpha-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5-1.0 cm(-1)) and induced surface pressure changes of 2 mN m(-1) in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic alpha-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

  6. N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

    Science.gov (United States)

    Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

    2009-09-15

    The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

  7. Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

    Science.gov (United States)

    Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

    2012-01-01

    A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

  8. Allergenic relevance of nonspecific lipid transfer proteins 2: Identification and characterization of Api g 6 from celery tuber as representative of a novel IgE-binding protein family.

    Science.gov (United States)

    Vejvar, Eva; Himly, Martin; Briza, Peter; Eichhorn, Stephanie; Ebner, Christof; Hemmer, Wolfgang; Ferreira, Fatima; Gadermaier, Gabriele

    2013-11-01

    Apium graveolens represents a relevant food allergen source linked with severe systemic reactions. We sought to identify an IgE-binding nonspecific lipid transfer protein (nsLTP) in celery tuber. A low molecular weight protein exclusively present in celery tuber was purified and designated Api g 6. The entire protein sequence was obtained by MS and classified as member of the nsLTP2 family. Api g 6 is monomeric in solution with a molecular mass of 6936 Da. The alpha-helical disulfide bond-stabilized structure confers tremendous thermal stability (Tm > 90°C) and high resistance to gastrointestinal digestion. Endolysosomal degradation demonstrated low susceptibility and the presence of a dominant peptide cluster at the C-terminus. Thirty-eight percent of A. graveolens allergic patients demonstrated IgE reactivity to purified natural Api g 6 in ELISA and heat treatment did only partially reduce its allergenic activity. No correlation in IgE binding and limited cross-reactivity was observed with Api g 2 and Art v 3, nsLTP1 from celery stalks and mugwort pollen. Api g 6, a novel nsLTP2 from celery tuber represents the first well-characterized allergen in this protein family. Despite similar structural and physicochemical features as nsLTP1, immunological properties of Api g 6 are distinct which warrants its inclusion in molecule-based diagnosis of A. graveolens allergy. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Birkelund, S; Holm, A

    1996-01-01

    The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may......-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel...... retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect...

  10. DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

    Science.gov (United States)

    Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

    1995-09-05

    Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

  11. Evaluation of iTRAQ and SWATH-MS for the Quantification of Proteins Associated with Insulin Resistance in Human Duodenal Biopsy Samples.

    Directory of Open Access Journals (Sweden)

    Sylvie Bourassa

    Full Text Available Insulin resistance (IR is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 15 insulin sensitive (IS control patients matched for age and waist circumference. A total of 3886 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation mass spectrometry approach and 2290 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra. Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2 of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.

  12. Effect of hydrocarbon stapling on the properties of alpha-helical antimicrobial peptides isolated from the venom of hymenoptera

    Czech Academy of Sciences Publication Activity Database

    Chapuis, Hubert Jean; Slaninová, Jiřina; Bednárová, Lucie; Monincová, Lenka; Buděšínský, Miloš; Čeřovský, Václav

    2012-01-01

    Roč. 43, č. 5 (2012), s. 2047-2058 ISSN 0939-4451 R&D Projects: GA ČR GA203/08/0536 Institutional research plan: CEZ:AV0Z40550506 Keywords : antimicrobial peptides * stapled peptides * amphipathic helix * CD spectroscopy Subject RIV: CC - Organic Chemistry Impact factor: 3.914, year: 2012

  13. Redox proteomics changes in the fungal pathogen Trichosporon asahii on arsenic exposure: identification of protein responses to metal-induced oxidative stress in an environmentally-sampled isolate.

    Directory of Open Access Journals (Sweden)

    Sidra Ilyas

    Full Text Available Trichosporon asahii is a yeast pathogen implicated in opportunistic infections. Cultures of an isolate collected from industrial wastewater were exposed for 2 days to 100 mg/L sodium arsenite (NaAsO2 and cadmium (CdCl2. Both metals reduced glutathione transferase (GST activity but had no effect on superoxide dismutase or catalase. NaAsO2 exposure increased glutathione reductase activity while CdCl2 had no effect. Protein thiols were labeled with 5-iodoacetamido fluorescein followed by one dimensional electrophoresis which revealed extensive protein thiol oxidation in response to CdCl2 treatment but thiol reduction in response to NaAsO2. Two dimensional electrophoresis analyses showed that the intensity of some protein spots was enhanced on treatment as judged by SameSpots image analysis software. In addition, some spots showed decreased IAF fluorescence suggesting thiol oxidation. Selected spots were excised and tryptic digested for identification by MALDI-TOF/TOF MS. Twenty unique T. asahii proteins were identified of which the following proteins were up-regulated in response to NaAsO2: 3-isopropylmalate dehydrogenase, phospholipase B, alanine-glyoxylate aminotransferase, ATP synthase alpha chain, 20S proteasome beta-type subunit Pre3p and the hypothetical proteins A1Q1_08001, A1Q2_03020, A1Q1_06950, A1Q1_06913. In addition, the following showed decreased thiol-associated fluorescence consistent with thiol oxidation; aconitase; aldehyde reductase I; phosphoglycerate kinase; translation elongation factor 2; heat shock protein 70 and hypothetical protein A1Q2_04745. Some proteins showed both increase in abundance coupled with decrease in IAF fluorescence; 3-hydroxyisobutyryl-CoA hydrolase; homoserine dehydrogenase Hom6 and hypothetical proteins A1Q2_03020 and A1Q1_00754. Targets implicated in redox response included 10 unique metabolic enzymes, heat shock proteins, a component of the 20S proteasome and translation elongation factor 2. These data

  14. Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

    Science.gov (United States)

    Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

    2001-05-01

    Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

  15. Expansion of the octarepeat domain alters the misfolding pathway but not the folding pathway of the prion protein.

    Science.gov (United States)

    Leliveld, S Rutger; Stitz, Lothar; Korth, Carsten

    2008-06-10

    A misfolded conformation of the prion protein (PrP), PrP (Sc), is the essential component of prions, the infectious agents that cause transmissible neurodegenerative diseases. Insertional mutations that lead to an increase in the number of octarepeats (ORs) in PrP are linked to familial human prion disease. In this study, we investigated how expansion of the OR domain causes PrP to favor a prion-like conformation. Therefore, we compared the conformational and aggregation modulating properties of wild-type versus expanded OR domains, either as a fusion construct with the protein G B1 domain (GB1-OR) or as an integral part of full-length mouse PrP (MoPrP). Using circular dichroism spectroscopy, we first demonstrated that ORs are not unfolded but exist as an ensemble of three distinct conformers: polyproline helix-like, beta-turn, and "Trp-related". Domain expansion had little effect on the conformation of GB1-OR fusion proteins. When part of MoPrP however, OR domain expansion changed PrP's folding landscape, not by hampering the production of native alpha-helical monomers but by greatly reducing the propensity to form amyloid and by altering the assembly of misfolded, beta-rich aggregates. These features may relate to subtle pH-dependent conformational differences between wild-type and mutant monomers. In conclusion, we propose that PrP insertional mutations are pathogenic because they enhance specific misfolding pathways of PrP rather than by undermining native folding. This idea was supported by a trial bioassay in transgenic mice overexpressing wild-type MoPrP, where intracerebral injection of recombinant MoPrP with an expanded OR domain but not wild-type MoPrP caused prion disease.

  16. Continuous-flow liquid microjunction surface sampling probe connected on-line with high-performance liquid chromatography/mass spectrometry for spatially resolved analysis of small molecules and proteins.

    Science.gov (United States)

    Van Berkel, Gary J; Kertesz, Vilmos

    2013-06-30

    A continuous-flow liquid microjunction surface sampling probe extracts soluble material from surfaces for direct ionization and detection by mass spectrometry. Demonstrated here is the on-line coupling of such a probe with high-performance liquid chromatography/mass spectrometry (HPLC/MS) enabling extraction, separation and detection of small molecules and proteins from surfaces in a spatially resolved (~0.5 mm diameter spots) manner. A continuous-flow liquid microjunction surface sampling probe was connected to a six-port, two-position valve for extract collection and injection to an HPLC column. A QTRAP® 5500 hybrid triple quadrupole linear ion trap equipped with a Turbo V™ ion source operated in positive electrospray ionization (ESI) mode was used for all experiments. The system operation was tested with the extraction, separation and detection of propranolol and associated metabolites from drug dosed tissues, caffeine from a coffee bean, cocaine from paper currency, and proteins from dried sheep blood spots on paper. Confirmed in the tissue were the parent drug and two different hydroxypropranolol glucuronides. The mass spectrometric response for these compounds from different locations in the liver showed an increase with increasing extraction time (5, 20 and 40 s). For on-line separation and detection/identification of extracted proteins from dried sheep blood spots, two major protein peaks dominated the chromatogram and could be correlated with the expected masses for the hemoglobin α and β chains. Spatially resolved sampling, separation, and detection of small molecules and proteins from surfaces can be accomplished using a continuous-flow liquid microjunction surface sampling probe coupled on-line with HPLC/MS detection. Published in 2013. This article is a U.S. Government work and is in the public domain in the USA.

  17. Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone-treated smooth muscle tissues for phos-tag SDS-PAGE.

    Science.gov (United States)

    Takeya, Kosuke; Kaneko, Toshiyuki; Miyazu, Motoi; Takai, Akira

    2018-01-01

    Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC 20 ) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC 20 , actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4-5 fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC 20 s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Evaluation of growth performance, serum biochemistry and haematological parameters on broiler birds fed with raw and processed samples of Entada scandens, Canavalia gladiata and Canavalia ensiformis seed meal as an alternative protein source.

    Science.gov (United States)

    Sasipriya, Gopalakrishnan; Siddhuraju, Perumal

    2013-03-01

    The experiment was carried out to investigate the inclusion of underutilised legumes, Entada scandens, Canavalia gladiata and Canavalia ensiformis, seed meal in soybean-based diet in broilers. The utilisation of these wild legumes is limited by the presence of antinutrient compounds. Processing methods like soaking followed by autoclaving in sodium bicarbonate solution in E. scandens and C. gladiata and soaking followed by autoclaving in ash solution in C. ensiformis were adopted. The proximate composition of raw and processed samples of E. scandens, C. gladiata and C. ensiformis were determined. The protein content was enhanced in processed sample of E. scandens (46 %) and C. ensiformis (16 %). This processing method had reduced the maximum number of antinutrients such as tannins (10-100 %), trypsin inhibitor activity (99 %), chymotrypsin inhibitor activity (72-100 %), canavanine (60-62 %), amylase inhibitor activity (73-100 %), saponins (78-92 %), phytic acid (19-40 %) and lectins. Hence, the raw samples at 15 % and processed samples at 15 and 30 % were replaced with soybean protein in commercial broiler diet respectively. Birds fed with 30 % processed samples of E. scandens, C. gladiata and C. ensiformis showed significantly similar results of growth performance, carcass characteristics, organ weight, haematological parameters and serum biochemical parameters (cholesterol, protein, bilirubin, albumin, globulin and liver and kidney function parameters) without any adverse effects after 42 days of supplementation. The proper utilisation of these underutilised legumes may act as an alternative protein ingredient in poultry diets.

  19. Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.

    Science.gov (United States)

    Massiah, M A; Starich, M R; Paschall, C; Summers, M F; Christensen, A M; Sundquist, W I

    1994-11-25

    The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away

  20. Orientation-dependent backbone-only residue pair scoring functions for fixed backbone protein design

    Directory of Open Access Journals (Sweden)

    Bordner Andrew J

    2010-04-01

    Full Text Available Abstract Background Empirical scoring functions have proven useful in protein structure modeling. Most such scoring functions depend on protein side chain conformations. However, backbone-only scoring functions do not require computationally intensive structure optimization and so are well suited to protein design, which requires fast score evaluation. Furthermore, scoring functions that account for the distinctive relative position and orientation preferences of residue pairs are expected to be more accurate than those that depend only on the separation distance. Results Residue pair scoring functions for fixed backbone protein design were derived using only backbone geometry. Unlike previous studies that used spherical harmonics to fit 2D angular distributions, Gaussian Mixture Models were used to fit the full 3D (position only and 6D (position and orientation distributions of residue pairs. The performance of the 1D (residue separation only, 3D, and 6D scoring functions were compared by their ability to identify correct threading solutions for a non-redundant benchmark set of protein backbone structures. The threading accuracy was found to steadily increase with increasing dimension, with the 6D scoring function achieving the highest accuracy. Furthermore, the 3D and 6D scoring functions were shown to outperform side chain-dependent empirical potentials from three other studies. Next, two computational methods that take advantage of the speed and pairwise form of these new backbone-only scoring functions were investigated. The first is a procedure that exploits available sequence data by averaging scores over threading solutions for homologs. This was evaluated by applying it to the challenging problem of identifying interacting transmembrane alpha-helices and found to further improve prediction accuracy. The second is a protein design method for determining the optimal sequence for a backbone structure by applying Belief Propagation

  1. Improved prediction of gestational hypertension by inclusion of placental growth factor and pregnancy associated plasma protein-a in a sample of Ghanaian women

    NARCIS (Netherlands)

    Antwi, Edward; Klipstein-Grobusch, Kerstin; Browne, Joyce L; Schielen, Peter C; Koram, Kwadwo A; Agyepong, Irene A; Grobbee, Diederick E

    2018-01-01

    BACKGROUND: We assessed whether adding the biomarkers Pregnancy Associated Plasma Protein-A (PAPP-A) and Placental Growth Factor (PlGF) to maternal clinical characteristics improved the prediction of a previously developed model for gestational hypertension in a cohort of Ghanaian pregnant women.

  2. Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer

    Czech Academy of Sciences Publication Activity Database

    Dowling, P.; Hughes, D. J.; Larkin, A.M.; Meiller, J.; Henry, M.; Meleady, P.; Lynch, V.; Pardini, B.; Naccarati, A.; Levý, M.; Vodička, Pavel; Neary, P.; Clynes, M.

    2015-01-01

    Roč. 441, feb. (2015), s. 133-141 ISSN 0009-8981 Institutional support: RVO:68378041 Keywords : biomarkers * colorectal cancer * proteomics * mass spectrometry * 14-3-3 proteins * pyruvate kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.799, year: 2015

  3. Stage-specific analysis of plasma protein profiles in ovarian cancer: Difference in-gel electrophoresis analysis of pooled clinical samples

    Directory of Open Access Journals (Sweden)

    Mark J Bailey

    2013-01-01

    Full Text Available Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4. The expression of TPM4 increased by 2-fold in Stage 2 before returning to "normal" levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage.

  4. Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain.

    Science.gov (United States)

    Resnick, D; Chatterton, J E; Schwartz, K; Slayter, H; Krieger, M

    1996-10-25

    Structures of secreted forms of the human type I and II class A macrophage scavenger receptors were studied using biochemical and biophysical methods. Proteolytic analysis was used to determine the intramolecular disulfide bonds in the type I-specific scavenger receptor cysteine-rich (SRCR) domain: Cys2-Cys7, Cys3-Cys8, and Cys5-Cys6. This pattern is likely to be shared by the highly homologous domains in the many other members of the SRCR domain superfamily. Electron microscopy using rotary shadowing and negative staining showed that the type I and II receptors are extended molecules whose contour lengths are approximately 440 A. They comprised two adjacent fibrous segments, an alpha-helical coiled-coil ( approximately 230 A, including a contribution from the N-terminal spacer domain) and a collagenous triple helix ( approximately 210 A). The type I molecules also contained a C-terminal globular structure ( approximately 58 x 76 A) composed of three SRCR domains. The fibrous domains were joined by an extremely flexible hinge. The angle between these domains varied from 0 to 180 degrees and depended on the conditions of sample preparation. Unexpectedly, at physiologic pH, the prevalent angle seen using rotary shadowing was 0 degrees , resulting in a structure that is significantly more compact than previously suggested. The apparent juxtaposition of the fibrous domains at neutral pH provides a framework for future structure-function studies of these unusual multiligand receptors.

  5. (¹⁵N ± ¹³C') edited (4, 3)D-H(CC)CONH TOCSY and (4, 3)D-NOESY HNCO experiments for unambiguous side chain and NOE assignments of proteins with high shift degeneracy.

    Science.gov (United States)

    Kumar, Dinesh; Arora, Ashish

    2011-11-01

    Well-resolved and unambiguous through-bond correlations and NOE data are crucial for high-quality protein structure determination by NMR. In this context, we present here (4, 3)D reduced dimensionality (RD) experiments: H(CC)CONH TOCSY and NOESY HNCO--which instead of (15)N shifts exploit the linear combination of (15)N(i) and (13)C'(i-1) shifts (where i is a residue number) to resolve the through-bond (1)H-(1)H correlations and through-space (1)H-(1)H NOEs. The strategy makes use of the fact that (15)N and (13)C' chemical shifts when combined linearly provide a dispersion which is better compared to those of the individual chemical shifts. The extended dispersion thus available in these experiments will help to obtain the unambiguous side chain and accurate NOE assignments especially for medium-sized alpha-helical or partially unstructured proteins [molecular weight (MW) between 12-15 kDa] as well as higher MW (between 15-25 kDa) folded proteins where spectral overlap renders inaccurate and ambiguous NOEs. Further, these reduced dimensionality experiments in combination with routinely used (15)N and (13)C' edited TOCSY and NOESY experiments will provide an alternative way for high-quality NMR structure determination of large unstable proteins (with very high shift degeneracy), which are not at all amenable to 4D NMR. The utility of these experiments has been demonstrated here using (13)C/(15)N labeled ubiquitin (76 aa) protein. Copyright © 2011 John Wiley & Sons, Ltd.

  6. 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.

  7. 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.

  8. Quantitative measurement of exchange dynamics in proteins via {sup 13}C relaxation dispersion of {sup 13}CHD{sub 2}-labeled samples

    Energy Technology Data Exchange (ETDEWEB)

    Rennella, Enrico; Schuetz, Anne K.; Kay, Lewis E., E-mail: kay@pound.med.utoronto.ca [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)

    2016-06-15

    Methyl groups have emerged as powerful probes of protein dynamics with timescales from picoseconds to seconds. Typically, studies involving high molecular weight complexes exploit {sup 13}CH{sub 3}- or {sup 13}CHD{sub 2}-labeling in otherwise highly deuterated proteins. The {sup 13}CHD{sub 2} label offers the unique advantage of providing {sup 13}C, {sup 1}H and {sup 2}H spin probes, however a disadvantage has been the lack of an experiment to record {sup 13}C Carr–Purcell–Meiboom–Gill relaxation dispersion that monitors millisecond time-scale dynamics, implicated in a wide range of biological processes. Herein we develop an experiment that eliminates artifacts that would normally result from the scalar coupling between {sup 13}C and {sup 2}H spins that has limited applications in the past. The utility of the approach is established with a number of applications, including measurement of ms dynamics of a disease mutant of a 320 kDa p97 complex.

  9. Adsorption of acidic, basic, and neutral proteins from aqueous samples using Fe3O4 magnetic nanoparticles modified with an ionic liquid

    International Nuclear Information System (INIS)

    Kamran, S.; Asadi, M.; Absalan, G.

    2013-01-01

    We have prepared and characterized Fe 3 O 4 nanoparticles and their binary mixtures (IL-Fe 3 O 4 ) with 1-hexyl-3-methylimidazolium bromide as ionic liquid for use in the adsorption of lysozyme (LYS), bovine serum albumin (BSA), and myoglobin (MYO). The optimum operational conditions for the adsorption of proteins (at 0.05-2.0 mg mL -1 ) were 4.0 mg mL -1 of nanoparticles and a contact time of 10 min. The maximum adsorption capacities are 455, 182 and 143 mg for LYS, BSA, and MYO per gram of adsorbent, respectively. The Langmuir model better fits the adsorption isotherms, with adsorption constants of 0.003, 0.015 and 0.008 L mg -1 , in order, for LYS, BSA, MYO. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. The adsorption processes are endothermic. The proteins can be desorbed from the nanoparticles by using NaCl solution at pH 9.5, and the nanoparticles thus can be recycled. (author)

  10. 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

  11. Measurement of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 after delayed separation of whole blood samples.

    NARCIS (Netherlands)

    Hartog, H.; Graaf, W.T.A. van der; Wesseling, J.; Veer, E. van der; Boezen, H.M.

    2008-01-01

    OBJECTIVES: Epidemiological studies benefit from unbiased blood specimens collected with minimal cost and effort of blood collection and storage. We evaluated the stability of IGF-1 and IGFBP-3 in whole blood samples stored at room temperature to justify delays in blood processing. DESIGN AND

  12. Measurement of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 after delayed separation of whole blood samples

    NARCIS (Netherlands)

    Hartog, Hermien; van der Graaf, Winette T. A.; Wesseling, Jelle; van der Veer, Eveline; Boezen, H. Marike

    Objectives: Epidemiological studies benefit from unbiased blood specimens collected with minimal cost and effort of blood collection and storage. We evaluated the stability of IGF-1 and IGFBP-3 in whole blood samples stored at room temperature to justify delays in blood processing. Design and

  13. The efficiency of microbial protein production from tropical forages and its measurement using spot samples of urine and CrEDTA clearance from the plasma

    International Nuclear Information System (INIS)

    King, A.; Poppi, D.P.; McLennan, S.R.

    1999-01-01

    The efficiency of microbial crude protein production (MCP) expressed as g MCP/kg digestible organic matter (DOM) was much lower (P 0.75 /d). The fractional disappearance rate of CrEDTA from the plasma was similar for cattle consuming either hay (1.26-1.54%/min). The glomerular filtration rate (GFR) estimated from urinary creatinine excretion was significantly different (289 L/d vs 793 L/d, P vs R respectively). The ratio of predicted allantoin clearance determined by reference to plasma volume and FDR of CrEDTA to the actual urinary excretion was 6.7 and 9.6 for R and P respectively. This difference meant that clearance of CrEDTA could not be used as a technique in association with plasma concentration of allantoin to estimate urinary excretion of allantoin. (author)

  14. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  15. Boat sampling

    International Nuclear Information System (INIS)

    Citanovic, M.; Bezlaj, H.

    1994-01-01

    This presentation describes essential boat sampling activities: on site boat sampling process optimization and qualification; boat sampling of base material (beltline region); boat sampling of weld material (weld No. 4); problems accompanied with weld crown varieties, RPV shell inner radius tolerance, local corrosion pitting and water clarity. The equipment used for boat sampling is described too. 7 pictures

  16. Graph sampling

    OpenAIRE

    Zhang, L.-C.; Patone, M.

    2017-01-01

    We synthesise the existing theory of graph sampling. We propose a formal definition of sampling in finite graphs, and provide a classification of potential graph parameters. We develop a general approach of Horvitz–Thompson estimation to T-stage snowball sampling, and present various reformulations of some common network sampling methods in the literature in terms of the outlined graph sampling theory.

  17. A simple and highly sensitive on-line column extraction liquid chromatography-tandem mass spectrometry method for the determination of protein-unbound tacrolimus in human plasma samples.

    Science.gov (United States)

    Bittersohl, Heike; Schniedewind, Björn; Christians, Uwe; Luppa, Peter B

    2018-04-27

    Therapeutic drug monitoring (TDM) of the immunosuppressive drug tacrolimus is essential to avoid side effects and rejection of the allograft after transplantation. In the blood circulation, tacrolimus is largely located inside erythrocytes or bound to plasma proteins and less than 0.1% is protein-unbound (free). One basic principle of clinical pharmacology is that only free drug is pharmacologically active and monitoring this portion has the potential to better reflect the drug effect than conventional measurements of total tacrolimus in whole blood. To address this, a highly sensitive and straightforward on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, validated and applied to patient plasma samples. The sample preparation included ultracentrifugation and addition of the stable isotope labeled drug analogue D2,13C-tacrolimus, followed by on-line sample extraction and measurement using a Sciex QTRAP ® 6500 in the multiple reaction monitoring mode. Due to very low concentrations of protein-unbound tacrolimus, it was important to develop a highly sensitive, precise and accurate assay. Here, we first report the efficient formation of tacrolimus lithium adduct ions, which greatly increased assay sensitivity. A lower limit of quantification (LLOQ) of 1 pg/mL (10 fg on column) was achieved and the assay was linear between 1 and 200 pg/mL. There was no carry-over detected. The inaccuracy ranged from -9.8 to 7.4% and the greatest imprecision was 7.5%. The matrix factor was found to be smaller than 1.1%. In summary, this method represents a suitable tool to investigate the potential clinical value of free tacrolimus monitoring in organ transplant recipients. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins

    Energy Technology Data Exchange (ETDEWEB)

    Barb, Adam W., E-mail: abarb@iastate.edu; Subedi, Ganesh P. [Iowa State University, Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology (United States)

    2016-01-15

    Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb{sup 3+} with high affinity (0.70 and 0.13 μM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (−0.221 to 0.081 ppm) and residual dipolar couplings (−7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb{sup 3+}){sub 2} complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems.

  19. An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins

    International Nuclear Information System (INIS)

    Barb, Adam W.; Subedi, Ganesh P.

    2016-01-01

    Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb 3+ with high affinity (0.70 and 0.13 μM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (−0.221 to 0.081 ppm) and residual dipolar couplings (−7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb 3+ ) 2 complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems

  20. Genetic variants associated with altered plasma levels of C-reactive protein are not associated with late-life cognitive ability in four Scottish samples.

    Science.gov (United States)

    Marioni, Riccardo E; Deary, Ian J; Murray, Gordon D; Lowe, Gordon D O; Rafnsson, Snorri B; Strachan, Mark W J; Luciano, Michelle; Houlihan, Lorna M; Gow, Alan J; Harris, Sarah E; Stewart, Marlene C; Rumley, Ann; Fowkes, F Gerry R; Price, Jackie F

    2010-01-01

    It is unknown whether the relationship between raised inflammatory biomarker levels and late-life cognitive ability is causal. We explored this issue by testing the association between genetic regulators of plasma C-reactive protein (CRP) and cognition. Data were analysed from four cohorts based in central Scotland (Total N = 4,782). Associations were tested between variants in the CRP gene and both plasma CRP levels and a battery of neuropsychological tests, including a vocabulary-based estimate of peak prior cognitive ability and a general (summary) cognitive factor score, or 'g'. CRP levels were associated with a number of variants in the CRP gene (SNPs), including rs1205, rs1130864, rs1800947, and rs1417938 (P range 4.2e-06 to 0.041). Higher CRP levels were also associated with vocabulary-adjusted cognitive ability, used here to estimate lifetime cognitive change (P range 1.7e-04 to 0.038). After correction for multiple testing and adjustment for age and sex, no statistically significant associations were found between the SNPs and cognition. CRP is unlikely to be a causal determinant of late-life cognitive ability.

  1. Validation of protein intake assessed from weighed dietary records against protein estimated from 24 h urine samples in children, adolescents and young adults participating in the Dortmund Nutritional and Longitudinally Designed (DONALD) Study

    DEFF Research Database (Denmark)

    Bokhof, Beate; Günther, Anke L B; Berg-Beckhoff, Gabriele

    2010-01-01

    from a simultaneously collected 24 h urine sample. DESIGN: Cross-sectional analyses including 439 participants of the Dortmund Nutritional and Longitudinally Designed (DONALD) Study from four age groups (3-4, 7-8, 11-13 and 18-23 years). Mean differences, Pearson correlation coefficients (r), cross.......5 (95 % CI -18.7, -8.3) g/d at age 18-23 years. Correlation coefficients were r = 0.7 for the total study sample and ranged from r = 0.5 to 0.6 in the different age groups. Both methods classified 85 % into the same/adjacent quartile for the whole study group (83-86 % for the different age groups) and 2...

  2. A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

    Science.gov (United States)

    Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

    2012-01-01

    In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

  3. Balanced sampling

    NARCIS (Netherlands)

    Brus, D.J.

    2015-01-01

    In balanced sampling a linear relation between the soil property of interest and one or more covariates with known means is exploited in selecting the sampling locations. Recent developments make this sampling design attractive for statistical soil surveys. This paper introduces balanced sampling

  4. Ensemble Sampling

    OpenAIRE

    Lu, Xiuyuan; Van Roy, Benjamin

    2017-01-01

    Thompson sampling has emerged as an effective heuristic for a broad range of online decision problems. In its basic form, the algorithm requires computing and sampling from a posterior distribution over models, which is tractable only for simple special cases. This paper develops ensemble sampling, which aims to approximate Thompson sampling while maintaining tractability even in the face of complex models such as neural networks. Ensemble sampling dramatically expands on the range of applica...

  5. Several genes encoding ribosomal proteins are over-expressed in prostate-cancer cell lines: confirmation of L7a and L37 over-expression in prostate-cancer tissue samples.

    Science.gov (United States)

    Vaarala, M H; Porvari, K S; Kyllönen, A P; Mustonen, M V; Lukkarinen, O; Vihko, P T

    1998-09-25

    A cDNA library specific for mRNA over-expressed in prostate cancer was generated by subtractive hybridization of transcripts originating from prostatic hyperplasia and cancer tissues. cDNA encoding ribosomal proteins L4, L5, L7a, L23a, L30, L37, S14 and S18 was found to be present among 100 analyzed clones. Levels of ribosomal mRNA were significantly higher at least in one of the prostate-cancer cell lines, LNCaP, DU-145 and PC-3, than in hyperplastic tissue, as determined by slot-blot hybridization. Furthermore, L23a- and S14-transcript levels were significantly elevated in PC-3 cells as compared with those in the normal prostate epithelial cell line PrEC. Generally, dramatic changes in the mRNA content of the ribosomal proteins were not detected, the most evident over-expression being that of L37 mRNA, which was 3.4 times more abundant in LNCaP cells than in hyperplastic prostate tissue. The over-expression of L7a and L37 mRNA was confirmed in prostate-cancer tissue samples by in situ hybridization. Elevated cancer-related expression of L4 and L30 has not been reported, but levels of the other ribosomal proteins are known to be increased in several types of cancers. These results therefore suggest that prostate cancer is comparable with other types of cancers, in that a larger pool of some ribosomal proteins is gained during the transformation process, by an unknown mechanism.

  6. Liquid phase separation of proteins based on electrophoretic effects in an electrospray setup during sample introduction into a gas-phase electrophoretic mobility molecular analyzer (CE–GEMMA/CE–ES–DMA)

    Science.gov (United States)

    Weiss, Victor U.; Kerul, Lukas; Kallinger, Peter; Szymanski, Wladyslaw W.; Marchetti-Deschmann, Martina; Allmaier, Günter

    2014-01-01

    Nanoparticle characterization is gaining importance in food technology, biotechnology, medicine, and pharmaceutical industry. An instrument to determine particle electrophoretic mobility (EM) diameters in the single-digit to double-digit nanometer range receiving increased attention is the gas-phase electrophoretic mobility molecular analyzer (GEMMA) separating electrophoretically single charged analytes in the gas-phase at ambient pressure. A fused-silica capillary is used for analyte transfer to the gas-phase by means of a nano electrospray (ES) unit. The potential of this capillary to separate analytes electrophoretically in the liquid phase due to different mobilities is, at measurement conditions recommended by the manufacturer, eliminated due to elevated pressure applied for sample introduction. Measurements are carried out upon constant feeding of analytes to the system. Under these conditions, aggregate formation is observed for samples including high amounts of non-volatile components or complex samples. This makes the EM determination of individual species sometimes difficult, if not impossible. With the current study we demonstrate that liquid phase electrophoretic separation of proteins (as exemplary analytes) occurs in the capillary (capillary zone electrophoresis, CE) of the nano ES unit of the GEMMA. This finding was consecutively applied for on-line desalting allowing EM diameter determination of analytes despite a high salt concentration within samples. The present study is to our knowledge the first report on the use of the GEMMA to determine EM diameters of analytes solubilized in the ES incompatible electrolyte solutions by the intended use of electrophoresis (in the liquid phase) during sample delivery. Results demonstrate the proof of concept of such an approach and additionally illustrate the high potential of a future on-line coupling of a capillary electrophoresis to a GEMMA instrument. PMID:25109866

  7. Laser sampling

    International Nuclear Information System (INIS)

    Gorbatenko, A A; Revina, E I

    2015-01-01

    The review is devoted to the major advances in laser sampling. The advantages and drawbacks of the technique are considered. Specific features of combinations of laser sampling with various instrumental analytical methods, primarily inductively coupled plasma mass spectrometry, are discussed. Examples of practical implementation of hybrid methods involving laser sampling as well as corresponding analytical characteristics are presented. The bibliography includes 78 references

  8. Soil sampling

    International Nuclear Information System (INIS)

    Fortunati, G.U.; Banfi, C.; Pasturenzi, M.

    1994-01-01

    This study attempts to survey the problems associated with techniques and strategies of soil sampling. Keeping in mind the well defined objectives of a sampling campaign, the aim was to highlight the most important aspect of representativeness of samples as a function of the available resources. Particular emphasis was given to the techniques and particularly to a description of the many types of samplers which are in use. The procedures and techniques employed during the investigations following the Seveso accident are described. (orig.)

  9. Language sampling

    DEFF Research Database (Denmark)

    Rijkhoff, Jan; Bakker, Dik

    1998-01-01

    This article has two aims: [1] to present a revised version of the sampling method that was originally proposed in 1993 by Rijkhoff, Bakker, Hengeveld and Kahrel, and [2] to discuss a number of other approaches to language sampling in the light of our own method. We will also demonstrate how our...... sampling method is used with different genetic classifications (Voegelin & Voegelin 1977, Ruhlen 1987, Grimes ed. 1997) and argue that —on the whole— our sampling technique compares favourably with other methods, especially in the case of exploratory research....

  10. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian, E-mail: jianzhou@scut.edu.cn

    2016-07-30

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  11. pi-Turns: types, systematics and the context of their occurrence in protein structures.

    Science.gov (United States)

    Dasgupta, Bhaskar; Chakrabarti, Pinak

    2008-09-22

    For a proper understanding of protein structure and folding it is important to know if a polypeptide segment adopts a conformation inherent in the sequence or it depends on the context of its flanking secondary structures. Turns of various lengths have been studied and characterized starting from three-residue gamma-turn to six-residue pi-turn. The Schellman motif occurring at the C-terminal end of alpha-helices is a classical example of hydrogen bonded pi-turn involving residues at (i) and (i+5) positions. Hydrogen bonded and non-hydrogen bonded beta- and alpha-turns have been identified previously; likewise, a systematic characterization of pi-turns would provide valuable insight into turn structures. An analysis of protein structures indicates that at least 20% of pi-turns occur independent of the Schellman motif. The two categories of pi-turns, designated as pi-HB and SCH, have been further classified on the basis of backbone conformation and both have AAAa as the major class. They differ in the residue usage at position (i+1), the former having a large preference for Pro that is absent in the latter. As in the case of shorter length beta- and alpha-turns, pi-turns have also been identified not only on the basis of the existence of hydrogen bond, but also using the distance between terminal C alpha-atoms, and this resulted in a comparable number of non-hydrogen-bonded pi-turns (pi-NHB). The presence of shorter beta- and alpha-turns within all categories of pi-turns, the subtle variations in backbone torsion angles along the turn residues, the location of the turns in the context of tertiary structures have been studied. pi-turns have been characterized, first using hydrogen bond and the distance between C alpha atoms of the terminal residues, and then using backbone torsion angles. While the Schellman motif has a structural role in helix termination, many of the pi-HB turns, being located on surface cavities, have functional role and there is also sequence

  12. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    International Nuclear Information System (INIS)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-01-01

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  13. Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

    Directory of Open Access Journals (Sweden)

    Hiu-Kwan Chow

    Full Text Available Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim protein Arnt fused to the leucine zipper (LZ dimerization domain from bZIP (basic region-leucine zipper protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H, transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed, as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d 148.9 nM and 40.2 nM, respectively, but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa. Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

  14. Sample preparation

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    Sample preparation prior to HPLC analysis is certainly one of the most important steps to consider in trace or ultratrace analysis. For many years scientists have tried to simplify the sample preparation process. It is rarely possible to inject a neat liquid sample or a sample where preparation may not be any more complex than dissolution of the sample in a given solvent. The last process alone can remove insoluble materials, which is especially helpful with the samples in complex matrices if other interactions do not affect extraction. Here, it is very likely a large number of components will not dissolve and are, therefore, eliminated by a simple filtration process. In most cases, the process of sample preparation is not as simple as dissolution of the component interest. At times, enrichment is necessary, that is, the component of interest is present in very large volume or mass of material. It needs to be concentrated in some manner so a small volume of the concentrated or enriched sample can be injected into HPLC. 88 refs

  15. Sampling Development

    Science.gov (United States)

    Adolph, Karen E.; Robinson, Scott R.

    2011-01-01

    Research in developmental psychology requires sampling at different time points. Accurate depictions of developmental change provide a foundation for further empirical studies and theories about developmental mechanisms. However, overreliance on widely spaced sampling intervals in cross-sectional and longitudinal designs threatens the validity of…

  16. Effects of spermine NONOate and ATP on the thermal stability of hemoglobin

    Directory of Open Access Journals (Sweden)

    Bassam Rasha

    2012-08-01

    Full Text Available Abstract Background Minor changes in protein structure induced by small organic and inorganic molecules can result in significant metabolic effects. The effects can be even more profound if the molecular players are chemically active and present in the cell in considerable amounts. The aim of our study was to investigate effects of a nitric oxide donor (spermine NONOate, ATP and sodium/potassium environment on the dynamics of thermal unfolding of human hemoglobin (Hb. The effect of these molecules was examined by means of circular dichroism spectrometry (CD in the temperature range between 25°C and 70°C. The alpha-helical content of buffered hemoglobin samples (0.1 mg/ml was estimated via ellipticity change measurements at a heating rate of 1°C/min. Results Major results were: 1 spermine NONOate persistently decreased the hemoglobin unfolding temperature Tuirrespectively of the Na + /K + environment, 2 ATP instead increased the unfolding temperature by 3°C in both sodium-based and potassium-based buffers and 3 mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions. Moreover, the presence of potassium facilitated a partial unfolding of alpha-helical structures even at room temperature. Conclusion The obtained data might shed more light on molecular mechanisms and biophysics involved in the regulation of protein activity by small solutes in the cell.

  17. Environmental sampling

    International Nuclear Information System (INIS)

    Puckett, J.M.

    1998-01-01

    Environmental Sampling (ES) is a technology option that can have application in transparency in nuclear nonproliferation. The basic process is to take a sample from the environment, e.g., soil, water, vegetation, or dust and debris from a surface, and through very careful sample preparation and analysis, determine the types, elemental concentration, and isotopic composition of actinides in the sample. The sample is prepared and the analysis performed in a clean chemistry laboratory (CCL). This ES capability is part of the IAEA Strengthened Safeguards System. Such a Laboratory is planned to be built by JAERI at Tokai and will give Japan an intrinsic ES capability. This paper presents options for the use of ES as a transparency measure for nuclear nonproliferation

  18. Changes in persistent contaminant concentration and CYP1A1 protein expression in biopsy samples from northern bottlenose whales, Hyperoodon ampullatus, following the onset of nearby oil and gas development

    Energy Technology Data Exchange (ETDEWEB)

    Hooker, Sascha K. [Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1 (Canada); Sea Mammal Research Unit, University of St Andrews, FIFE KY16 8YG (United Kingdom)], E-mail: s.hooker@st-andrews.ac.uk; Metcalfe, Tracy L.; Metcalfe, Chris D. [Environmental and Resource Studies, Trent University, Peterborough, Ontario K9J 7B8 (Canada); Angell, Carolyn M.; Wilson, Joanna Y.; Moore, Michael J. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Whitehead, Hal [Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1 (Canada)

    2008-03-15

    A small population of endangered northern bottlenose whales (Hyperoodon ampullatus) inhabits 'The Gully' a Marine Protected Area on the Scotian Shelf, eastern Canada. Amid concerns regarding nearby oil and gas development, we took 36 skin and blubber biopsy samples in 1996-1997 (prior to major development) and 2002-2003 (five years after development began), and three samples from a population in the Davis Strait, Labrador in 2003. These were analysed for cytochrome P4501A1 (CYP1A1) protein expression (n = 36), and for persistent contaminants (n = 23). CYP1A1 showed generally low expression in whales from The Gully, but higher levels during 2003, potentially coincident with recorded oil spills, and higher levels in Davis Strait whales. A range of PCB congeners and organochlorine compounds were detected, with concentrations similar to other North Atlantic odontocetes. Concentrations were higher in whales from The Gully than from the Davis Strait, with significant increases in 4,4'-DDE and trans-nonachlor in 2002-2003 relative to 1996-1997. - Whale contaminants highlight concerns from oil and gas development near a marine protected area.

  19. Changes in persistent contaminant concentration and CYP1A1 protein expression in biopsy samples from northern bottlenose whales, Hyperoodon ampullatus, following the onset of nearby oil and gas development

    International Nuclear Information System (INIS)

    Hooker, Sascha K.; Metcalfe, Tracy L.; Metcalfe, Chris D.; Angell, Carolyn M.; Wilson, Joanna Y.; Moore, Michael J.; Whitehead, Hal

    2008-01-01

    A small population of endangered northern bottlenose whales (Hyperoodon ampullatus) inhabits 'The Gully' a Marine Protected Area on the Scotian Shelf, eastern Canada. Amid concerns regarding nearby oil and gas development, we took 36 skin and blubber biopsy samples in 1996-1997 (prior to major development) and 2002-2003 (five years after development began), and three samples from a population in the Davis Strait, Labrador in 2003. These were analysed for cytochrome P4501A1 (CYP1A1) protein expression (n = 36), and for persistent contaminants (n = 23). CYP1A1 showed generally low expression in whales from The Gully, but higher levels during 2003, potentially coincident with recorded oil spills, and higher levels in Davis Strait whales. A range of PCB congeners and organochlorine compounds were detected, with concentrations similar to other North Atlantic odontocetes. Concentrations were higher in whales from The Gully than from the Davis Strait, with significant increases in 4,4'-DDE and trans-nonachlor in 2002-2003 relative to 1996-1997. - Whale contaminants highlight concerns from oil and gas development near a marine protected area

  20. Spherical sampling

    CERN Document Server

    Freeden, Willi; Schreiner, Michael

    2018-01-01

    This book presents, in a consistent and unified overview, results and developments in the field of today´s spherical sampling, particularly arising in mathematical geosciences. Although the book often refers to original contributions, the authors made them accessible to (graduate) students and scientists not only from mathematics but also from geosciences and geoengineering. Building a library of topics in spherical sampling theory it shows how advances in this theory lead to new discoveries in mathematical, geodetic, geophysical as well as other scientific branches like neuro-medicine. A must-to-read for everybody working in the area of spherical sampling.

  1. Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from Pyrococcus furiosus.

    Directory of Open Access Journals (Sweden)

    Pascal F Egea

    Full Text Available In all organisms the Signal Recognition Particle (SRP, binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu. The 2.5 A resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely alpha-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 A resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19*SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP

  2. Fluidic sampling

    International Nuclear Information System (INIS)

    Houck, E.D.

    1992-01-01

    This paper covers the development of the fluidic sampler and its testing in a fluidic transfer system. The major findings of this paper are as follows. Fluidic jet samples can dependably produce unbiased samples of acceptable volume. The fluidic transfer system with a fluidic sampler in-line will transfer water to a net lift of 37.2--39.9 feet at an average ratio of 0.02--0.05 gpm (77--192 cc/min). The fluidic sample system circulation rate compares very favorably with the normal 0.016--0.026 gpm (60--100 cc/min) circulation rate that is commonly produced for this lift and solution with the jet-assisted airlift sample system that is normally used at ICPP. The volume of the sample taken with a fluidic sampler is dependant on the motive pressure to the fluidic sampler, the sample bottle size and on the fluidic sampler jet characteristics. The fluidic sampler should be supplied with fluid having the motive pressure of the 140--150 percent of the peak vacuum producing motive pressure for the jet in the sampler. Fluidic transfer systems should be operated by emptying a full pumping chamber to nearly empty or empty during the pumping cycle, this maximizes the solution transfer rate

  3. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge.

    Science.gov (United States)

    Perryman, Alexander L; Santiago, Daniel N; Forli, Stefano; Martins, Diogo Santos; Olson, Arthur J

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  4. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge

    Science.gov (United States)

    Perryman, Alexander L.; Santiago, Daniel N.; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J.

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  5. Sampling methods

    International Nuclear Information System (INIS)

    Loughran, R.J.; Wallbrink, P.J.; Walling, D.E.; Appleby, P.G.

    2002-01-01

    Methods for the collection of soil samples to determine levels of 137 Cs and other fallout radionuclides, such as excess 210 Pb and 7 Be, will depend on the purposes (aims) of the project, site and soil characteristics, analytical capacity, the total number of samples that can be analysed and the sample mass required. The latter two will depend partly on detector type and capabilities. A variety of field methods have been developed for different field conditions and circumstances over the past twenty years, many of them inherited or adapted from soil science and sedimentology. The use of them inherited or adapted from soil science and sedimentology. The use of 137 Cs in erosion studies has been widely developed, while the application of fallout 210 Pb and 7 Be is still developing. Although it is possible to measure these nuclides simultaneously, it is common for experiments to designed around the use of 137 Cs along. Caesium studies typically involve comparison of the inventories found at eroded or sedimentation sites with that of a 'reference' site. An accurate characterization of the depth distribution of these fallout nuclides is often required in order to apply and/or calibrate the conversion models. However, depending on the tracer involved, the depth distribution, and thus the sampling resolution required to define it, differs. For example, a depth resolution of 1 cm is often adequate when using 137 Cs. However, fallout 210 Pb and 7 Be commonly has very strong surface maxima that decrease exponentially with depth, and fine depth increments are required at or close to the soil surface. Consequently, different depth incremental sampling methods are required when using different fallout radionuclides. Geomorphic investigations also frequently require determination of the depth-distribution of fallout nuclides on slopes and depositional sites as well as their total inventories

  6. Personalizing Protein Nourishment

    Science.gov (United States)

    DALLAS, DAVID C.; SANCTUARY, MEGAN R.; QU, YUNYAO; KHAJAVI, SHABNAM HAGHIGHAT; VAN ZANDT, ALEXANDRIA E.; DYANDRA, MELISSA; FRESE, STEVEN A.; BARILE, DANIELA; GERMAN, J. BRUCE

    2016-01-01

    Proteins are not equally digestible—their proteolytic susceptibility varies by their source and processing method. Incomplete digestion increases colonic microbial protein fermentation (putrefaction), which produces toxic metabolites that can induce inflammation in vitro and have been associated with inflammation in vivo. Individual humans differ in protein digestive capacity based on phenotypes, particularly disease states. To avoid putrefaction-induced intestinal inflammation, protein sources and processing methods must be tailored to the consumer’s digestive capacity. This review explores how food processing techniques alter protein digestibility and examines how physiological conditions alter digestive capacity. Possible solutions to improving digestive function or matching low digestive capacity with more digestible protein sources are explored. Beyond the ileal digestibility measurements of protein digestibility, less invasive, quicker and cheaper techniques for monitoring the extent of protein digestion and fermentation are needed to personalize protein nourishment. Biomarkers of protein digestive capacity and efficiency can be identified with the toolsets of peptidomics, metabolomics, microbial sequencing and multiplexed protein analysis of fecal and urine samples. By monitoring individual protein digestive function, the protein component of diets can be tailored via protein source and processing selection to match individual needs to minimize colonic putrefaction and, thus, optimize gut health. PMID:26713355

  7. Molecular basis for endotoxin neutralization by amphipathic peptides derived from the alpha-helical cationic core-region of NK-lysin.

    Science.gov (United States)

    Brandenburg, Klaus; Garidel, Patrick; Fukuoka, Satoshi; Howe, Jörg; Koch, Michel H J; Gutsmann, Thomas; Andrä, Jörg

    2010-08-01

    An analysis of the interaction of the NK-lysin derived peptide NK-2 and of analogs thereof with bacterial lipopolysaccharide (LPS, endotoxin) was performed to determine the most important biophysical parameters for an effective LPS neutralization. We used microcalorimetry, FTIR spectroscopy, Zeta potential measurements, and small-angle X-ray scattering to analyze the peptide:LPS binding enthalpy, the accessible LPS surface charge, the fluidity of the LPS hydrocarbon chains, their phase transition enthalpy change, the aggregate structure of LPS, and how these parameters are modulated by the peptides. We conclude that (i) a high peptide:LPS binding affinity, which is facilitated by electrostatic and hydrophobic interactions and which leads to a positive Zeta potential, (ii) the formation of peptide-enriched domains, which destabilize the lipid packing, demonstrated by a drastic decrease of phase transition enthalpy change of LPS, and (iii) the multilamellarization of the LPS aggregate structure are crucial for an effective endotoxin neutralization by cationic peptides.

  8. Digital Microfluidics Sample Analyzer

    Science.gov (United States)

    Pollack, Michael G.; Srinivasan, Vijay; Eckhardt, Allen; Paik, Philip Y.; Sudarsan, Arjun; Shenderov, Alex; Hua, Zhishan; Pamula, Vamsee K.

    2010-01-01

    Three innovations address the needs of the medical world with regard to microfluidic manipulation and testing of physiological samples in ways that can benefit point-of-care needs for patients such as premature infants, for which drawing of blood for continuous tests can be life-threatening in their own right, and for expedited results. A chip with sample injection elements, reservoirs (and waste), droplet formation structures, fluidic pathways, mixing areas, and optical detection sites, was fabricated to test the various components of the microfluidic platform, both individually and in integrated fashion. The droplet control system permits a user to control droplet microactuator system functions, such as droplet operations and detector operations. Also, the programming system allows a user to develop software routines for controlling droplet microactuator system functions, such as droplet operations and detector operations. A chip is incorporated into the system with a controller, a detector, input and output devices, and software. A novel filler fluid formulation is used for the transport of droplets with high protein concentrations. Novel assemblies for detection of photons from an on-chip droplet are present, as well as novel systems for conducting various assays, such as immunoassays and PCR (polymerase chain reaction). The lab-on-a-chip (a.k.a., lab-on-a-printed-circuit board) processes physiological samples and comprises a system for automated, multi-analyte measurements using sub-microliter samples of human serum. The invention also relates to a diagnostic chip and system including the chip that performs many of the routine operations of a central labbased chemistry analyzer, integrating, for example, colorimetric assays (e.g., for proteins), chemiluminescence/fluorescence assays (e.g., for enzymes, electrolytes, and gases), and/or conductometric assays (e.g., for hematocrit on plasma and whole blood) on a single chip platform.

  9. Phylogenetic analysis of G1P[8] and G12P[8] rotavirus A samples obtained in the pre- and post-vaccine periods, and molecular modeling of VP4 and VP7 proteins.

    Science.gov (United States)

    Almeida, Tâmera Nunes Vieira; de Sousa, Teresinha Teixeira; da Silva, Roosevelt Alves; Fiaccadori, Fabíola Souza; Souza, Menira; Badr, Kareem Rady; de Paula Cardoso, Divina das Dôres

    2017-09-01

    Reduction in morbimortality rates for acute gastroenteritis (AGE) by Rotavirus A (RVA) has been observed after the introduction of vaccines, however the agent continues to circulate. The present study described the genomic characterization of the 11 dsRNA segments of two RVA samples G1P[8] obtained in the pre- and post-vaccination periods and one of G12P[8] sample (post-vaccine), compared to Rotarix™ vaccine. Analysis by molecular sequencing of the samples showed that the three samples belonged to genogroup I. In addition, the analysis of VP7 gene revealed that the samples G1 (pre-vaccine), G1 (post-vaccine) and G12 were characterized as lineages II, I and III, respectively. Regarding to VP4 and NSP4 gene it was observed that all samples belonged to lineage III, whereas for VP6 gene, the sample of the pre- and post-vaccine belonged to the lineage IV and I, respectively. Considering the VP7 gene, it was observed high nucleotide and amino acid identity for the two G1 samples when compared to Rotarix™ vaccine and lesser identity for the G12 sample. In relation to antigenic epitope of VP7 greater modifications were observed for the G12 sample in the 7-2 epitope that was confirmed by molecular modeling. On the other hand, for VP4, some changes in the 8-1 and 8-3 antigenic epitopes was observed for the three samples. This data could be interpreted as a low selective pressure exerted by vaccination in relation to G1P[8] samples and lesser protection in relation to G12P[8]. Thus, the continuous monitoring of RVA circulating samples remains important. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. 24-hour urine protein

    Science.gov (United States)

    ... your provider may be able to order a test that is done on just one urine sample (protein-to-creatinine ratio). Normal Results The normal ... Some labs use different measurements or test different samples. Talk to your provider about the meaning of your specific test ... Abnormal results may be due to: A group ...

  11. Enhanced sampling algorithms.

    Science.gov (United States)

    Mitsutake, Ayori; Mori, Yoshiharu; Okamoto, Yuko

    2013-01-01

    In biomolecular systems (especially all-atom models) with many degrees of freedom such as proteins and nucleic acids, there exist an astronomically large number of local-minimum-energy states. Conventional simulations in the canonical ensemble are of little use, because they tend to get trapped in states of these energy local minima. Enhanced conformational sampling techniques are thus in great demand. A simulation in generalized ensemble performs a random walk in potential energy space and can overcome this difficulty. From only one simulation run, one can obtain canonical-ensemble averages of physical quantities as functions of temperature by the single-histogram and/or multiple-histogram reweighting techniques. In this article we review uses of the generalized-ensemble algorithms in biomolecular systems. Three well-known methods, namely, multicanonical algorithm, simulated tempering, and replica-exchange method, are described first. Both Monte Carlo and molecular dynamics versions of the algorithms are given. We then present various extensions of these three generalized-ensemble algorithms. The effectiveness of the methods is tested with short peptide and protein systems.

  12. Quantification analysis of the expression of tumor-associated proteins in serum samples from patients with ovarian cancer and those with other tumor location. Possibilities of their use in the diagnosis and estimation of the extent of a tumorous process

    Directory of Open Access Journals (Sweden)

    T. S. Bobrova

    2012-01-01

    Full Text Available The specific features of the expression of tumor-associated proteins (TAP were immunologically studied in the sera of patients with ovarian cancer (OC and other tumor location by means of immune sera (As or monoclonal antibodies (MAb to find out whether they could be used to diagnose and estimate the extent of a tumorous process.MAb 1 (to HEp-2 cell membrane proteins, larynx cancer, Ac4 (to a pool of two ovarian cystadenocarcinomas, and MAb 3 (to affinity-pu- rified proteins of the apparently intact human gastric mucosa were used to examine the sera of patients with OC and other tumor location and positive responsiveness was detected in 82, ~100, and 77 % of cases, respectively. The differences in the expression of TAP in the patients versus healthy donors were shown to be statistically significant (p = 0.0001; p = 0.015; p = 0.01, respectively.The sensitivity of quantifying ELISA in detecting TAP was 78 and 85 % in patients with Stages I–II and III–IV OC, respectively; ~100 and 89 % in patients with breast cancer and in those with gastrointestinal tract cancer, respectively; and 60 and 14 % in patients with lymphopro- liferative diseases and healthy donors, respectively. Comparison of TAP detection rates in the authors’ test systems with multiplex testing with a biochip array of 12 tumor markers has shown that these test systems are at the world standard level.

  13. 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.

  14. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  15. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  16. Whey Protein

    Science.gov (United States)

    ... reliable information about the safety of taking whey protein if you are pregnant or breast feeding. Stay on the safe side and avoid use. Milk allergy: If you are allergic to cow's milk, avoid using whey protein.

  17. Discovery and validation of plasma-protein biomarker panels for the detection of colorectal cancer and advanced adenoma in a Danish collection of samples from patients referred for diagnostic colonoscopy

    DEFF Research Database (Denmark)

    Blume, John E.; Wilhelmsen, Michael; Benz, Ryan W.

    2016-01-01

    and utilization of such a resource is an important step in the development of blood-based biomarker tests for colorectal cancer.Methods: We have created a subject data and biological sample resource, Endoscopy II, which is based on 4698 individuals referred for diagnostic colonoscopy in Denmark between May 2010...

  18. Modern survey sampling

    CERN Document Server

    Chaudhuri, Arijit

    2014-01-01

    Exposure to SamplingAbstract Introduction Concepts of Population, Sample, and SamplingInitial RamificationsAbstract Introduction Sampling Design, Sampling SchemeRandom Numbers and Their Uses in Simple RandomSampling (SRS)Drawing Simple Random Samples with and withoutReplacementEstimation of Mean, Total, Ratio of Totals/Means:Variance and Variance EstimationDetermination of Sample SizesA.2 Appendix to Chapter 2 A.More on Equal Probability Sampling A.Horvitz-Thompson EstimatorA.SufficiencyA.LikelihoodA.Non-Existence Theorem More Intricacies Abstract Introduction Unequal Probability Sampling StrategiesPPS Sampling Exploring Improved WaysAbstract Introduction Stratified Sampling Cluster SamplingMulti-Stage SamplingMulti-Phase Sampling: Ratio and RegressionEstimationviiviii ContentsControlled SamplingModeling Introduction Super-Population ModelingPrediction Approach Model-Assisted Approach Bayesian Methods Spatial SmoothingSampling on Successive Occasions: Panel Rotation Non-Response and Not-at-Homes Weighting Adj...

  19. Electrophoretic transfer protein zymography.

    Science.gov (United States)

    Pan, Daniel; Hill, Adam P; Kashou, Anthony; Wilson, Karl A; Tan-Wilson, Anna

    2011-04-15

    Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS-PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Systematic sampling with errors in sample locations

    DEFF Research Database (Denmark)

    Ziegel, Johanna; Baddeley, Adrian; Dorph-Petersen, Karl-Anton

    2010-01-01

    analysis using point process methods. We then analyze three different models for the error process, calculate exact expressions for the variances, and derive asymptotic variances. Errors in the placement of sample points can lead to substantial inflation of the variance, dampening of zitterbewegung......Systematic sampling of points in continuous space is widely used in microscopy and spatial surveys. Classical theory provides asymptotic expressions for the variance of estimators based on systematic sampling as the grid spacing decreases. However, the classical theory assumes that the sample grid...... is exactly periodic; real physical sampling procedures may introduce errors in the placement of the sample points. This paper studies the effect of errors in sample positioning on the variance of estimators in the case of one-dimensional systematic sampling. First we sketch a general approach to variance...

  1. Quantification of the value of C-reactive protein and its relationship with periodontal disease and cardiovascular risk in a sample of patients from the Facultad de Odontologia de la Universidad de Costa Rica: preliminary study

    International Nuclear Information System (INIS)

    Bermudez Mora, Gustavo; Gonzalez Leon, Yerlin; Jimenez Hernandez, Melissa; Padron Giraldino, Lieny; Rojas Villagra, Andrea

    2013-01-01

    Plasma P-CR concentration was measured and the value obtained was related to the presence and severity of periodontal and cardiovascular disease. A cross-sectional descriptive study was carried out, with the sample of 30 patients of different gender, age, type of periodontal disease and systemic condition. A serological test was performed on the subjects and analyzed quantitatively, using the measuring parameter of the Roche / Hitachi cobas 111 device, which automatically calculates the concentration of the P-CR in each sample. The results lack a statistically significant relationship of gender, age or degree of periodontal disease. The average value of P-CR was obtained from the study (3.72 mg / L) corresponds to a high risk of cardiovascular disease based on the parameters established by the American Heart Association. The only significant factor was the presence of associated systemic diseases in patients with high levels of P-CR, in comparison with those who suffer from some type of ailment. (author) [es

  2. Signal sampling circuit

    NARCIS (Netherlands)

    Louwsma, S.M.; Vertregt, Maarten

    2011-01-01

    A sampling circuit for sampling a signal is disclosed. The sampling circuit comprises a plurality of sampling channels adapted to sample the signal in time-multiplexed fashion, each sampling channel comprising a respective track-and-hold circuit connected to a respective analogue to digital

  3. Signal sampling circuit

    NARCIS (Netherlands)

    Louwsma, S.M.; Vertregt, Maarten

    2010-01-01

    A sampling circuit for sampling a signal is disclosed. The sampling circuit comprises a plurality of sampling channels adapted to sample the signal in time-multiplexed fashion, each sampling channel comprising a respective track-and-hold circuit connected to a respective analogue to digital

  4. Conformational analysis of the partially disordered measles virus N(TAIL)-XD complex by SDSL EPR spectroscopy.

    Science.gov (United States)

    Kavalenka, Aleh; Urbancic, Iztok; Belle, Valérie; Rouger, Sabrina; Costanzo, Stéphanie; Kure, Sandra; Fournel, André; Longhi, Sonia; Guigliarelli, Bruno; Strancar, Janez

    2010-03-17

    To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) both free and in complex with the X domain (XD, aa 459-507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted N(TAIL) variants, we showed that the 490-500 region of N(TAIL) is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the alpha-helical sampling of the free form. In addition, we showed that the 505-525 region of N(TAIL) conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the N(TAIL)-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488-525 region of N(TAIL), in particular the unfolding of the alpha-helical region when the temperature was increased from 281 K to 310 K. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  6. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  7. Urine sample preparation for proteomic analysis.

    Science.gov (United States)

    Olszowy, Pawel; Buszewski, Boguslaw

    2014-10-01

    Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross-reactions that should be taken into consideration. The incorporation of new post-translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Influence of physical and chemical properties of HTSXT-FTIR samples on the quality of prediction models developed to determine absolute concentrations of total proteins, carbohydrates and triglycerides: a preliminary study on the determination of their absolute concentrations in fresh microalgal biomass.

    Science.gov (United States)

    Serrano León, Esteban; Coat, Rémy; Moutel, Benjamin; Pruvost, Jérémy; Legrand, Jack; Gonçalves, Olivier

    2014-11-01

    Absolute concentrations of total macromolecules (triglycerides, proteins and carbohydrates) in microorganisms can be rapidly measured by FTIR spectroscopy, but caution is needed to avoid non-specific experimental bias. Here, we assess the limits within which this approach can be used on model solutions of macromolecules of interest. We used the Bruker HTSXT-FTIR system. Our results show that the solid deposits obtained after the sampling procedure present physical and chemical properties that influence the quality of the absolute concentration prediction models (univariate and multivariate). The accuracy of the models was degraded by a factor of 2 or 3 outside the recommended concentration interval of 0.5-35 µg spot(-1). Change occurred notably in the sample hydrogen bond network, which could, however, be controlled using an internal probe (pseudohalide anion). We also demonstrate that for aqueous solutions, accurate prediction of total carbohydrate quantities (in glucose equivalent) could not be made unless a constant amount of protein was added to the model solution (BSA). The results of the prediction model for more complex solutions, here with two components: glucose and BSA, were very encouraging, suggesting that this FTIR approach could be used as a rapid quantification method for mixtures of molecules of interest, provided the limits of use of the HTSXT-FTIR method are precisely known and respected. This last finding opens the way to direct quantification of total molecules of interest in more complex matrices.

  9. Validation of EIA sampling methods - bacterial and biochemical analysis

    Digital Repository Service at National Institute of Oceanography (India)

    Sheelu, G.; LokaBharathi, P.A.; Nair, S.; Raghukumar, C.; Mohandass, C.

    to temporal factors. Paired T-test between pre- and post-disturbance samples suggested that the above methods of sampling and variables like TC, protein and TOC could be used for monitoring disturbance....

  10. On the Sampling

    OpenAIRE

    Güleda Doğan

    2017-01-01

    This editorial is on statistical sampling, which is one of the most two important reasons for editorial rejection from our journal Turkish Librarianship. The stages of quantitative research, the stage in which we are sampling, the importance of sampling for a research, deciding on sample size and sampling methods are summarised briefly.

  11. Information sampling behavior with explicit sampling costs

    Science.gov (United States)

    Juni, Mordechai Z.; Gureckis, Todd M.; Maloney, Laurence T.

    2015-01-01

    The decision to gather information should take into account both the value of information and its accrual costs in time, energy and money. Here we explore how people balance the monetary costs and benefits of gathering additional information in a perceptual-motor estimation task. Participants were rewarded for touching a hidden circular target on a touch-screen display. The target’s center coincided with the mean of a circular Gaussian distribution from which participants could sample repeatedly. Each “cue” — sampled one at a time — was plotted as a dot on the display. Participants had to repeatedly decide, after sampling each cue, whether to stop sampling and attempt to touch the hidden target or continue sampling. Each additional cue increased the participants’ probability of successfully touching the hidden target but reduced their potential reward. Two experimental conditions differed in the initial reward associated with touching the hidden target and the fixed cost per cue. For each condition we computed the optimal number of cues that participants should sample, before taking action, to maximize expected gain. Contrary to recent claims that people gather less information than they objectively should before taking action, we found that participants over-sampled in one experimental condition, and did not significantly under- or over-sample in the other. Additionally, while the ideal observer model ignores the current sample dispersion, we found that participants used it to decide whether to stop sampling and take action or continue sampling, a possible consequence of imperfect learning of the underlying population dispersion across trials. PMID:27429991

  12. How Sample Size Affects a Sampling Distribution

    Science.gov (United States)

    Mulekar, Madhuri S.; Siegel, Murray H.

    2009-01-01

    If students are to understand inferential statistics successfully, they must have a profound understanding of the nature of the sampling distribution. Specifically, they must comprehend the determination of the expected value and standard error of a sampling distribution as well as the meaning of the central limit theorem. Many students in a high…

  13. [Nutritional value of the proteins of carboxydobacteria].

    Science.gov (United States)

    Volova, T G; Vlasova, N V; Barashkov, V A

    1980-01-01

    The biological value of proteins from 3 carboxidobacterial strains was assessed on the basis of the protein amino acid content, consumption by the test-organism T. pyriformis and protein susceptibility to proteolytic enzymes in vitro. Carboxidobacterial proteins are characterized by a full-value amino acid composition and contain large amounts of all indispensable amino acids. This indicates that on the whole these proteins are quality ones. Experiments with the test-organisms have shown, however, that the relative biological value of the protein samples significantly yields to casein. The total digestibility of carboxidobacterial proteins by pepsin and trypsin in vitro is close to that of wheat vegetable proteins and lower as compared with that of casein. Carboxidobacterial proteins may be thus attributed to a protein group being an intermediate one between casein and wheat vegetable proteins.

  14. Radioactivity in environmental samples

    International Nuclear Information System (INIS)

    Fornaro, Laura

    2001-01-01

    The objective of this practical work is to familiarize the student with radioactivity measures in environmental samples. For that were chosen samples a salt of natural potassium, a salt of uranium or torio and a sample of drinkable water

  15. DNA Sampling Hook

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The DNA Sampling Hook is a significant improvement on a method of obtaining a tissue sample from a live fish in situ from an aquatic environment. A tissue sample...

  16. Iowa Geologic Sampling Points

    Data.gov (United States)

    Iowa State University GIS Support and Research Facility — Point locations of geologic samples/files in the IGS repository. Types of samples include well cuttings, outcrop samples, cores, drillers logs, measured sections,...

  17. Network and adaptive sampling

    CERN Document Server

    Chaudhuri, Arijit

    2014-01-01

    Combining the two statistical techniques of network sampling and adaptive sampling, this book illustrates the advantages of using them in tandem to effectively capture sparsely located elements in unknown pockets. It shows how network sampling is a reliable guide in capturing inaccessible entities through linked auxiliaries. The text also explores how adaptive sampling is strengthened in information content through subsidiary sampling with devices to mitigate unmanageable expanding sample sizes. Empirical data illustrates the applicability of both methods.

  18. Avaliação de métodos espectrofotométricos para determinação de proteína em amostras de lagoas de estabilização Evaluation of spectrophotometric methods for protein determination in waste stabilization ponds sample

    Directory of Open Access Journals (Sweden)

    Adriana Cristina Poli Miwa

    2008-06-01

    Full Text Available Esta pesquisa teve como objetivo principal comparar cinco métodos espectrofotométricos para determinação de proteínas em amostras provenientes de estações de tratamento de efluentes sanitários. O intuito foi definir uma metodologia de aplicação rápida, fácil e confiável para este tipo de amostra. As lagoas de estabilização, como sistemas de tratamento biológico, têm como principais constituintes proteínas, carboidratos e lipídeos, mas também apresentam muitos compostos interferentes, como por exemplo, uréia, detergentes e compostos fenólicos, que podem prejudicar a quantificação de tais parâmetros. Os métodos analisados foram Lowry, Biureto, Bradford e Ácido bicinconínico. O método de Lowry mostrou-se mais adequado às características da amostra, com boa reprodutibilidade, reagente específico, custo moderado e ausência de substancias interferentes.This research had as main objective to compare five spectrophotometric methods for protein determination in samples proceeding from sanitary effluent of treatment plant. Intention was to define a methodology that is of fast and easy and reliable application for this type of sample. The stabilization ponds, as systems of biological treatment, have as main constituent proteins, carbohydrates and lipids, but also they present many interfering composites, for example, phenolic urea, detergents and composites, that can harm the quantification of such parameters. The analyzed methods had been Lowry, Biuret, Bradford and Acid bicinconinic. The method of Lowry revealed more adequate to the characteristics of the sample, with good reproducibility, specific reagent, moderate cost and absence of interfering substance.

  19. 'Boomerang'-like insertion of a fusogenic peptide in a lipid membrane revealed by solid-state 19F NMR.

    Science.gov (United States)

    Afonin, Sergii; Dürr, Ulrich H N; Glaser, Ralf W; Ulrich, Anne S

    2004-02-01

    Solid state (19)F NMR revealed the conformation and alignment of the fusogenic peptide sequence B18 from the sea urchin fertilization protein bindin embedded in flat phospholipid bilayers. Single (19)F labels were introduced into nine distinct positions along the wild-type sequence by substituting each hydrophobic amino acid, one by one, with L-4-fluorophenylglycine. Their anisotropic chemical shifts were measured in uniaxially oriented membrane samples and used as orientational constraints to model the peptide structure in the membrane-bound state. Previous (1)H NMR studies of B18 in 30% TFE and in detergent micelles had shown that the peptide structure consists of two alpha-helical segments that are connected by a flexible hinge. This helix-break-helix motif was confirmed here by the solid-state (19)F NMR data, while no other secondary structure (beta-sheet, 3(10)-helix) was compatible with the set of orientational constraints. For both alpha-helical segments we found that the helical conformation extends all the way to the respective N- and C-termini of the peptide. Analysis of the corresponding tilt and azimuthal rotation angles showed that the N-terminal helix of B18 is immersed obliquely into the bilayer (at a tilt angle tau approximately 54 degrees), whereas the C-terminus is peripherally aligned (tau approximately 91 degrees). The azimuthal orientation of the two segments is consistent with the amphiphilic distribution of side-chains. The observed 'boomerang'-like mode of insertion into the membrane may thus explain how peptide binding leads to lipid dehydration and acyl chain perturbation as a prerequisite for bilayer fusion to occur. Copyright 2004 John Wiley & Sons, Ltd.

  20. Sample preparation guidelines for two-dimensional electrophoresis.

    Science.gov (United States)

    Posch, Anton

    2014-12-01

    Sample preparation is one of the key technologies for successful two-dimensional electrophoresis (2DE). Due to the great diversity of protein sample types and sources, no single sample preparation method works with all proteins; for any sample the optimum procedure must be determined empirically. This review is meant to provide a broad overview of the most important principles in sample preparation in order to avoid a multitude of possible pitfalls. Sample preparation protocols from the expert in the field were screened and evaluated. On the basis of these protocols and my own comprehensive practical experience important guidelines are given in this review. The presented guidelines will facilitate straightforward protocol development for researchers new to gel-based proteomics. In addition the available choices are rationalized in order to successfully prepare a protein sample for 2DE separations. The strategies described here are not limited to 2DE and can also be applied to other protein separation techniques.

  1. Sampling procedures and tables

    International Nuclear Information System (INIS)

    Franzkowski, R.

    1980-01-01

    Characteristics, defects, defectives - Sampling by attributes and by variables - Sample versus population - Frequency distributions for the number of defectives or the number of defects in the sample - Operating characteristic curve, producer's risk, consumer's risk - Acceptable quality level AQL - Average outgoing quality AOQ - Standard ISQ 2859 - Fundamentals of sampling by variables for fraction defective. (RW)

  2. Protein intake does not increase vastus lateralis muscle protein synthesis during cycling

    DEFF Research Database (Denmark)

    Hulston, CJ; Wolsk, Emil; Grøndahl, Thomas Sahl

    2011-01-01

    PURPOSE: This study aimed to investigate the effect of protein ingestion on leg protein turnover and vastus lateralis muscle protein synthesis during bicycle exercise and recovery. METHODS: Eight healthy males participated in two experiments in which they ingested either a carbohydrate solution...... sampling, and blood flow measurements. Muscle protein synthesis was calculated from the incorporation of l-[ring-C6]phenylalanine into protein. RESULTS: Consuming protein during exercise increased leg protein synthesis and decreased net leg protein breakdown; however, protein ingestion did not increase...... protein synthesis within the highly active vastus lateralis muscle (0.029%·h(-1), ± 0.004%·h(-1), and 0.030%·h(-1), ± 0.003%·h(-1), in CHO and CHO + P, respectively; P = 0.88). In contrast, consuming protein, during exercise and recovery, increased postexercise vastus lateralis muscle protein synthesis...

  3. Effective sample labeling

    International Nuclear Information System (INIS)

    Rieger, J.T.; Bryce, R.W.

    1990-01-01

    Ground-water samples collected for hazardous-waste and radiological monitoring have come under strict regulatory and quality assurance requirements as a result of laws such as the Resource Conservation and Recovery Act. To comply with these laws, the labeling system used to identify environmental samples had to be upgraded to ensure proper handling and to protect collection personnel from exposure to sample contaminants and sample preservatives. The sample label now used as the Pacific Northwest Laboratory is a complete sample document. In the event other paperwork on a labeled sample were lost, the necessary information could be found on the label

  4. Enhanced conformational sampling using enveloping distribution sampling.

    Science.gov (United States)

    Lin, Zhixiong; van Gunsteren, Wilfred F

    2013-10-14

    To lessen the problem of insufficient conformational sampling in biomolecular simulations is still a major challenge in computational biochemistry. In this article, an application of the method of enveloping distribution sampling (EDS) is proposed that addresses this challenge and its sampling efficiency is demonstrated in simulations of a hexa-β-peptide whose conformational equilibrium encompasses two different helical folds, i.e., a right-handed 2.7(10∕12)-helix and a left-handed 3(14)-helix, separated by a high energy barrier. Standard MD simulations of this peptide using the GROMOS 53A6 force field did not reach convergence of the free enthalpy difference between the two helices even after 500 ns of simulation time. The use of soft-core non-bonded interactions in the centre of the peptide did enhance the number of transitions between the helices, but at the same time led to neglect of relevant helical configurations. In the simulations of a two-state EDS reference Hamiltonian that envelops both the physical peptide and the soft-core peptide, sampling of the conformational space of the physical peptide ensures that physically relevant conformations can be visited, and sampling of the conformational space of the soft-core peptide helps to enhance the transitions between the two helices. The EDS simulations sampled many more transitions between the two helices and showed much faster convergence of the relative free enthalpy of the two helices compared with the standard MD simulations with only a slightly larger computational effort to determine optimized EDS parameters. Combined with various methods to smoothen the potential energy surface, the proposed EDS application will be a powerful technique to enhance the sampling efficiency in biomolecular simulations.

  5. Optimized preparation of urine samples for two-dimensional electrophoresis and initial application to patient samples

    DEFF Research Database (Denmark)

    Lafitte, Daniel; Dussol, Bertrand; Andersen, Søren

    2002-01-01

    OBJECTIVE: We optimized of the preparation of urinary samples to obtain a comprehensive map of urinary proteins of healthy subjects and then compared this map with the ones obtained with patient samples to show that the pattern was specific of their kidney disease. DESIGN AND METHODS: The urinary...

  6. Sampling in practice

    DEFF Research Database (Denmark)

    Esbensen, Kim Harry; Petersen, Lars

    2005-01-01

    A basic knowledge of the Theory of Sampling (TOS) and a set of only eight sampling unit operations is all the practical sampler needs to ensure representativeness of samples extracted from all kinds of lots: production batches, - truckloads, - barrels, sub-division in the laboratory, sampling...... in nature and in the field (environmental sampling, forestry, geology, biology), from raw materials or manufactory processes etc. We here can only give a brief introduction to the Fundamental Sampling Principle (FSP) and these eight Sampling Unit Operations (SUO’s). Always respecting FSP and invoking only...... the necessary SUO’s (dependent on the practical situation) is the only prerequisite needed for eliminating all sampling bias and simultaneously minimizing sampling variance, and this is in addition a sure guarantee for making the final analytical results trustworthy. No reliable conclusions can be made unless...

  7. Modeling of allergen proteins found in sea food products Modelagem de proteínas alergênicas em frutos do mar

    Directory of Open Access Journals (Sweden)

    Nataly Galán-Freyle

    2012-06-01

    Full Text Available Shellfish are a source of food allergens, and their consumption is the cause of severe allergic reactions in humans. Tropomyosins, a family of muscle proteins, have been identified as the major allergens in shellfish and mollusks species. Nevertheless, few experimentally determined three-dimensional structures are available in the Protein Data Base (PDB. In this study, 3D models of several homologous of tropomyosins present in marine shellfish and mollusk species (Chaf 1, Met e1, Hom a1, Per v1, and Pen a1 were constructed, validated, and their immunoglobulin E binding epitopes were identified using bioinformatics tools. All protein models for these allergens consisted of long alpha-helices. Chaf 1, Met e1, and Hom a1 had six conserved regions with sequence similarities to known epitopes, whereas Per v1 and Pen a1 contained only one. Lipophilic potentials of identified epitopes revealed a high propensity of hydrophobic amino acids in the immunoglobulin E binding site. This information could be useful to design tropomyosin-specific immunotherapy for sea food allergies.Os mariscos são fontes de alérgenos alimentares e seu consumo é a causa de graves reações alérgicas em humanos. Tropomiosinas, uma família de proteínas musculares, foram identificadas como os principais alérgenos em espécies de crustáceos e moluscos. No entanto, poucas estruturas experimentais tridimensionais estão disponíveis no Protein Data Base (PDB. Neste trabalho, modelos 3D de vários homólogos de tropomiosinas presentes em moluscos marinhos e espécies de moluscos (Chaf 1, Met e1, Hom a1, v1 Per e Pen a1 foram construídas, validadas e seus epítopos de ligação de imunoglobulina E (IgE foram identificados, utilizando ferramentas de bioinformática. Todos os modelos de proteína para esses alérgenos consistiam em longas alfa-hélices. Chaf 1, Met e1, e Hom a1 apresentaram seis regiões conservadas com similaridades de sequência para epítopos conhecidos

  8. Sampling of ore

    International Nuclear Information System (INIS)

    Boehme, R.C.; Nicholas, B.L.

    1987-01-01

    This invention relates to a method of an apparatus for ore sampling. The method includes the steps of periodically removing a sample of the output material of a sorting machine, weighing each sample so that each is of the same weight, measuring a characteristic such as the radioactivity, magnetivity or the like of each sample, subjecting at least an equal portion of each sample to chemical analysis to determine the mineral content of the sample and comparing the characteristic measurement with desired mineral content of the chemically analysed portion of the sample to determine the characteristic/mineral ratio of the sample. The apparatus includes an ore sample collector, a deflector for deflecting a sample of ore particles from the output of an ore sorter into the collector and means for moving the deflector from a first position in which it is clear of the particle path from the sorter to a second position in which it is in the particle path at predetermined time intervals and for predetermined time periods to deflect the sample particles into the collector. The apparatus conveniently includes an ore crusher for comminuting the sample particle, a sample hopper means for weighing the hopper, a detector in the hopper for measuring a characteristic such as radioactivity, magnetivity or the like of particles in the hopper, a discharge outlet from the hopper and means for feeding the particles from the collector to the crusher and then to the hopper

  9. Fish protein fingerprint in whole muscle samples of yellow perch ...

    African Journals Online (AJOL)

    Many studies have shown the impact of environmental and/or genetic factors on the growth and development of various fish species. However, the role of genes supporting the cellular and molecular mechanisms responsible for fish was to compare whole muscle proteomic profiles of large versus small growth yellow perch ...

  10. Genetic Sample Inventory

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This database archives genetic tissue samples from marine mammals collected primarily from the U.S. east coast. The collection includes samples from field programs,...

  11. Superposition Enhanced Nested Sampling

    Directory of Open Access Journals (Sweden)

    Stefano Martiniani

    2014-08-01

    Full Text Available The theoretical analysis of many problems in physics, astronomy, and applied mathematics requires an efficient numerical exploration of multimodal parameter spaces that exhibit broken ergodicity. Monte Carlo methods are widely used to deal with these classes of problems, but such simulations suffer from a ubiquitous sampling problem: The probability of sampling a particular state is proportional to its entropic weight. Devising an algorithm capable of sampling efficiently the full phase space is a long-standing problem. Here, we report a new hybrid method for the exploration of multimodal parameter spaces exhibiting broken ergodicity. Superposition enhanced nested sampling combines the strengths of global optimization with the unbiased or athermal sampling of nested sampling, greatly enhancing its efficiency with no additional parameters. We report extensive tests of this new approach for atomic clusters that are known to have energy landscapes for which conventional sampling schemes suffer from broken ergodicity. We also introduce a novel parallelization algorithm for nested sampling.

  12. Chorionic villus sampling

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003406.htm Chorionic villus sampling To use the sharing features on this page, please enable JavaScript. Chorionic villus sampling (CVS) is a test some pregnant women have ...

  13. Sampling on Quasicrystals

    OpenAIRE

    Grepstad, Sigrid

    2011-01-01

    We prove that quasicrystals are universal sets of stable sampling in any dimension. Necessary and sufficient density conditions for stable sampling and interpolation sets in one dimension are studied in detail.

  14. Genetic Sample Inventory - NRDA

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This database archives genetic tissue samples from marine mammals collected in the North-Central Gulf of Mexico from 2010-2015. The collection includes samples from...

  15. Test sample handling apparatus

    International Nuclear Information System (INIS)

    1981-01-01

    A test sample handling apparatus using automatic scintillation counting for gamma detection, for use in such fields as radioimmunoassay, is described. The apparatus automatically and continuously counts large numbers of samples rapidly and efficiently by the simultaneous counting of two samples. By means of sequential ordering of non-sequential counting data, it is possible to obtain precisely ordered data while utilizing sample carrier holders having a minimum length. (U.K.)

  16. Laboratory Sampling Guide

    Science.gov (United States)

    2012-05-11

    environment, and by ingestion of foodstuffs that have incorporated C-14 by photosynthesis . Like tritium, C-14 is a very low energy beta emitter and is... bacterial growth and to minimize development of solids in the sample. • Properly identify each sample container with name, SSN, and collection start and...sampling in the same cardboard carton. The sample may be kept cool or frozen during collection to control odor and bacterial growth. • Once

  17. High speed network sampling

    OpenAIRE

    Rindalsholt, Ole Arild

    2005-01-01

    Master i nettverks- og systemadministrasjon Classical Sampling methods play an important role in the current practice of Internet measurement. With today’s high speed networks, routers cannot manage to generate complete Netflow data for every packet. They have to perform restricted sampling. This thesis summarizes some of the most important sampling schemes and their applications before diving into an analysis on the effect of sampling Netflow records.

  18. Mars Sample Handling Functionality

    Science.gov (United States)

    Meyer, M. A.; Mattingly, R. L.

    2018-04-01

    The final leg of a Mars Sample Return campaign would be an entity that we have referred to as Mars Returned Sample Handling (MRSH.) This talk will address our current view of the functional requirements on MRSH, focused on the Sample Receiving Facility (SRF).

  19. IAEA Sampling Plan

    Energy Technology Data Exchange (ETDEWEB)

    Geist, William H. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-09-15

    The objectives for this presentation are to describe the method that the IAEA uses to determine a sampling plan for nuclear material measurements; describe the terms detection probability and significant quantity; list the three nuclear materials measurement types; describe the sampling method applied to an item facility; and describe multiple method sampling.

  20. Developing Water Sampling Standards

    Science.gov (United States)

    Environmental Science and Technology, 1974

    1974-01-01

    Participants in the D-19 symposium on aquatic sampling and measurement for water pollution assessment were informed that determining the extent of waste water stream pollution is not a cut and dry procedure. Topics discussed include field sampling, representative sampling from storm sewers, suggested sampler features and application of improved…

  1. Protein nanoparticles for therapeutic protein delivery.

    Science.gov (United States)

    Herrera Estrada, L P; Champion, J A

    2015-06-01

    Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

  2. Generalized sampling in Julia

    DEFF Research Database (Denmark)

    Jacobsen, Christian Robert Dahl; Nielsen, Morten; Rasmussen, Morten Grud

    2017-01-01

    Generalized sampling is a numerically stable framework for obtaining reconstructions of signals in different bases and frames from their samples. For example, one can use wavelet bases for reconstruction given frequency measurements. In this paper, we will introduce a carefully documented toolbox...... for performing generalized sampling in Julia. Julia is a new language for technical computing with focus on performance, which is ideally suited to handle the large size problems often encountered in generalized sampling. The toolbox provides specialized solutions for the setup of Fourier bases and wavelets....... The performance of the toolbox is compared to existing implementations of generalized sampling in MATLAB....

  3. Physicochemical properties and mineral and protein content of honey samples from Ceará state, Northeastern Brazil Propriedades físico-químicas, minerais e teor de proteínas em amostras de méis do estado do Ceará, nordeste do Brasil

    Directory of Open Access Journals (Sweden)

    Maria da Conceição Tavares Cavalcanti Liberato

    2013-03-01

    Full Text Available This study evaluated the physicochemical properties and protein and mineral content of honey samples from Ceará State, Northeastern Brazil, one of the major honey exporters in the country. Nutritional importance of the minerals detected was also analyzed. Physicochemical properties were examined according to the AOAC and CAC official methods; the protein content was determined using the Bradford method, and the minerals were analyzed by atomic absorption spectrometry. All analyses were performed in triplicate. The levels of macrominerals sodium (Na, potassium (K, calcium (Ca, and magnesium (Mg varied from 1.80-47.20, 21.30-1513.30, 14.58-304.82, and 2.48-28.33 mg/kg, respectively, and the trace elements iron (Fe, copper (Cu, manganese (Mn, zinc (Zn, selenium (Se, and chromium (Cr varied from 0.12-8.76, 0.07-1.29, 0.06-1.96, 0.07-1.85 mg/kg, 0.36 × 10-3-62.00 × 10-3 and 22.50 × 10-3-170.33 × 10-3 µg/kg, respectively. Myracrodruon urundeuva honey sample had high contents of macrominerals (Na, K, Ca, and Mg. Protein content of the Anacardium occidentale honey sample was the highest (1121.00 µg/g among the samples analyzed. Among the minerals detected in the honey samples, K showed the highest concentration, followed by Ca, Na, and Mg. The presence of trace elements can show environmental contamination. The honey samples studied were free of trace elements contamination, except for Mn; the Piptadenia moniliformis was the only honey sample that was in compliance with the law requirements. The variations of the chemical constituents in the honey samples are probably related to differences in the floral origin and mineral and protein contents and confirm the nutritional importance of Ceará State honey.Este trabalho avaliou propriedades físico-químicas, teores de proteína e minerais em méis do Ceará, um dos principais exportadores do País. Também foi analisada a importância nutricional com relação aos minerais detectados. As

  4. Development of aptamers against unpurified proteins.

    Science.gov (United States)

    Goto, Shinichi; Tsukakoshi, Kaori; Ikebukuro, Kazunori

    2017-12-01

    SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples. © 2017 Wiley Periodicals, Inc.

  5. The Lunar Sample Compendium

    Science.gov (United States)

    Meyer, Charles

    2009-01-01

    The Lunar Sample Compendium is a succinct summary of the data obtained from 40 years of study of Apollo and Luna samples of the Moon. Basic petrographic, chemical and age information is compiled, sample-by-sample, in the form of an advanced catalog in order to provide a basic description of each sample. The LSC can be found online using Google. The initial allocation of lunar samples was done sparingly, because it was realized that scientific techniques would improve over the years and new questions would be formulated. The LSC is important because it enables scientists to select samples within the context of the work that has already been done and facilitates better review of proposed allocations. It also provides back up material for public displays, captures information found only in abstracts, grey literature and curatorial databases and serves as a ready access to the now-vast scientific literature.

  6. Image Sampling with Quasicrystals

    Directory of Open Access Journals (Sweden)

    Mark Grundland

    2009-07-01

    Full Text Available We investigate the use of quasicrystals in image sampling. Quasicrystals produce space-filling, non-periodic point sets that are uniformly discrete and relatively dense, thereby ensuring the sample sites are evenly spread out throughout the sampled image. Their self-similar structure can be attractive for creating sampling patterns endowed with a decorative symmetry. We present a brief general overview of the algebraic theory of cut-and-project quasicrystals based on the geometry of the golden ratio. To assess the practical utility of quasicrystal sampling, we evaluate the visual effects of a variety of non-adaptive image sampling strategies on photorealistic image reconstruction and non-photorealistic image rendering used in multiresolution image representations. For computer visualization of point sets used in image sampling, we introduce a mosaic rendering technique.

  7. Urine sample collection protocols for bioassay samples

    Energy Technology Data Exchange (ETDEWEB)

    MacLellan, J.A.; McFadden, K.M.

    1992-11-01

    In vitro radiobioassay analyses are used to measure the amount of radioactive material excreted by personnel exposed to the potential intake of radioactive material. The analytical results are then used with various metabolic models to estimate the amount of radioactive material in the subject`s body and the original intake of radioactive material. Proper application of these metabolic models requires knowledge of the excretion period. It is normal practice to design the bioassay program based on a 24-hour excretion sample. The Hanford bioassay program simulates a total 24-hour urine excretion sample with urine collection periods lasting from one-half hour before retiring to one-half hour after rising on two consecutive days. Urine passed during the specified periods is collected in three 1-L bottles. Because the daily excretion volume given in Publication 23 of the International Commission on Radiological Protection (ICRP 1975, p. 354) for Reference Man is 1.4 L, it was proposed to use only two 1-L bottles as a cost-saving measure. This raised the broader question of what should be the design capacity of a 24-hour urine sample kit.

  8. Urine sample collection protocols for bioassay samples

    Energy Technology Data Exchange (ETDEWEB)

    MacLellan, J.A.; McFadden, K.M.

    1992-11-01

    In vitro radiobioassay analyses are used to measure the amount of radioactive material excreted by personnel exposed to the potential intake of radioactive material. The analytical results are then used with various metabolic models to estimate the amount of radioactive material in the subject's body and the original intake of radioactive material. Proper application of these metabolic models requires knowledge of the excretion period. It is normal practice to design the bioassay program based on a 24-hour excretion sample. The Hanford bioassay program simulates a total 24-hour urine excretion sample with urine collection periods lasting from one-half hour before retiring to one-half hour after rising on two consecutive days. Urine passed during the specified periods is collected in three 1-L bottles. Because the daily excretion volume given in Publication 23 of the International Commission on Radiological Protection (ICRP 1975, p. 354) for Reference Man is 1.4 L, it was proposed to use only two 1-L bottles as a cost-saving measure. This raised the broader question of what should be the design capacity of a 24-hour urine sample kit.

  9. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Science.gov (United States)

    Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

    2015-03-03

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  10. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  11. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  12. Optimization of protein buffer cocktails using Thermofluor.

    Science.gov (United States)

    Reinhard, Linda; Mayerhofer, Hubert; Geerlof, Arie; Mueller-Dieckmann, Jochen; Weiss, Manfred S

    2013-02-01

    The stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. A quick and easy approach to identify a buffer composition which increases the stability and possibly the conformational homogeneity of a protein sample is the fluorescence-based thermal-shift assay (Thermofluor). Here, a novel 96-condition screen for Thermofluor experiments is presented which consists of buffer and additive parts. The buffer screen comprises 23 different buffers and the additive screen includes small-molecule additives such as salts and nucleotide analogues. The utilization of small-molecule components which increase the thermal stability of a protein sample frequently results in a protein preparation of higher quality and quantity and ultimately also increases the chances of the protein crystallizing.

  13. Multielemental analysis of milk samples

    International Nuclear Information System (INIS)

    Omar Al-Dayel; Jameel Al-Hefne; Didarul A Chowdhury; Turki Al-Ajyan

    2002-01-01

    Milk is a basic food since it provides essential nutrients (proteins, lipids, carbohydrates) and micronutrients (minerals, Vitamins, enzymes). In fact, in formula milk essential elements have been usually added in order to satisfy nutritional requirements. However, too high additions of these elements can produce detrimental effects on human health. More important, milk can also constitute a source of exposure to toxic elements, especially dangerous for infants. Method is presented for the multielemental analysis of a wide range of elements in milk samples. The aim of this work is the development of a multielemental method for the analysis of major, minor and trace essential and toxic elements in milk. Several milk samples with different origins were collected from the Saudi Arabia markets and analyzed by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). For preparation of the samples for analysis, they were digested by closed vessel microwave digestion system with H 2 O 2 /HNO 3 . About 40 elements were determined. A reference material was analysed for the validation of the proposed method. (Author)

  14. Sample size methodology

    CERN Document Server

    Desu, M M

    2012-01-01

    One of the most important problems in designing an experiment or a survey is sample size determination and this book presents the currently available methodology. It includes both random sampling from standard probability distributions and from finite populations. Also discussed is sample size determination for estimating parameters in a Bayesian setting by considering the posterior distribution of the parameter and specifying the necessary requirements. The determination of the sample size is considered for ranking and selection problems as well as for the design of clinical trials. Appropria

  15. Statistical sampling strategies

    International Nuclear Information System (INIS)

    Andres, T.H.

    1987-01-01

    Systems assessment codes use mathematical models to simulate natural and engineered systems. Probabilistic systems assessment codes carry out multiple simulations to reveal the uncertainty in values of output variables due to uncertainty in the values of the model parameters. In this paper, methods are described for sampling sets of parameter values to be used in a probabilistic systems assessment code. Three Monte Carlo parameter selection methods are discussed: simple random sampling, Latin hypercube sampling, and sampling using two-level orthogonal arrays. Three post-selection transformations are also described: truncation, importance transformation, and discretization. Advantages and disadvantages of each method are summarized

  16. Statistical distribution sampling

    Science.gov (United States)

    Johnson, E. S.

    1975-01-01

    Determining the distribution of statistics by sampling was investigated. Characteristic functions, the quadratic regression problem, and the differential equations for the characteristic functions are analyzed.

  17. Big Data, Small Sample.

    Science.gov (United States)

    Gerlovina, Inna; van der Laan, Mark J; Hubbard, Alan

    2017-05-20

    Multiple comparisons and small sample size, common characteristics of many types of "Big Data" including those that are produced by genomic studies, present specific challenges that affect reliability of inference. Use of multiple testing procedures necessitates calculation of very small tail probabilities of a test statistic distribution. Results based on large deviation theory provide a formal condition that is necessary to guarantee error rate control given practical sample sizes, linking the number of tests and the sample size; this condition, however, is rarely satisfied. Using methods that are based on Edgeworth expansions (relying especially on the work of Peter Hall), we explore the impact of departures of sampling distributions from typical assumptions on actual error rates. Our investigation illustrates how far the actual error rates can be from the declared nominal levels, suggesting potentially wide-spread problems with error rate control, specifically excessive false positives. This is an important factor that contributes to "reproducibility crisis". We also review some other commonly used methods (such as permutation and methods based on finite sampling inequalities) in their application to multiple testing/small sample data. We point out that Edgeworth expansions, providing higher order approximations to the sampling distribution, offer a promising direction for data analysis that could improve reliability of studies relying on large numbers of comparisons with modest sample sizes.

  18. Sampling system and method

    Science.gov (United States)

    Decker, David L.; Lyles, Brad F.; Purcell, Richard G.; Hershey, Ronald Lee

    2013-04-16

    The present disclosure provides an apparatus and method for coupling conduit segments together. A first pump obtains a sample and transmits it through a first conduit to a reservoir accessible by a second pump. The second pump further conducts the sample from the reservoir through a second conduit.

  19. Simple street tree sampling

    Science.gov (United States)

    David J. Nowak; Jeffrey T. Walton; James Baldwin; Jerry. Bond

    2015-01-01

    Information on street trees is critical for management of this important resource. Sampling of street tree populations provides an efficient means to obtain street tree population information. Long-term repeat measures of street tree samples supply additional information on street tree changes and can be used to report damages from catastrophic events. Analyses of...

  20. Sampling or gambling

    Energy Technology Data Exchange (ETDEWEB)

    Gy, P.M.

    1981-12-01

    Sampling can be compared to no other technique. A mechanical sampler must above all be selected according to its aptitude for supressing or reducing all components of the sampling error. Sampling is said to be correct when it gives all elements making up the batch of matter submitted to sampling an uniform probability of being selected. A sampler must be correctly designed, built, installed, operated and maintained. When the conditions of sampling correctness are not strictly respected, the sampling error can no longer be controlled and can, unknown to the user, be unacceptably large: the sample is no longer representative. The implementation of an incorrect sampler is a form of gambling and this paper intends to show that at this game the user is nearly always the loser in the long run. The users' and the manufacturers' interests may diverge and the standards which should safeguard the users' interests very often fail to do so by tolerating or even recommending incorrect techniques such as the implementation of too narrow cutters traveling too fast through the stream to be sampled.

  1. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  2. LC-MS/MS determination of 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279) in dog plasma with high-throughput protein precipitation sample preparation.

    Science.gov (United States)

    Kim, Joseph; Flick, Jeanette; Reimer, Michael T; Rodila, Ramona; Wang, Perry G; Zhang, Jun; Ji, Qin C; El-Shourbagy, Tawakol A

    2007-11-01

    As an effective DPP-IV inhibitor, 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279), is an investigational drug candidate under development at Abbott Laboratories for potential treatment of type 2 diabetes. In order to support the development of ABT-279, multiple analytical methods for an accurate, precise and selective concentration determination of ABT-279 in different matrices were developed and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. The analytical method for ABT-279 in dog plasma was validated in parallel to other validations for ABT-279 determination in different matrices. In order to shorten the sample preparation time and increase method precision, an automated multi-channel liquid handler was used to perform high-throughput protein precipitation and all other liquid transfers. The separation was performed through a Waters YMC ODS-AQ column (2.0 x 150 mm, 5 microm, 120 A) with a mobile phase of 20 mm ammonium acetate in 20% acetonitrile at a flow rate of 0.3 mL/min. Data collection started at 2.2 min and continued for 2.0 min. The validated linear dynamic range in dog plasma was between 3.05 and 2033.64 ng/mL using a 50 microL sample volume. The achieved r(2) coefficient of determination from three consecutive runs was between 0.998625 and 0.999085. The mean bias was between -4.1 and 4.3% for all calibration standards including lower limit of quantitation. The mean bias was between -8.0 and 0.4% for the quality control samples. The precision, expressed as a coefficient of variation (CV), was < or =4.1% for all levels of quality control samples. The validation results demonstrated that the high-throughput method was accurate, precise and selective for the determination of ABT-279 in dog plasma. The validated method was also employed to support two toxicology studies. The passing rate was 100% for all 49 runs from

  3. Sample pretretment in microsystems

    DEFF Research Database (Denmark)

    Perch-Nielsen, Ivan R.

    2003-01-01

    : Sample preparation → DNA amplification → DNA analysis. The overall goal of the project is integration of as many as possible of these steps. This thesis covers mainly pretreatment in a microchip. Some methods for sample pretreatment have been tested. Most conventional is fluorescence activated cell sort......When a sample, e.g. from a patient, is processed using conventional methods, the sample must be transported to the laboratory where it is analyzed, after which the results is sent back. By integrating the separate steps of the analysis in a micro total analysis system (μTAS), results can...... be obtained fast and better. Preferably with all the processes from sample to signal moved to the bedside of the patient. Of course there is still much to learn and study in the process of miniaturization. DNA analysis is one process subject to integration. There are roughly three steps in a DNA analysis...

  4. Biological sample collector

    Science.gov (United States)

    Murphy, Gloria A [French Camp, CA

    2010-09-07

    A biological sample collector is adapted to a collect several biological samples in a plurality of filter wells. A biological sample collector may comprise a manifold plate for mounting a filter plate thereon, the filter plate having a plurality of filter wells therein; a hollow slider for engaging and positioning a tube that slides therethrough; and a slide case within which the hollow slider travels to allow the tube to be aligned with a selected filter well of the plurality of filter wells, wherein when the tube is aligned with the selected filter well, the tube is pushed through the hollow slider and into the selected filter well to sealingly engage the selected filter well and to allow the tube to deposit a biological sample onto a filter in the bottom of the selected filter well. The biological sample collector may be portable.

  5. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  6. Building biochips: a protein production pipeline

    Science.gov (United States)

    de Carvalho-Kavanagh, Marianne G. S.; Albala, Joanna S.

    2004-06-01

    Protein arrays are emerging as a practical format in which to study proteins in high-throughput using many of the same techniques as that of the DNA microarray. The key advantage to array-based methods for protein study is the potential for parallel analysis of thousands of samples in an automated, high-throughput fashion. Building protein arrays capable of this analysis capacity requires a robust expression and purification system capable of generating hundreds to thousands of purified recombinant proteins. We have developed a method to utilize LLNL-I.M.A.G.E. cDNAs to generate recombinant protein libraries using a baculovirus-insect cell expression system. We have used this strategy to produce proteins for analysis of protein/DNA and protein/protein interactions using protein microarrays in order to understand the complex interactions of proteins involved in homologous recombination and DNA repair. Using protein array techniques, a novel interaction between the DNA repair protein, Rad51B, and histones has been identified.

  7. PFP Wastewater Sampling Facility

    International Nuclear Information System (INIS)

    Hirzel, D.R.

    1995-01-01

    This test report documents the results obtained while conducting operational testing of the sampling equipment in the 225-WC building, the PFP Wastewater Sampling Facility. The Wastewater Sampling Facility houses equipment to sample and monitor the PFP's liquid effluents before discharging the stream to the 200 Area Treated Effluent Disposal Facility (TEDF). The majority of the streams are not radioactive and discharges from the PFP Heating, Ventilation, and Air Conditioning (HVAC). The streams that might be contaminated are processed through the Low Level Waste Treatment Facility (LLWTF) before discharging to TEDF. The sampling equipment consists of two flow-proportional composite samplers, an ultrasonic flowmeter, pH and conductivity monitors, chart recorder, and associated relays and current isolators to interconnect the equipment to allow proper operation. Data signals from the monitors are received in the 234-5Z Shift Office which contains a chart recorder and alarm annunciator panel. The data signals are also duplicated and sent to the TEDF control room through the Local Control Unit (LCU). Performing the OTP has verified the operability of the PFP wastewater sampling system. This Operability Test Report documents the acceptance of the sampling system for use

  8. Contributions to sampling statistics

    CERN Document Server

    Conti, Pier; Ranalli, Maria

    2014-01-01

    This book contains a selection of the papers presented at the ITACOSM 2013 Conference, held in Milan in June 2013. ITACOSM is the bi-annual meeting of the Survey Sampling Group S2G of the Italian Statistical Society, intended as an international  forum of scientific discussion on the developments of theory and application of survey sampling methodologies and applications in human and natural sciences. The book gathers research papers carefully selected from both invited and contributed sessions of the conference. The whole book appears to be a relevant contribution to various key aspects of sampling methodology and techniques; it deals with some hot topics in sampling theory, such as calibration, quantile-regression and multiple frame surveys, and with innovative methodologies in important topics of both sampling theory and applications. Contributions cut across current sampling methodologies such as interval estimation for complex samples, randomized responses, bootstrap, weighting, modeling, imputati...

  9. Waste classification sampling plan

    International Nuclear Information System (INIS)

    Landsman, S.D.

    1998-01-01

    The purpose of this sampling is to explain the method used to collect and analyze data necessary to verify and/or determine the radionuclide content of the B-Cell decontamination and decommissioning waste stream so that the correct waste classification for the waste stream can be made, and to collect samples for studies of decontamination methods that could be used to remove fixed contamination present on the waste. The scope of this plan is to establish the technical basis for collecting samples and compiling quantitative data on the radioactive constituents present in waste generated during deactivation activities in B-Cell. Sampling and radioisotopic analysis will be performed on the fixed layers of contamination present on structural material and internal surfaces of process piping and tanks. In addition, dose rate measurements on existing waste material will be performed to determine the fraction of dose rate attributable to both removable and fixed contamination. Samples will also be collected to support studies of decontamination methods that are effective in removing the fixed contamination present on the waste. Sampling performed under this plan will meet criteria established in BNF-2596, Data Quality Objectives for the B-Cell Waste Stream Classification Sampling, J. M. Barnett, May 1998

  10. Sample Return Robot

    Data.gov (United States)

    National Aeronautics and Space Administration — This Challenge requires demonstration of an autonomous robotic system to locate and collect a set of specific sample types from a large planetary analog area and...

  11. Ecotoxicology statistical sampling

    International Nuclear Information System (INIS)

    Saona, G.

    2012-01-01

    This presentation introduces to general concepts in toxicology sample designs such as the distribution of organic or inorganic contaminants, a microbiological contamination, and the determination of the position in an eco toxicological bioassays ecosystem.

  12. Mini MAX - Medicaid Sample

    Data.gov (United States)

    U.S. Department of Health & Human Services — To facilitate wider use of MAX, CMS contracted with Mathematica to convene a technical expert panel (TEP) and determine the feasibility of creating a sample file for...

  13. Operational air sampling report

    International Nuclear Information System (INIS)

    Lyons, C.L.

    1994-03-01

    Nevada Test Site vertical shaft and tunnel events generate beta/gamma fission products. The REECo air sampling program is designed to measure these radionuclides at various facilities supporting these events. The current testing moratorium and closure of the Decontamination Facility has decreased the scope of the program significantly. Of the 118 air samples collected in the only active tunnel complex, only one showed any airborne fission products. Tritiated water vapor concentrations were very similar to previously reported levels. The 206 air samples collected at the Area-6 decontamination bays and laundry were again well below any Derived Air Concentration calculation standard. Laboratory analyses of these samples were negative for any airborne fission products

  14. Collecting Samples for Testing

    Science.gov (United States)

    ... Creatinine Ratio Valproic Acid Vancomycin Vanillylmandelic Acid (VMA) VAP Vitamin A Vitamin B12 and Folate Vitamin D ... that used for CSF in that they require aspiration of a sample of the fluid through a ...

  15. Roadway sampling evaluation.

    Science.gov (United States)

    2014-09-01

    The Florida Department of Transportation (FDOT) has traditionally required that all sampling : and testing of asphalt mixtures be at the Contractors production facility. With recent staffing cuts, as : well as budget reductions, FDOT has been cons...

  16. Soil Gas Sampling

    Science.gov (United States)

    Field Branches Quality System and Technical Procedures: This document describes general and specific procedures, methods and considerations to be used and observed when collecting soil gas samples for field screening or laboratory analysis.

  17. Soil Sampling Operating Procedure

    Science.gov (United States)

    EPA Region 4 Science and Ecosystem Support Division (SESD) document that describes general and specific procedures, methods, and considerations when collecting soil samples for field screening or laboratory analysis.

  18. Statistical sampling plans

    International Nuclear Information System (INIS)

    Jaech, J.L.

    1984-01-01

    In auditing and in inspection, one selects a number of items by some set of procedures and performs measurements which are compared with the operator's values. This session considers the problem of how to select the samples to be measured, and what kinds of measurements to make. In the inspection situation, the ultimate aim is to independently verify the operator's material balance. The effectiveness of the sample plan in achieving this objective is briefly considered. The discussion focuses on the model plant

  19. Two phase sampling

    CERN Document Server

    Ahmad, Zahoor; Hanif, Muhammad

    2013-01-01

    The development of estimators of population parameters based on two-phase sampling schemes has seen a dramatic increase in the past decade. Various authors have developed estimators of population using either one or two auxiliary variables. The present volume is a comprehensive collection of estimators available in single and two phase sampling. The book covers estimators which utilize information on single, two and multiple auxiliary variables of both quantitative and qualitative nature. Th...

  20. Novel Protein Microarray Technology to Examine Men with Prostate Cancer

    National Research Council Canada - National Science Library

    Lilja, Hans

    2005-01-01

    The authors developed a novel macro and nanoporous silicon surface for protein microarrays to facilitate high-throughput biomarker discovery, and high-density protein-chip array analyses of complex biological samples...

  1. Uranium tailings sampling manual

    International Nuclear Information System (INIS)

    Feenstra, S.; Reades, D.W.; Cherry, J.A.; Chambers, D.B.; Case, G.G.; Ibbotson, B.G.

    1985-01-01

    The purpose of this manual is to describe the requisite sampling procedures for the application of uniform high-quality standards to detailed geotechnical, hydrogeological, geochemical and air quality measurements at Canadian uranium tailings disposal sites. The selection and implementation of applicable sampling procedures for such measurements at uranium tailings disposal sites are complicated by two primary factors. Firstly, the physical and chemical nature of uranium mine tailings and effluent is considerably different from natural soil materials and natural waters. Consequently, many conventional methods for the collection and analysis of natural soils and waters are not directly applicable to tailings. Secondly, there is a wide range in the physical and chemical nature of uranium tailings. The composition of the ore, the milling process, the nature of tailings depositon, and effluent treatment vary considerably and are highly site-specific. Therefore, the definition and implementation of sampling programs for uranium tailings disposal sites require considerable evaluation, and often innovation, to ensure that appropriate sampling and analysis methods are used which provide the flexibility to take into account site-specific considerations. The following chapters describe the objective and scope of a sampling program, preliminary data collection, and the procedures for sampling of tailings solids, surface water and seepage, tailings pore-water, and wind-blown dust and radon

  2. Reactor water sampling device

    International Nuclear Information System (INIS)

    Sakamaki, Kazuo.

    1992-01-01

    The present invention concerns a reactor water sampling device for sampling reactor water in an in-core monitor (neutron measuring tube) housing in a BWR type reactor. The upper end portion of a drain pipe of the reactor water sampling device is attached detachably to an in-core monitor flange. A push-up rod is inserted in the drain pipe vertically movably. A sampling vessel and a vacuum pump are connected to the lower end of the drain pipe. A vacuum pump is operated to depressurize the inside of the device and move the push-up rod upwardly. Reactor water in the in-core monitor housing flows between the drain pipe and the push-up rod and flows into the sampling vessel. With such a constitution, reactor water in the in-core monitor housing can be sampled rapidly with neither opening the lid of the reactor pressure vessel nor being in contact with air. Accordingly, operator's exposure dose can be reduced. (I.N.)

  3. Wet gas sampling

    Energy Technology Data Exchange (ETDEWEB)

    Welker, T.F.

    1997-07-01

    The quality of gas has changed drastically in the past few years. Most gas is wet with hydrocarbons, water, and heavier contaminants that tend to condense if not handled properly. If a gas stream is contaminated with condensables, the sampling of that stream must be done in a manner that will ensure all of the components in the stream are introduced into the sample container as the composite. The sampling and handling of wet gas is extremely difficult under ideal conditions. There are no ideal conditions in the real world. The problems related to offshore operations and other wet gas systems, as well as the transportation of the sample, are additional problems that must be overcome if the analysis is to mean anything to the producer and gatherer. The sampling of wet gas systems is decidedly more difficult than sampling conventional dry gas systems. Wet gas systems were generally going to result in the measurement of one heating value at the inlet of the pipe and a drastic reduction in the heating value of the gas at the outlet end of the system. This is caused by the fallout or accumulation of the heavier products that, at the inlet, may be in the vapor state in the pipeline; hence, the high gravity and high BTU. But, in fact, because of pressure and temperature variances, these liquids condense and form a liquid that is actually running down the pipe as a stream or is accumulated in drips to be blown from the system. (author)

  4. Lunar Sample Compendium

    Science.gov (United States)

    Meyer, Charles

    2005-01-01

    The purpose of the Lunar Sample Compendium will be to inform scientists, astronauts and the public about the various lunar samples that have been returned from the Moon. This Compendium will be organized rock by rock in the manor of a catalog, but will not be as comprehensive, nor as complete, as the various lunar sample catalogs that are available. Likewise, this Compendium will not duplicate the various excellent books and reviews on the subject of lunar samples (Cadogen 1981, Heiken et al. 1991, Papike et al. 1998, Warren 2003, Eugster 2003). However, it is thought that an online Compendium, such as this, will prove useful to scientists proposing to study individual lunar samples and should help provide backup information for lunar sample displays. This Compendium will allow easy access to the scientific literature by briefly summarizing the significant findings of each rock along with the documentation of where the detailed scientific data are to be found. In general, discussion and interpretation of the results is left to the formal reviews found in the scientific literature. An advantage of this Compendium will be that it can be updated, expanded and corrected as need be.

  5. Tissue Sampling Guides for Porcine Biomedical Models.

    Science.gov (United States)

    Albl, Barbara; Haesner, Serena; Braun-Reichhart, Christina; Streckel, Elisabeth; Renner, Simone; Seeliger, Frank; Wolf, Eckhard; Wanke, Rüdiger; Blutke, Andreas

    2016-04-01

    This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from ∼50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology; immunohistochemistry;in situhybridization; electron microscopy; and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results. © The Author(s) 2016.

  6. Protein Detection with Aptamer Biosensors

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    2008-07-01

    Full Text Available Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers.

  7. Nonuniform sampling by quantiles

    Science.gov (United States)

    Craft, D. Levi; Sonstrom, Reilly E.; Rovnyak, Virginia G.; Rovnyak, David

    2018-03-01

    A flexible strategy for choosing samples nonuniformly from a Nyquist grid using the concept of statistical quantiles is presented for broad classes of NMR experimentation. Quantile-directed scheduling is intuitive and flexible for any weighting function, promotes reproducibility and seed independence, and is generalizable to multiple dimensions. In brief, weighting functions are divided into regions of equal probability, which define the samples to be acquired. Quantile scheduling therefore achieves close adherence to a probability distribution function, thereby minimizing gaps for any given degree of subsampling of the Nyquist grid. A characteristic of quantile scheduling is that one-dimensional, weighted NUS schedules are deterministic, however higher dimensional schedules are similar within a user-specified jittering parameter. To develop unweighted sampling, we investigated the minimum jitter needed to disrupt subharmonic tracts, and show that this criterion can be met in many cases by jittering within 25-50% of the subharmonic gap. For nD-NUS, three supplemental components to choosing samples by quantiles are proposed in this work: (i) forcing the corner samples to ensure sampling to specified maximum values in indirect evolution times, (ii) providing an option to triangular backfill sampling schedules to promote dense/uniform tracts at the beginning of signal evolution periods, and (iii) providing an option to force the edges of nD-NUS schedules to be identical to the 1D quantiles. Quantile-directed scheduling meets the diverse needs of current NUS experimentation, but can also be used for future NUS implementations such as off-grid NUS and more. A computer program implementing these principles (a.k.a. QSched) in 1D- and 2D-NUS is available under the general public license.

  8. 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...

  9. AND/OR Importance Sampling

    OpenAIRE

    Gogate, Vibhav; Dechter, Rina

    2012-01-01

    The paper introduces AND/OR importance sampling for probabilistic graphical models. In contrast to importance sampling, AND/OR importance sampling caches samples in the AND/OR space and then extracts a new sample mean from the stored samples. We prove that AND/OR importance sampling may have lower variance than importance sampling; thereby providing a theoretical justification for preferring it over importance sampling. Our empirical evaluation demonstrates that AND/OR importance sampling is ...

  10. Neutron protein crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Niimura, Nobuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-10-01

    X-ray diffraction of single crystal has enriched the knowledge of various biological molecules such as proteins, DNA, t-RNA, viruses, etc. It is difficult to make structural analysis of hydrogen atoms in a protein using X-ray crystallography, whereas neutron diffraction seems usable to directly determine the location of those hydrogen atoms. Here, neutron diffraction method was applied to structural analysis of hen egg-white lysozyme. Since the crystal size of a protein to analyze is generally small (5 mm{sup 3} at most), the neutron beam at the sample position in monochromator system was set to less than 5 x 5 mm{sup 2} and beam divergence to 0.4 degree or less. Neutron imaging plate with {sup 6}Li or Gd mixed with photostimulated luminescence material was used and about 2500 Bragg reflections were recorded in one crystal setting. A total of 38278 reflections for 2.0 A resolution were collected in less than 10 days. Thus, stereo views of Trp-111 omit map around the indol ring of Trp-111 was presented and the three-dimensional arrangement of 696H and 264D atoms in the lysozyme molecules was determined using the omit map. (M.N.)

  11. Sample collection and documentation

    International Nuclear Information System (INIS)

    Cullings, Harry M.; Fujita, Shoichiro; Watanabe, Tadaaki; Yamashita, Tomoaki; Tanaka, Kenichi; Endo, Satoru; Shizuma, Kiyoshi; Hoshi, Masaharu; Hasai, Hiromi

    2005-01-01

    Beginning within a few weeks after the bombings and periodically during the intervening decades, investigators in Hiroshima and Nagasaki have collected samples of materials that were in the cities at the time of the bombings. Although some early efforts were not driven by specific measurement objectives, many others were. Even some of the very earliest samples collected in 1945 were based on carefully conceived research plans and detailed specifications for samples appropriate to particular retrospective measurements, i.e., of particular residual quantities remaining from exposure to the neutrons and gamma rays from the bombs. This chapter focuses mainly on the work of groups at two institutions that have actively collaborated since the 1980s in major collection efforts and have shared samples among themselves and with other investigators: the Radiation Effects Research Foundation (RERF) and its predecessor the Atomic Bomb Casualty Commission (ABCC), and Hiroshima University. In addition, a number of others are listed, who also contributed to the literature by their collection of samples. (J.P.N.)

  12. Current strategies for protein production and purification enabling membrane protein structural biology.

    Science.gov (United States)

    Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

    2016-12-01

    Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

  13. Linearization of the Bradford Protein Assay

    OpenAIRE

    Ernst, Orna; Zor, Tsaffrir

    2010-01-01

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

  14. Effects of Whey, Caseinate, or Milk Protein Ingestion on Muscle Protein Synthesis after Exercise.

    Science.gov (United States)

    Kanda, Atsushi; Nakayama, Kyosuke; Sanbongi, Chiaki; Nagata, Masashi; Ikegami, Shuji; Itoh, Hiroyuki

    2016-06-03

    Whey protein (WP) is characterized as a "fast" protein and caseinate (CA) as a "slow" protein according to their digestion and absorption rates. We hypothesized that co-ingestion of milk proteins (WP and CA) may be effective for prolonging the muscle protein synthesis response compared to either protein alone. We therefore compared the effect of ingesting milk protein (MP) to either WP or CA alone on muscle protein synthesis after exercise in rats. We also compared the effects of these milk-derived proteins to a control, soy protein (SP). Male Sprague-Dawley rats swam for two hours. Immediately after exercise, one of the following four solutions was administered: WP, CA, MP, or SP. Individual rats were euthanized at designated postprandial time points and triceps muscle samples collected for measurement of the protein fractional synthesis rate (FSR). FSR tended to increase in all groups post-ingestion, although the initial peaks of FSR occurred at different times (WP, peak time = 60 min, FSR = 7.76%/day; MP, peak time = 90 min, FSR = 8.34%/day; CA, peak time = 120 min, FSR = 7.85%/day). Milk-derived proteins caused significantly greater increases (p protein synthesis to occur at different times (WP, fast; MP, intermediate; CA, slow) and the dairy proteins have a superior effect on muscle protein synthesis after exercise compared with SP.

  15. Groundwater sampling: Chapter 5

    Science.gov (United States)

    Wang, Qingren; Munoz-Carpena, Rafael; Foster, Adam; Migliaccio, Kati W.; Li, Yuncong; Migliaccio, Kati

    2011-01-01

    About the book: As water quality becomes a leading concern for people and ecosystems worldwide, it must be properly assessed in order to protect water resources for current and future generations. Water Quality Concepts, Sampling, and Analyses supplies practical information for planning, conducting, or evaluating water quality monitoring programs. It presents the latest information and methodologies for water quality policy, regulation, monitoring, field measurement, laboratory analysis, and data analysis. The book addresses water quality issues, water quality regulatory development, monitoring and sampling techniques, best management practices, and laboratory methods related to the water quality of surface and ground waters. It also discusses basic concepts of water chemistry and hydrology related to water sampling and analysis; instrumentation; water quality data analysis; and evaluation and reporting results.

  16. INEL Sample Management Office

    International Nuclear Information System (INIS)

    Watkins, C.

    1994-01-01

    The Idaho National Engineering Laboratory (INEL) Sample Management Office (SMO) was formed as part of the EG ampersand G Idaho Environmental Restoration Program (ERP) in June, 1990. Since then, the SMO has been recognized and sought out by other prime contractors and programs at the INEL. Since December 1991, the DOE-ID Division Directors for the Environmental Restoration Division and Waste Management Division supported the expansion of the INEL ERP SMO into the INEL site wide SMO. The INEL SMO serves as a point of contact for multiple environmental analytical chemistry and laboratory issues (e.g., capacity, capability). The SMO chemists work with project managers during planning to help develop data quality objectives, select appropriate analytical methods, identify special analytical services needs, identify a source for the services, and ensure that requirements for sampling and analysis (e.g., preservations, sample volumes) are clear and technically accurate. The SMO chemists also prepare work scope statements for the laboratories performing the analyses

  17. Independent random sampling methods

    CERN Document Server

    Martino, Luca; Míguez, Joaquín

    2018-01-01

    This book systematically addresses the design and analysis of efficient techniques for independent random sampling. Both general-purpose approaches, which can be used to generate samples from arbitrary probability distributions, and tailored techniques, designed to efficiently address common real-world practical problems, are introduced and discussed in detail. In turn, the monograph presents fundamental results and methodologies in the field, elaborating and developing them into the latest techniques. The theory and methods are illustrated with a varied collection of examples, which are discussed in detail in the text and supplemented with ready-to-run computer code. The main problem addressed in the book is how to generate independent random samples from an arbitrary probability distribution with the weakest possible constraints or assumptions in a form suitable for practical implementation. The authors review the fundamental results and methods in the field, address the latest methods, and emphasize the li...

  18. Radioactive air sampling methods

    CERN Document Server

    Maiello, Mark L

    2010-01-01

    Although the field of radioactive air sampling has matured and evolved over decades, it has lacked a single resource that assimilates technical and background information on its many facets. Edited by experts and with contributions from top practitioners and researchers, Radioactive Air Sampling Methods provides authoritative guidance on measuring airborne radioactivity from industrial, research, and nuclear power operations, as well as naturally occuring radioactivity in the environment. Designed for industrial hygienists, air quality experts, and heath physicists, the book delves into the applied research advancing and transforming practice with improvements to measurement equipment, human dose modeling of inhaled radioactivity, and radiation safety regulations. To present a wide picture of the field, it covers the international and national standards that guide the quality of air sampling measurements and equipment. It discusses emergency response issues, including radioactive fallout and the assets used ...

  19. Interactive Sample Book (ISB)

    DEFF Research Database (Denmark)

    Heimdal, Elisabeth Jacobsen; Lenau, Torben Anker; Guglielmi, Michel

    2009-01-01

    supervisor Torben A. Lenau. Inspiration to use smart materials Interactive textiles are still quite an unknown phenomenon to many. It is thus often difficult to communicate what kind of potentials lie within these materials. This is why the ISB project was started, as a practice based research project...... and senses in relation to integrated decoration and function primarily to indoor applications. The result of the project will be a number of interactive textiles, to be gathered in an interactive sample book (ISB), in a similar way as the sample books of wallpapers one can take home from the shop and choose...... from. In other words, it is a kind of display material, which in a simple manner can illustrate how different techniques and smart materials work. The sample book should display a number of possibilities where sensor technology, smart materials and textiles are mixed to such an extent that the textile...

  20. Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation

    DEFF Research Database (Denmark)

    Malmström, Johan; Larsen, Kristoffer; Hansson, Lennart

    2002-01-01

    -dimensional electrophoresis was interfaced to miniaturized sample preparation techniques using microcapillary extraction. Four protein groups were identified; cytoskeletal, adhesion, scavenger and metabolic proteins. These patient's proteomes showed a high degree of heterogeneity between patients but larger homogeneity...

  1. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

    We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

  2. The C-terminal tetrapeptide HWFW of the Chlorella HUP1 hexose/H+-symporter is essential for full activity and an alpha-helical structure of the C-terminus

    Czech Academy of Sciences Publication Activity Database

    Graál, R.; Robl, I.; Opekarová, Miroslava; Tanner, W.

    2000-01-01

    Roč. 468, - (2000), s. 225-230 ISSN 0014-5793 R&D Projects: GA ČR GA204/99/0492 Grant - others:Deutsche Forschungsgemeinschaft(DE) SFB521; NATO Collaborative Research Grant(NQ) LST.CLG974804 Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.440, year: 2000

  3. The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens [version 2; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2015-06-01

    Full Text Available The therapeutic potential of α-helical anti-microbial peptides (AH-AMP to combat pathogens is fast gaining prominence. Based on recently published open access software for characterizing α-helical peptides (PAGAL, we elucidate a search methodology (SCALPEL that leverages the massive structural data pre-existing in the PDB database to obtain AH-AMPs belonging to the host proteome. We provide in vitro validation of SCALPEL on plant pathogens (Xylella fastidiosa, Xanthomonas arboricola and Liberibacter crescens by identifying AH-AMPs that mirror the function and properties of cecropin B, a well-studied AH-AMP. The identified peptides include a linear AH-AMP present within the existing structure of phosphoenolpyruvate carboxylase (PPC20, and an AH-AMP mimicing the properties of the two α-helices of cecropin B from chitinase (CHITI25. The minimum inhibitory concentration of these peptides are comparable to that of cecropin B, while anionic peptides used as control failed to show any inhibitory effect on these pathogens. Substitute therapies in place of conventional chemotherapies using membrane permeabilizing peptides like these might also prove effective to target cancer cells. The use of native structures from the same organism could possibly ensure that administration of such peptides will be better tolerated and not elicit an adverse immune response. We suggest a similar approach to target Ebola epitopes, enumerated using PAGAL recently, by selecting suitable peptides from the human proteome, especially in wake of recent reports of cationic amphiphiles inhibiting virus entry and infection.

  4. Analysis of monazite samples

    International Nuclear Information System (INIS)

    Kartiwa Sumadi; Yayah Rohayati

    1996-01-01

    The 'monazit' analytical program has been set up for routine work of Rare Earth Elements analysis in the monazite and xenotime minerals samples. Total relative error of the analysis is very low, less than 2.50%, and the reproducibility of counting statistic and stability of the instrument were very excellent. The precision and accuracy of the analytical program are very good with the maximum percentage relative are 5.22% and 1.61%, respectively. The mineral compositions of the 30 monazite samples have been also calculated using their chemical constituents, and the results were compared to the grain counting microscopic analysis

  5. RAPID PROCESSING OF ARCHIVAL TISSUE SAMPLES FOR PROTEOMIC ANALYSIS USING PRESSURE-CYCLING TECHNOLOGY

    Directory of Open Access Journals (Sweden)

    Vinuth N. Puttamallesh1,2

    2017-06-01

    Full Text Available Advent of mass spectrometry based proteomics has revolutionized our ability to study proteins from biological specimen in a high-throughput manner. Unlike cell line based studies, biomedical research involving tissue specimen is often challenging due to limited sample availability. In addition, investigation of clinically relevant research questions often requires enormous amount of time for sample collection prospectively. Formalin fixed paraffin embedded (FFPE archived tissue samples are a rich source of tissue specimen for biomedical research. However, there are several challenges associated with analysing FFPE samples. Protein cross-linking and degradation of proteins particularly affects proteomic analysis. We demonstrate that barocycler that uses pressure-cycling technology enables efficient protein extraction and processing of small amounts of FFPE tissue samples for proteomic analysis. We identified 3,525 proteins from six 10µm esophageal squamous cell carcinoma (ESCC tissue sections. Barocycler allows efficient protein extraction and proteolytic digestion of proteins from FFPE tissue sections at par with conventional methods.

  6. Parathyroid hormone-related protein blood test

    Science.gov (United States)

    ... ency/article/003691.htm Parathyroid hormone-related protein blood test To use the sharing features on this page, ... measures the level of a hormone in the blood, called parathyroid hormone-related protein. How the Test is Performed A blood sample is needed . How ...

  7. Determination of soluble protein contents from RVNRL

    International Nuclear Information System (INIS)

    Wan Manshol Wan Zin; Nurulhuda Othman

    1996-01-01

    This project was carried out to determine the soluble protein contents on RVNRL film vulcanisates, with respect to the RVNRL storage time, gamma irradiation dose absorbed by the latex and the effect of different leaching time and leaching conditions. These three factors are important in the hope to determine the best possible mean of minimizing the soluble protein contents in products made from RVNRL. Within the nine months storage period employed in the study, the results show that, the longer the storage period the less the soluble protein extracted from the film samples. Gamma irradiation dose absorbed by the samples, between 5.3 kGy to 25.2 kGy seems to influence the soluble protein contents of the RVNRL films vulcanisates. The higher the dose the more was the soluble protein extracted from the film samples. At an absorbed dose of 5.3 kGy and 25.2 kGy, the soluble contents were 0. 198 mg/ml and 0.247 mg/ml respectively. At a fixed leaching temperature, the soluble proteins increases with leaching time and at a fixed leaching time, the soluble proteins increases with leaching temperature. ne highest extractable protein contents was determined at a leaching time of 10 minutes and leaching temperature of 90'C The protein analysis were done by using Modified Lowry Method

  8. Improved segmental isotope labeling of proteins and application to a larger protein

    International Nuclear Information System (INIS)

    Otomo, Takanori; Teruya, Kenta; Uegaki, Koichi; Yamazaki, Toshio; Kyogoku, Yoshimasa

    1999-01-01

    A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13 C/ 15 N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples

  9. Liquid waste sampling device

    International Nuclear Information System (INIS)

    Kosuge, Tadashi

    1998-01-01

    A liquid pumping pressure regulator is disposed on the midway of a pressure control tube which connects the upper portion of a sampling pot and the upper portion of a liquid waste storage vessel. With such a constitution, when the pressure in the sampling pot is made negative, and liquid wastes are sucked to the liquid pumping tube passing through the sampling pot, the difference between the pressure on the entrance of the liquid pumping pressure regulator of the pressure regulating tube and the pressure at the bottom of the liquid waste storage vessel is made constant. An opening degree controlling meter is disposed to control the degree of opening of a pressure regulating valve for sending actuation pressurized air to the liquid pumping pressure regulator. Accordingly, even if the liquid level of liquid wastes in the liquid waste storage vessel is changed, the height for the suction of the liquid wastes in the liquid pumping tube can be kept constant. With such procedures, sampling can be conducted correctly, and the discharge of the liquid wastes to the outside can be prevented. (T.M.)

  10. IXM gas sampling procedure

    International Nuclear Information System (INIS)

    Pingel, L.A.

    1995-01-01

    Ion Exchange Modules (IXMs) are used at the 105-KE and -KW Fuel Storage Basins to control radionuclide concentrations in the water. A potential safety concern relates to production of hydrogen gas by radiolysis of the water trapped in the ion exchange media of spent IXMs. This document provides a procedure for sampling the gases in the head space of the IXM

  11. Request for wood samples

    NARCIS (Netherlands)

    NN,

    1977-01-01

    In recent years the wood collection at the Rijksherbarium was greatly expanded following a renewed interest in wood anatomy as an aid for solving classification problems. Staff members of the Rijksherbarium added to the collection by taking interesting wood samples with them from their expeditions

  12. Check Sample Abstracts.

    Science.gov (United States)

    Alter, David; Grenache, David G; Bosler, David S; Karcher, Raymond E; Nichols, James; Rajadhyaksha, Aparna; Camelo-Piragua, Sandra; Rauch, Carol; Huddleston, Brent J; Frank, Elizabeth L; Sluss, Patrick M; Lewandrowski, Kent; Eichhorn, John H; Hall, Janet E; Rahman, Saud S; McPherson, Richard A; Kiechle, Frederick L; Hammett-Stabler, Catherine; Pierce, Kristin A; Kloehn, Erica A; Thomas, Patricia A; Walts, Ann E; Madan, Rashna; Schlesinger, Kathie; Nawgiri, Ranjana; Bhutani, Manoop; Kanber, Yonca; Abati, Andrea; Atkins, Kristen A; Farrar, Robert; Gopez, Evelyn Valencerina; Jhala, Darshana; Griffin, Sonya; Jhala, Khushboo; Jhala, Nirag; Bentz, Joel S; Emerson, Lyska; Chadwick, Barbara E; Barroeta, Julieta E; Baloch, Zubair W; Collins, Brian T; Middleton, Owen L; Davis, Gregory G; Haden-Pinneri, Kathryn; Chu, Albert Y; Keylock, Joren B; Ramoso, Robert; Thoene, Cynthia A; Stewart, Donna; Pierce, Arand; Barry, Michelle; Aljinovic, Nika; Gardner, David L; Barry, Michelle; Shields, Lisa B E; Arnold, Jack; Stewart, Donna; Martin, Erica L; Rakow, Rex J; Paddock, Christopher; Zaki, Sherif R; Prahlow, Joseph A; Stewart, Donna; Shields, Lisa B E; Rolf, Cristin M; Falzon, Andrew L; Hudacki, Rachel; Mazzella, Fermina M; Bethel, Melissa; Zarrin-Khameh, Neda; Gresik, M Vicky; Gill, Ryan; Karlon, William; Etzell, Joan; Deftos, Michael; Karlon, William J; Etzell, Joan E; Wang, Endi; Lu, Chuanyi M; Manion, Elizabeth; Rosenthal, Nancy; Wang, Endi; Lu, Chuanyi M; Tang, Patrick; Petric, Martin; Schade, Andrew E; Hall, Geraldine S; Oethinger, Margret; Hall, Geraldine; Picton, Avis R; Hoang, Linda; Imperial, Miguel Ranoa; Kibsey, Pamela; Waites, Ken; Duffy, Lynn; Hall, Geraldine S; Salangsang, Jo-Anne M; Bravo, Lulette Tricia C; Oethinger, Margaret D; Veras, Emanuela; Silva, Elvia; Vicens, Jimena; Silva, Elvio; Keylock, Joren; Hempel, James; Rushing, Elizabeth; Posligua, Lorena E; Deavers, Michael T; Nash, Jason W; Basturk, Olca; Perle, Mary Ann; Greco, Alba; Lee, Peng; Maru, Dipen; Weydert, Jamie Allen; Stevens, Todd M; Brownlee, Noel A; Kemper, April E; Williams, H James; Oliverio, Brock J; Al-Agha, Osama M; Eskue, Kyle L; Newlands, Shawn D; Eltorky, Mahmoud A; Puri, Puja K; Royer, Michael C; Rush, Walter L; Tavora, Fabio; Galvin, Jeffrey R; Franks, Teri J; Carter, James Elliot; Kahn, Andrea Graciela; Lozada Muñoz, Luis R; Houghton, Dan; Land, Kevin J; Nester, Theresa; Gildea, Jacob; Lefkowitz, Jerry; Lacount, Rachel A; Thompson, Hannis W; Refaai, Majed A; Quillen, Karen; Lopez, Ana Ortega; Goldfinger, Dennis; Muram, Talia; Thompson, Hannis

    2009-02-01

    The following abstracts are compiled from Check Sample exercises published in 2008. These peer-reviewed case studies assist laboratory professionals with continuing medical education and are developed in the areas of clinical chemistry, cytopathology, forensic pathology, hematology, microbiology, surgical pathology, and transfusion medicine. Abstracts for all exercises published in the program will appear annually in AJCP.

  13. Large scale sample management and data analysis via MIRACLE

    DEFF Research Database (Denmark)

    Block, Ines; List, Markus; Pedersen, Marlene Lemvig

    Reverse-phase protein arrays (RPPAs) allow sensitive quantification of relative protein abundance in thousands of samples in parallel. In the past years the technology advanced based on improved methods and protocols concerning sample preparation and printing, antibody selection, optimization...... of staining conditions and mode of signal analysis. However, the sample management and data analysis still poses challenges because of the high number of samples, sample dilutions,