Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

DEFF Research Database (Denmark)

Kragelund, B B; Poulsen, K; Andersen, K V

1999-01-01

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability...

Stability and structure of the membrane protein transporter Ffh is modulated by substrates and lipids

DEFF Research Database (Denmark)

Reinau, Marika Ejby; Otzen, Daniel

2009-01-01

the apoprotein. Escherichia coli lipid and DOPG (and to a smaller extent DOPC) increase Ffh's α-helical content, possibly related to Ffh's role in guiding membrane proteins to the membrane. Binding is largely mediated by electrostatic interactions but does not protect Ffh against trypsinolysis. We conclude...... that Ffh is a structurally flexible and dynamic protein whose stability is significantly modulated by the environment. © 2009 Elsevier Inc. All rights reserved....

Protein stability and enzyme activity at extreme biological temperatures

International Nuclear Information System (INIS)

Feller, Georges

2010-01-01

Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 0 C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

Self-Assembling Peptide Surfactants A6K and A6D Adopt a-Helical Structures Useful for Membrane Protein Stabilization

Directory of Open Access Journals (Sweden)

Furen Zhuang

2011-10-01

Full Text Available Elucidation of membrane protein structures have been greatly hampered by difficulties in producing adequately large quantities of the functional protein and stabilizing them. A6D and A6K are promising solutions to the problem and have recently been used for the rapid production of membrane-bound G protein-coupled receptors (GPCRs. We propose that despite their short lengths, these peptides can adopt α-helical structures through interactions with micelles formed by the peptides themselves. These α-helices are then able to stabilize α-helical motifs which many membrane proteins contain. We also show that A6D and A6K can form β-sheets and appear as weak hydrogels at sufficiently high concentrations. Furthermore, A6D and A6K together in sodium dodecyl sulfate (SDS can form expected β-sheet structures via a surprising α-helical intermediate.

Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

Science.gov (United States)

Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

2015-10-15

Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Thermodynamic effects of proline introduction on protein stability.

Science.gov (United States)

Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C; Varadarajan, Raghavan

2007-02-01

The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive. Copyright 2006 Wiley-Liss, Inc.

Dissecting the critical factors for thermodynamic stability of modular proteins using molecular modeling approach.

Directory of Open Access Journals (Sweden)

Yuno Lee

Full Text Available Repeat proteins have recently attracted much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural and biophysical features. In particular, repeat proteins show high stability against temperature and chaotic agents. Despite many studies, structural features for the stability of repeat proteins remain poorly understood. Here we present an interesting result from in silico analyses pursuing the factors which affect the stability of repeat proteins. Previously developed repebody structure based on variable lymphocytes receptors (VLRs which consists of leucine-rich repeat (LRR modules was used as initial structure for the present study. We constructed extra six repebody structures with varying numbers of repeat modules and those structures were used for molecular dynamics simulations. For the structures, the intramolecular interactions including backbone H-bonds, van der Waals energy, and hydrophobicity were investigated and then the radius of gyration, solvent-accessible surface area, ratio of secondary structure, and hydration free energy were also calculated to find out the relationship between the number of LRR modules and stability of the protein. Our results show that the intramolecular interactions lead to more compact structure and smaller surface area of the repebodies, which are critical for the stability of repeat proteins. The other features were also well compatible with the experimental results. Based on our observations, the repebody-5 was proposed as the best structure from the all repebodies in structure optimization process. The present study successfully demonstrated that our computer-based molecular modeling approach can significantly contribute to the experiment-based protein engineering challenge.

Ion pairs in non-redundant protein structures

Indian Academy of Sciences (India)

Ion pairs contribute to several functions including the activity of catalytic triads, fusion of viral membranes, stability in thermophilic proteins and solvent–protein interactions. Furthermore, they have the ability to affect the stability of protein structures and are also a part of the forces that act to hold monomers together.

Insights into the role of hydration in protein structure and stability obtained through hydrostatic pressure studies

Directory of Open Access Journals (Sweden)

C.A. Royer

2005-08-01

Full Text Available A thorough understanding of protein structure and stability requires that we elucidate the molecular basis for the effects of both temperature and pressure on protein conformational transitions. While temperature effects are relatively well understood and the change in heat capacity upon unfolding has been reasonably well parameterized, the state of understanding of pressure effects is much less advanced. Ultimately, a quantitative parameterization of the volume changes (at the basis of pressure effects accompanying protein conformational transitions will be required. The present report introduces a qualitative hypothesis based on available model compound data for the molecular basis of volume change upon protein unfolding and its dependence on temperature.

Prospects in the use of aptamers for characterizing the structure and stability of bioactive proteins and peptides in food.

Science.gov (United States)

Agyei, Dominic; Acquah, Caleb; Tan, Kei Xian; Hii, Hieng Kok; Rajendran, Subin R C K; Udenigwe, Chibuike C; Danquah, Michael K

2018-01-01

Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d ) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed. Graphical abstract Advancing bioactive proteins and peptide functionality via aptameric ligands.

Thermal precipitation fluorescence assay for protein stability screening.

Science.gov (United States)

Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Analysis of protein stability and ligand interactions by thermal shift assay.

Science.gov (United States)

Huynh, Kathy; Partch, Carrie L

2015-02-02

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

Towards a "Golden Standard" for computing globin stability: Stability and structure sensitivity of myoglobin mutants.

Science.gov (United States)

Kepp, Kasper P

2015-10-01

Fast and accurate computation of protein stability is increasingly important for e.g. protein engineering and protein misfolding diseases, but no consensus methods exist for important proteins such as globins, and performance may depend on the type of structural input given. This paper reports benchmarking of six protein stability calculators (POPMUSIC 2.1, I-Mutant 2.0, I-Mutant 3.0, CUPSAT, SDM, and mCSM) against 134 experimental stability changes for mutations of sperm-whale myoglobin. Six different high-resolution structures were used to test structure sensitivity that may impair protein calculations. The trend accuracy of the methods decreased as I-Mutant 2.0 (R=0.64-0.65), SDM (R=0.57-0.60), POPMUSIC2.1 (R=0.54-0.57), I-Mutant 3.0 (R=0.53-0.55), mCSM (R=0.35-0.47), and CUPSAT (R=0.25-0.48). The mean signed errors increased as SDMMean absolute errors increased as I-Mutant 2.0Structural sensitivity increased as I-Mutant 3.0 (0.05)proteins. The results also emphasize the importance of high-quality crystal structures and reveal structure-dependent effects even in the near-atomic resolution limit. Copyright © 2015 Elsevier B.V. All rights reserved.

TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

Science.gov (United States)

Feller, Georges

2010-08-01

Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

Directory of Open Access Journals (Sweden)

Alessandra Pasquo

Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

Towards a "Golden Standard" for computing globin stability: Stability and structure sensitivity of myoglobin mutants

DEFF Research Database (Denmark)

Kepp, Kasper Planeta

2015-01-01

Fast and accurate computation of protein stability is increasingly important for e.g. protein engineering and protein misfolding diseases, but no consensus methods exist for important proteins such as globins, and performance may depend on the type of structural input given. This paper reports be...

CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function.

Science.gov (United States)

Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

2013-07-01

The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.

Overcoming bottlenecks in the membrane protein structural biology pipeline.

Science.gov (United States)

Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

2016-06-15

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Stability of Hyperthermophilic Proteins

DEFF Research Database (Denmark)

Stiefler-Jensen, Daniel

stability by randomly generate mutants and lengthy screening processes to identify the best new mutants. However, with the increase in available genomic sequences of thermophilic or hyperthermophilic organisms a world of enzymes with intrinsic high stability are now available. As these organisms are adapted...... to life at high temperatures so are their enzymes, as a result the high stability is accompanied by low activity at moderate temperatures. Thus, much effort had been put into decoding the mechanisms behind the high stability of the thermophilic enzymes. The hope is to enable scientist to design enzymes...... in the high stability of hyperthermophilic enzymes. The thesis starts with an introduction to the field of protein and enzyme stability with special focus on the thermophilic and hyperthermophilic enzymes and proteins. After the introduction three original research manuscripts present the experimental data...

New directions towards structure formation and stability of protein-rich foods from globular proteins

NARCIS (Netherlands)

Purwanti, N.; Goot, van der A.J.; Boom, R.M.; Vereijken, J.M.

2010-01-01

Concentrated protein-rich foods have strong potential to be developed in terms of health and well-being roles. Unfortunately, limitations in creating products with the rights texture and stability hinder the use of those products by consumers. Main reason is that the formation of micro- and

A rapid, ensemble and free energy based method for engineering protein stabilities.

Science.gov (United States)

Naganathan, Athi N

2013-05-02

Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnological applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mechanical model that accounts for the ensemble nature of folding. We validate the method by predicting the absolute changes in stability for 138 experimental mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing meosphile-thermophile protein pairs. In parallel, we devise a methodology to rapidly engineer mutations in silico which we benchmark against experimental mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnology industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.

Biophysical evaluation of aminoclay as an effective protectant for protein stabilization during freeze-drying and storage

Directory of Open Access Journals (Sweden)

Song JG

2016-12-01

Full Text Available Jae Geun Song, Sang Hoon Lee, Hyo-Kyung Han College of Pharmacy, Dongguk University, Goyang, South Korea Abstract: This study aimed to evaluate aminoclay (3-aminopropyl-functionalized magnesium phyllosilicate as an effective protectant for the stabilization of protein formulation in freeze-drying. Bovine serum albumin (BSA, as a model protein, was freeze-dried with aminoclay at various concentrations, and the effects of aminoclay on the structural stability of proteins were compared with those of the conventional stabilizers. The structural characteristics of the protein were determined by size exclusion chromatography (SEC, circular dichroism (CD, and Fourier transform infrared (FTIR spectroscopy. Furthermore, physicochemical and morphological characteristics were examined by X-ray powder diffraction (XRPD, differential scanning calorimetry (DSC, and scanning electron microscopy (SEM. XRPD and DSC patterns indicated that the glass transition temperature (Tg of the amorphous formulation of aminoclay mixed with proteins was gradually elevated as the concentration of aminoclay increased. FTIR and CD spectral analysis suggested that the protein structure was well maintained with aminoclay during the freeze-drying process and 3 months of storage at 4°C and 40°C. Furthermore, aminoclay conferred the greatest protection against aggregation and retained the monomer content of BSA even at a high temperature. The morphological characteristics of lyophilized proteins were also well conserved during the storage with aminoclay. These results suggested that aminoclay may be useful as an alternative stabilizer for maintaining the structural stability of protein formulations. Keywords: aminoclay, cryoprotectant, lyoprotectant, freeze-drying, protein, stability

Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration.

Science.gov (United States)

Rusinga, Farai I; Weis, David D

2017-08-01

The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well-understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L -1 and 400 g L -1 of Ficoll, a synthetic polysaccharide, using a recently-developed strong cation exchange-based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275-1282]. Transiently helical regions of ACTR exchanged faster in 300 g L -1 Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L -1 Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L -1 Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L -1 Ficoll is a semi-dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468-1479. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Fast loop modeling for protein structures

Science.gov (United States)

Zhang, Jiong; Nguyen, Son; Shang, Yi; Xu, Dong; Kosztin, Ioan

2015-03-01

X-ray crystallography is the main method for determining 3D protein structures. In many cases, however, flexible loop regions of proteins cannot be resolved by this approach. This leads to incomplete structures in the protein data bank, preventing further computational study and analysis of these proteins. For instance, all-atom molecular dynamics (MD) simulation studies of structure-function relationship require complete protein structures. To address this shortcoming, we have developed and implemented an efficient computational method for building missing protein loops. The method is database driven and uses deep learning and multi-dimensional scaling algorithms. We have implemented the method as a simple stand-alone program, which can also be used as a plugin in existing molecular modeling software, e.g., VMD. The quality and stability of the generated structures are assessed and tested via energy scoring functions and by equilibrium MD simulations. The proposed method can also be used in template-based protein structure prediction. Work supported by the National Institutes of Health [R01 GM100701]. Computer time was provided by the University of Missouri Bioinformatics Consortium.

Increasing protein stability by improving beta-turns.

Science.gov (United States)

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization.

Science.gov (United States)

Xu, Jing; Mukherjee, Dipaloke; Chang, Sam K C

2018-02-01

This study investigated the effects of the ultrahigh pressure homogenization (pressure, protein concentration, oil phase fraction, pH, temperature, and ionic strength) and storage on the properties of nanoemulsions (100-500nm range), which were stabilized by laboratory-prepared soybean protein isolate (SPI), β-conglycinin (7S) and glycinin (11S). The nanoemulsions made with SPI, 7S and 11S proteins exhibited considerable stability over various ionic strengths (0-500mM NaCl), pH (7), thermal treatments (30-60°C) and storage (0-45days). The far-UV spectra of SPI, 7S, 11S dispersions, and SPI-, 7S-, 11S protein-stabilized nanoemulsions were analyzed for the protein structural changes following lipid removal. The ultra-high pressure homogenization changed the secondary structure of SPI, 7S, 11S proteins in the nanoemulsions, and enhanced their stability. This study demonstrated that SPI, 7S, and 11S proteins can be used as effective emulsifiers in nanoemulsions prepared by ultra-high pressure homogenization. Copyright © 2017. Published by Elsevier Ltd.

Enhanced Stability of a Protein with Increasing Temperature

DEFF Research Database (Denmark)

Vinther, Joachim Møllesøe; Kristensen, Søren M; Led, Jens J

2010-01-01

The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility ...

Isomeric Detergent Comparison for Membrane Protein Stability

DEFF Research Database (Denmark)

Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.

2016-01-01

and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta...... and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility....../stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane...

Characterization of the influence of 1-butyl-3-methylimidazolium chloride on the structure and thermal stability of green fluorescent protein

International Nuclear Information System (INIS)

Heller, William T.; O'Neill, Hugh Michael; Zhang, Qiu; Baker, Gary A.

2010-01-01

Ionic liquids (ILs) are finding a vast array of applications as novel solvents for a wide variety of processes that include enzymatic chemistry, particularly as more biocompatible ILs are designed and discovered. While it is assumed that a native or near-native structure is required for enzymatic activity, there is some evidence that ILs alter protein structure and oligomerization states in a manner than can negatively impact function. The IL 1-butyl-3-methylimidazolium chloride, (bmim)Cl, is a well-studied, water-miscible member of the popular 1-alkyl-3-methylimidazolium IL family. To improve our understanding of the impact of water-miscible ILs on proteins, we have characterized the structure and oligomerization state of green fluorescent protein (GFP) in aqueous solutions containing 25 and 50 vol % (bmim)Cl using a combination of optical spectroscopy and small-angle neutron scattering (SANS). Measurements were also performed as a function of temperature to provide insight into the effect of the IL on the thermal stability of GFP. While GFP exists as a dimer in water, the presence of 25 vol % (bmim)Cl causes GFP to transition to a monomeric state. The SANS data indicate that GFP is a great deal less compact in 50 vol % (bmim)Cl than in neat water, indicative of unfolding from the native structure. The oligomerization state of the protein in IL-containing aqueous solution changes from a dimer to a monomer in response to the IL, but does not change as a function of temperature in the IL-containing solution. The SANS and spectroscopic results also demonstrate that the addition of (bmim)Cl to the solution decreases the thermal stability of GFP, allowing the protein to unfold at lower temperatures than in aqueous solution.

Self-assembling peptides form nanodiscs that stabilize membrane proteins

DEFF Research Database (Denmark)

Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

2014-01-01

-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

Energy Technology Data Exchange (ETDEWEB)

Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

2012-08-21

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

Contribution of buried aspartic acid to the stability of the PDZ2 protein

International Nuclear Information System (INIS)

Jayasimha, Pruthvi; Shanmuganathan, Aranganathan; Suladze, Saba; Makhatadze, George I.

2012-01-01

Highlights: ► Buried Asp residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 salt bridges. ► Contribution of buried Asp to stability was estimated using model protein PDZ2. ► The energetic contribution of Asp56 to PDZ2 stability estimated to be 18 kJ · mol −1 . ► Findings are discussed in terms of contribution of Asp residues to protein stability. - Abstract: Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18 kJ · mol −1 . These findings are discussed in terms of contribution of buried ionizable groups to protein stability.

Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

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Perinelli, Diego R; Cespi, Marco; Pucciarelli, Stefania; Vincenzetti, Silvia; Casettari, Luca; Lam, Jenny K W; Logrippo, Serena; Canala, Elisa; Soliman, Mahmoud E; Bonacucina, Giulia; Palmieri, Giovanni F

2017-01-01

Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Oligomeric protein structure networks: insights into protein-protein interactions

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Brinda KV

2005-12-01

spots have also been made based on the results obtained from this analysis, which await experimental verification. Conclusion The construction and analysis of oligomeric protein structure networks and their comparison with monomeric protein structure networks provide insights into protein association. Further, the interface hubs identified using the present method can be effective targets for interface de-stabilizing mutations. We believe this analysis will significantly enhance our knowledge of the principles behind protein association and also aid in protein design.

Sampling Realistic Protein Conformations Using Local Structural Bias

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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...

Structural stability of DNA origami nanostructures in the presence of chaotropic agents.

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Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2016-05-21

DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.

Protein engineering of subtilisins to improve stability in detergent formulations.

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von der Osten, C; Branner, S; Hastrup, S; Hedegaard, L; Rasmussen, M D; Bisgård-Frantzen, H; Carlsen, S; Mikkelsen, J M

1993-03-01

Microbial proteases are used extensively in a large number of industrial processes and most importantly in detergent formulations facilitating the removal of proteinaceous stains. Site-directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. It is shown that in spite of significant structural homology between subtilisins subjected to protein engineering the effects of specific mutations can be quite different. Mutations that stabilize one subtilisin may destabilize another.

Combining structural modeling with ensemble machine learning to accurately predict protein fold stability and binding affinity effects upon mutation.

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Niklas Berliner

Full Text Available Advances in sequencing have led to a rapid accumulation of mutations, some of which are associated with diseases. However, to draw mechanistic conclusions, a biochemical understanding of these mutations is necessary. For coding mutations, accurate prediction of significant changes in either the stability of proteins or their affinity to their binding partners is required. Traditional methods have used semi-empirical force fields, while newer methods employ machine learning of sequence and structural features. Here, we show how combining both of these approaches leads to a marked boost in accuracy. We introduce ELASPIC, a novel ensemble machine learning approach that is able to predict stability effects upon mutation in both, domain cores and domain-domain interfaces. We combine semi-empirical energy terms, sequence conservation, and a wide variety of molecular details with a Stochastic Gradient Boosting of Decision Trees (SGB-DT algorithm. The accuracy of our predictions surpasses existing methods by a considerable margin, achieving correlation coefficients of 0.77 for stability, and 0.75 for affinity predictions. Notably, we integrated homology modeling to enable proteome-wide prediction and show that accurate prediction on modeled structures is possible. Lastly, ELASPIC showed significant differences between various types of disease-associated mutations, as well as between disease and common neutral mutations. Unlike pure sequence-based prediction methods that try to predict phenotypic effects of mutations, our predictions unravel the molecular details governing the protein instability, and help us better understand the molecular causes of diseases.

A generative, probabilistic model of local protein structure

DEFF Research Database (Denmark)

Boomsma, Wouter; Mardia, Kanti V.; Taylor, Charles C.

2008-01-01

Despite significant progress in recent years, protein structure prediction maintains its status as one of the prime unsolved problems in computational biology. One of the key remaining challenges is an efficient probabilistic exploration of the structural space that correctly reflects the relative...... conformational stabilities. Here, we present a fully probabilistic, continuous model of local protein structure in atomic detail. The generative model makes efficient conformational sampling possible and provides a framework for the rigorous analysis of local sequence-structure correlations in the native state...

Threading structural model of the manganese-stabilizing protein PsbO reveals presence of two possible beta-sandwich domains.

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Pazos, F; Heredia, P; Valencia, A; de las Rivas, J

2001-12-01

The manganese-stabilizing protein (PsbO) is an essential component of photosystem II (PSII) and is present in all oxyphotosynthetic organisms. PsbO allows correct water splitting and oxygen evolution by stabilizing the reactions driven by the manganese cluster. Despite its important role, its structure and detailed functional mechanism are still unknown. In this article we propose a structural model based on fold recognition and molecular modeling. This model has additional support from a study of the distribution of characteristics of the PsbO sequence family, such as the distribution of conserved, apolar, tree-determinants, and correlated positions. Our threading results consistently showed PsbO as an all-beta (beta) protein, with two homologous beta domains of approximately 120 amino acids linked by a flexible Proline-Glycine-Glycine (PGG) motif. These features are compatible with a general elongated and flexible architecture, in which the two domains form a sandwich-type structure with Greek key topology. The first domain is predicted to include 8 to 9 beta-strands, the second domain 6 to 7 beta-strands. An Ig-like beta-sandwich structure was selected as a template to build the 3-D model. The second domain has, between the strands, long-loops rich in Pro and Gly that are difficult to model. One of these long loops includes a highly conserved region (between P148 and P174) and a short alpha-helix (between E181 and N188)). These regions are characteristic parts of PsbO and show that the second domain is not so similar to the template. Overall, the model was able to account for much of the experimental data reported by several authors, and it would allow the detection of key residues and regions that are proposed in this article as essential for the structure and function of PsbO. Copyright 2001 Wiley-Liss, Inc.

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

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Dror, Adi; Shemesh, Einav; Dayan, Natali

2014-01-01

The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

High-quality Thermodynamic Data on the Stability Changes of Proteins Upon Single-site Mutations

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Pucci, Fabrizio, E-mail: fapucci@ulb.ac.be; Bourgeas, Raphaël, E-mail: rbourgeas@ulb.ac.be; Rooman, Marianne, E-mail: mrooman@ulb.ac.be [Department of BioModeling, BioInformatics and BioProcesses, Université Libre de Bruxelles, CP 165/61, Roosevelt Avenue 50, 1050 Brussels, Belgium and Interuniversity Institute of Bioinformatics in Brussels, CP 263, Triumph Bld, 1050 Brussels (Belgium)

2016-06-15

We have set up and manually curated a dataset containing experimental information on the impact of amino acid substitutions in a protein on its thermal stability. It consists of a repository of experimentally measured melting temperatures (T{sub m}) and their changes upon point mutations (ΔT{sub m}) for proteins having a well-resolved x-ray structure. This high-quality dataset is designed for being used for the training or benchmarking of in silico thermal stability prediction methods. It also reports other experimentally measured thermodynamic quantities when available, i.e., the folding enthalpy (ΔH) and heat capacity (ΔC{sub P}) of the wild type proteins and their changes upon mutations (ΔΔH and ΔΔC{sub P}), as well as the change in folding free energy (ΔΔG) at a reference temperature. These data are analyzed in view of improving our insights into the correlation between thermal and thermodynamic stabilities, the asymmetry between the number of stabilizing and destabilizing mutations, and the difference in stabilization potential of thermostable versus mesostable proteins.

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

Science.gov (United States)

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-08-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

Structure and Pathology of Tau Protein in Alzheimer Disease

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Michala Kolarova

2012-01-01

Full Text Available Alzheimer's disease (AD is the most common type of dementia. In connection with the global trend of prolonging human life and the increasing number of elderly in the population, the AD becomes one of the most serious health and socioeconomic problems of the present. Tau protein promotes assembly and stabilizes microtubules, which contributes to the proper function of neuron. Alterations in the amount or the structure of tau protein can affect its role as a stabilizer of microtubules as well as some of the processes in which it is implicated. The molecular mechanisms governing tau aggregation are mainly represented by several posttranslational modifications that alter its structure and conformational state. Hence, abnormal phosphorylation and truncation of tau protein have gained attention as key mechanisms that become tau protein in a pathological entity. Evidences about the clinicopathological significance of phosphorylated and truncated tau have been documented during the progression of AD as well as their capacity to exert cytotoxicity when expressed in cell and animal models. This paper describes the normal structure and function of tau protein and its major alterations during its pathological aggregation in AD.

Protein Comparability Assessments and Potential Applicability of High Throughput Biophysical Methods and Data Visualization Tools to Compare Physical Stability Profiles

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Mohammad A. Alsenaidy

2014-03-01

Full Text Available In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs, radar charts, and comparative signature diagrams (CSDs for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1 mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF. Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress. Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles.

Science.gov (United States)

Alsenaidy, Mohammad A; Jain, Nishant K; Kim, Jae H; Middaugh, C Russell; Volkin, David B

2014-01-01

In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes.

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Inés Plasencia

Full Text Available BACKGROUND: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. METHODOLOGY/PRINCIPAL FINDING: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-β-D-glucopyranoside (OG or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC, or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE, 1-palmitoyl-2oleoyl-phosphatidylethanolamine (POPE, 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS, and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°C. CONCLUSION/SIGNIFICANCE: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications.

Recent advances in the applications of ionic liquids in protein stability and activity: a review.

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Patel, Rajan; Kumari, Meena; Khan, Abbul Bashar

2014-04-01

Room temperatures ionic liquids are considered as miraculous solvents for biological system. Due to their inimitable properties and large variety of applications, they have been widely used in enzyme catalysis and protein stability and separation. The related information present in the current review is helpful to the researchers working in the field of biotechnology and biochemistry to design or choose an ionic liquid that can serve as a noble and selective solvent for any particular enzymatic reaction, protein preservation and other protein based applications. We have extensively analyzed the methods used for studying the protein-IL interaction which is useful in providing information about structural and conformational dynamics of protein. This can be helpful to develop and understanding about the effect of ionic liquids on stability and activity of proteins. In addition, the affect of physico-chemical properties of ionic liquids, viz. hydrogen bond capacity and hydrophobicity on protein stability are discussed.

Surface (glyco-)proteins: primary structure and crystallization under microgravity conditions

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Claus, H.; Akca, E.; Schultz, N.; Karbach, G.; Schlott, B.; Debaerdemaeker, T.; De Clercq, J.-P.; König, H.

2001-08-01

The Archaea comprise microorganisms that live under environmental extremes, like high temperature, low pH value or high salt concentration. Their cells are often covered by a single layer of (glyco)protein subunits (S-layer) in hexagonal arrangement. In order to get further hints about the molecular mechanisms of protein stabilization we compared the primary and secondary structures of archaeal S-layer (glyco)proteins. We found an increase of charged amino acids in the S-layer proteins of the extreme thermophilic species compared to their mesophilic counterparts. Our data and those of other authors suggest that ionic interactions, e.g., salt bridges seem to be played a major role in protein stabilization at high temperatures. Despite the differences in the growth optima and the predominance of some amino acids the primary structures of S-layers revealed also a significant degree of identity between phylogenetically related archaea. These obervations indicate that protein sequences of S-layers have been conserved during the evolution from extremely thermophilic to mesophilic life. To support these findings the three-dimensional structure of the S-layer proteins has to be elucidated. Recently, we described the first successful crystallization of an extreme thermophilic surface(glyco)protein under microgravity conditions.

Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

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Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

2016-01-01

While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

Hydrophobic environment is a key factor for the stability of thermophilic proteins.

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Gromiha, M Michael; Pathak, Manish C; Saraboji, Kadhirvel; Ortlund, Eric A; Gaucher, Eric A

2013-04-01

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter-residue interactions, ion-pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic-mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three-dimensional structures of elongation factor-Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the

The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins.

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Claudia Rodríguez-Almazán

Full Text Available The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.

The role of a topologically conserved isoleucine in glutathione transferase structure, stability and function

International Nuclear Information System (INIS)

Achilonu, Ikechukwu; Gildenhuys, Samantha; Fisher, Loren; Burke, Jonathan; Fanucchi, Sylvia; Sewell, B. Trevor; Fernandes, Manuel; Dirr, Heini W.

2010-01-01

The role of a topologically conserved isoleucine in the structure of glutathione transferase was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The common fold shared by members of the glutathione-transferase (GST) family has a topologically conserved isoleucine residue at the N-terminus of helix 3 which is involved in the packing of helix 3 against two β-strands in domain 1. The role of the isoleucine residue in the structure, function and stability of GST was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The X-ray structures of the I71A and I71V mutants resolved at 1.75 and 2.51 Å, respectively, revealed that the mutations do not alter the overall structure of the protein compared with the wild type. Urea-induced equilibrium unfolding studies using circular dichroism and tryptophan fluorescence suggest that the mutation of Ile71 to alanine or valine reduces the stability of the protein. A functional assay with 1-chloro-2,4-dinitrobenzene shows that the mutation does not significantly alter the function of the protein relative to the wild type. Overall, the results suggest that conservation of the topologically conserved Ile71 maintains the structural stability of the protein but does not play a significant role in catalysis and substrate binding

Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

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Zhao, Hua

2015-01-01

There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

Circular dichroism and fluorescence spectroscopy of cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 demonstrates that group I cations are particularly effective in providing structure and stability to this halophilic protein.

Directory of Open Access Journals (Sweden)

Christopher J Reed

Full Text Available Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1, which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0-3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.

Science.gov (United States)

Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A; Kruse, Andrew C; Nurva, Shailika; Loland, Claus J; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G; Guan, Lan; Gether, Ulrik; Byrne, Bernadette; Kobilka, Brian; Gellman, Samuel H

2010-12-01

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

Energy Technology Data Exchange (ETDEWEB)

Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

2007-03-01

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability

International Nuclear Information System (INIS)

Zhu, W.; Manjasetty, B.; Chance, M.

2007-01-01

The functional properties of proteins depend on their three-dimensional shapes. Protein structures can be determined by X-ray crystallography as a tool. The three-dimensional structure of the apo form of the Escherichia coli L-arabinose isomerase (ECAI) has recently been determined. ECAI is responsible for the initial stage of L-arabinose catabolism, converting arabinose into ribulose in vivo. This enzyme also plays a crucial role in catalyzing the conversion of galactose into tagatose (low calorie natural sugar) in vitro. ECAI utilizes Mn 2+ for its catalytic activity. Crystals of the ECAI + Mn 2+ complex helps to investigate the catalytic properties of the enzyme. Therefore, crystals of ECAI + Mn 2+ complex were grown using hanging drop vapor diffusion method at room temperature. Diffraction data were collected at X4C beamline, National Synchrotron Light Source, Brookhaven National Laboratory. The structure was solved by the molecular replacement technique and has been refined to Rwork of 0.23 at 2.8 (angstrom) resolution using X3A beamline computational facility. The structure was deposited to Protein Data Bank (PDB ID 2HXG). Mn 2+ ion was localized to the previously identified putative active site with octahedral coordination. Comparison of apo and holo form of ECAI structures permits the identification of structural features that are of importance to the intrinsic activity and heat stability of AI

Characterization of milk proteins-lutein complexes and the impact on lutein chemical stability.

Science.gov (United States)

Yi, Jiang; Fan, Yuting; Yokoyama, Wallace; Zhang, Yuzhu; Zhao, Liqing

2016-06-01

In this study, the interaction of WPI (whey protein isolate) and SC (sodium caseinate) with hydrophobic lutein was investigated through UV-vis spectroscopy and circular dichroism (CD) as well as fluorescence. The effects on lutein's chemical stability were also examined. The decrease of turbidity of lutein suggested that lutein's aqueous solubility was improved after binding with milk proteins. CD analysis indicated lutein had little impact on the secondary structures of both proteins. Different preparation methods have significant impacts on the binding constant. Fluorescence results indicated that WPI and SC interact with lutein by hydrophobic contacts. Milk proteins have protective effects on lutein against oxidation and decomposition, and SC showed better capability in protecting lutein from oxidation than WPI during 16 days storage. The lutein's chemical stability was increased with increasing of proteins concentration. The results indicated that milk proteins may act as effective carriers for lipophilic nutraceuticals. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stabilities and Dynamics of Protein Folding Nuclei by Molecular Dynamics Simulation

Science.gov (United States)

Song, Yong-Shun; Zhou, Xin; Zheng, Wei-Mou; Wang, Yan-Ting

2017-07-01

To understand how the stabilities of key nuclei fragments affect protein folding dynamics, we simulate by molecular dynamics (MD) simulation in aqueous solution four fragments cut out of a protein G, including one α-helix (seqB: KVFKQYAN), two β-turns (seqA: LNGKTLKG and seqC: YDDATKTF), and one β-strand (seqD: DGEWTYDD). The Markov State Model clustering method combined with the coarse-grained conformation letters method are employed to analyze the data sampled from 2-μs equilibrium MD simulation trajectories. We find that seqA and seqB have more stable structures than their native structures which become metastable when cut out of the protein structure. As expected, seqD alone is flexible and does not have a stable structure. Throughout our simulations, the native structure of seqC is stable but cannot be reached if starting from a structure other than the native one, implying a funnel-shape free energy landscape of seqC in aqueous solution. All the above results suggest that different nuclei have different formation dynamics during protein folding, which may have a major contribution to the hierarchy of protein folding dynamics. Supported by the National Basic Research Program of China under Grant No. 2013CB932804, the National Natural Science Foundation of China under Grant No. 11421063, and the CAS Biophysics Interdisciplinary Innovation Team Project

Controlled formation of emulsion gels stabilized by salted myofibrillar protein under malondialdehyde (MDA)-induced oxidative stress.

Science.gov (United States)

Zhou, Feibai; Sun, Weizheng; Zhao, Mouming

2015-04-15

This study presented the cold-set gelation of emulsions stabilized by salted myofibrillar protein (MP) under oxidative stress originated from malondialdehyde (MDA). Gel properties were compared over a range of MDA/NaCl concentrations including gel viscoelastic properties, strength, water-holding capacity (WHC), amount of protein entrapped, and microstructure. The oxidative stability of emulsion gels as indicated by lipid hydroperoxide was further determined and compared. Results indicated that emulsion stabilized by MP at swollen state under certain ionic strengths (0.2-0.6 M) was the premise of gel formation under MDA. In the presence of intermediate MDA concentrations (2.5-10 mM), the emulsion gels showed an improved elasticity, strength, WHC, and oxidative stability. This improvement should be mainly attributed to the enhanced protein-protein cross-linkings via MDA, which were homogeneously formed among absorbed and/or unabsorbed proteins, entrapping a greater amount and fractions of protein within network. Therefore, the oil droplets were better adherent to the gel matrix. Nevertheless, addition of high MDA concentrations (25-50 mM) led to the formation of excessive covalent bonds, which might break protein-protein bonds and trigger the desorption of protein from the interface. This ultimately caused "oil leak" phenomena as well as the collapse of gel structure and, thus, overall decreased gel properties and oxidative stability.

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.

Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

Science.gov (United States)

Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

2015-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients. PMID:26633385

Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

Directory of Open Access Journals (Sweden)

Saúl Gómez-Manzo

2015-12-01

Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency, and the G6PD Santa Maria and A+ (less severe deficiency (Class I, II and III, respectively affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients.

Degradation of structurally characterized proteins injected into HeLa cells. Basic measurements

International Nuclear Information System (INIS)

Rogers, S.W.; Rechsteiner, M.

1988-01-01

Thirty-five proteins of known x-ray structure were labeled by chloramine-T radioiodination or by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using red cell-mediated microinjection. Degradation rates of the injected proteins were then determined over the next 50 h by measuring the release of soluble isotope to the culture medium. Control experiments demonstrated that the measured rates were not compromised by proteolysis within RBCs, the presence of unfused RBCs, or degradation of protein released from RBCs to the medium. Degradation of some injected proteins was faster during the first 12 h after fusion than at later times, apparently a response of HeLa cells to trypsinization. However, all proteins exhibited first-order degradation rates between 24 and 48 h post injection. Except for seven proteins, stabilities measured during this interval were unaffected by the labeling procedure. Reductive methylation was used to choose among the seven discordant values, and half-lives for the 35 proteins ranged from 16 h for lysozyme to 214 h for yeast alcohol dehydrogenase. Since half-lives for six of the injected proteins closely match values obtained by in vivo measurements, we consider our estimates of the metabolic stabilities of the injected proteins to be generally accurate. Therefore, the half-lives obtained by microinjection should prove useful in the search for relationships between protein structure and intracellular stability

Improved glucose-neopentyl glycol (GNG) amphiphiles for membrane protein solubilization and stabilization.

Science.gov (United States)

Cho, Kyung Ho; Bae, Hyoung Eun; Das, Manabendra; Gellman, Samuel H; Chae, Pil Seok

2014-02-01

Membrane proteins are inherently amphipathic and undergo dynamic conformational changes for proper function within native membranes. Maintaining the functional structures of these biomacromolecules in aqueous media is necessary for structural studies but difficult to achieve with currently available tools, thus necessitating the development of novel agents with favorable properties. This study introduces several new glucose-neopentyl glycol (GNG) amphiphiles and reveals some agents that display favorable behaviors for the solubilization and stabilization of a large, multi-subunit membrane protein assembly. Furthermore, a detergent structure-property relationship that could serve as a useful guideline for the design of novel amphiphiles is discussed. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane proteins bind lipids selectively to modulate their structure and function.

Science.gov (United States)

Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane

Proteins with Novel Structure, Function and Dynamics

Science.gov (United States)

Pohorille, Andrew

2014-01-01

Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828-831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81-83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha-helix or beta-sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

Energy Technology Data Exchange (ETDEWEB)

Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A. [Instituto Leloir, Buenos Aires (Argentina); Braden, B.C. [Bowie State Univ., Maryland (United States)

2004-07-01

The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

International Nuclear Information System (INIS)

Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A.; Braden, B.C.

2004-01-01

The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

Structure-Energy Relationships of Halogen Bonds in Proteins.

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Scholfield, Matthew R; Ford, Melissa Coates; Carlsson, Anna-Carin C; Butta, Hawera; Mehl, Ryan A; Ho, P Shing

2017-06-06

The structures and stabilities of proteins are defined by a series of weak noncovalent electrostatic, van der Waals, and hydrogen bond (HB) interactions. In this study, we have designed and engineered halogen bonds (XBs) site-specifically to study their structure-energy relationship in a model protein, T4 lysozyme. The evidence for XBs is the displacement of the aromatic side chain toward an oxygen acceptor, at distances that are equal to or less than the sums of their respective van der Waals radii, when the hydroxyl substituent of the wild-type tyrosine is replaced by a halogen. In addition, thermal melting studies show that the iodine XB rescues the stabilization energy from an otherwise destabilizing substitution (at an equivalent noninteracting site), indicating that the interaction is also present in solution. Quantum chemical calculations show that the XB complements an HB at this site and that solvent structure must also be considered in trying to design molecular interactions such as XBs into biological systems. A bromine substitution also shows displacement of the side chain, but the distances and geometries do not indicate formation of an XB. Thus, we have dissected the contributions from various noncovalent interactions of halogens introduced into proteins, to drive the application of XBs, particularly in biomolecular design.

Structural entanglements in protein complexes

Science.gov (United States)

Zhao, Yani; Chwastyk, Mateusz; Cieplak, Marek

2017-06-01

We consider multi-chain protein native structures and propose a criterion that determines whether two chains in the system are entangled or not. The criterion is based on the behavior observed by pulling at both termini of each chain simultaneously in the two chains. We have identified about 900 entangled systems in the Protein Data Bank and provided a more detailed analysis for several of them. We argue that entanglement enhances the thermodynamic stability of the system but it may have other functions: burying the hydrophobic residues at the interface and increasing the DNA or RNA binding area. We also study the folding and stretching properties of the knotted dimeric proteins MJ0366, YibK, and bacteriophytochrome. These proteins have been studied theoretically in their monomeric versions so far. The dimers are seen to separate on stretching through the tensile mechanism and the characteristic unraveling force depends on the pulling direction.

Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

DEFF Research Database (Denmark)

Plasencia, Ines; Survery, Sabeen; Ibragimova, Sania

2011-01-01

Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize...... reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles...... and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPh...

Physicochemical stability of lycopene-loaded emulsions stabilized by plant or dairy proteins

NARCIS (Netherlands)

Ho, Kacie; Schroen, C.G.P.H.; San Martín-González, M.F.; Berton-Carabin, C.C.

2017-01-01

Lycopene is a lipophilic bioactive compound that can be challenging to deliver in vivo. To mediate this, delivery strategies, such as protein-stabilized oil-in-water (O/W) emulsions, have been suggested to improve the physicochemical stability and bioavailability of lycopene. In this research, the

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

OpenAIRE

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-01-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only...

Simulation of Protein Structure, Dynamics and Function in Organic Media

National Research Council Canada - National Science Library

Daggett, Valerie

1998-01-01

The overall goal of our ONR-sponsored research is to pursue realistic molecular modeling strudies pertinnent to the related properties of protein stability, dynamics, structure, function, and folding in aqueous solution...

A Subset of Autism-Associated Genes Regulate the Structural Stability of Neurons

Science.gov (United States)

Lin, Yu-Chih; Frei, Jeannine A.; Kilander, Michaela B. C.; Shen, Wenjuan; Blatt, Gene J.

2016-01-01

Autism spectrum disorder (ASD) comprises a range of neurological conditions that affect individuals’ ability to communicate and interact with others. People with ASD often exhibit marked qualitative difficulties in social interaction, communication, and behavior. Alterations in neurite arborization and dendritic spine morphology, including size, shape, and number, are hallmarks of almost all neurological conditions, including ASD. As experimental evidence emerges in recent years, it becomes clear that although there is broad heterogeneity of identified autism risk genes, many of them converge into similar cellular pathways, including those regulating neurite outgrowth, synapse formation and spine stability, and synaptic plasticity. These mechanisms together regulate the structural stability of neurons and are vulnerable targets in ASD. In this review, we discuss the current understanding of those autism risk genes that affect the structural connectivity of neurons. We sub-categorize them into (1) cytoskeletal regulators, e.g., motors and small RhoGTPase regulators; (2) adhesion molecules, e.g., cadherins, NCAM, and neurexin superfamily; (3) cell surface receptors, e.g., glutamatergic receptors and receptor tyrosine kinases; (4) signaling molecules, e.g., protein kinases and phosphatases; and (5) synaptic proteins, e.g., vesicle and scaffolding proteins. Although the roles of some of these genes in maintaining neuronal structural stability are well studied, how mutations contribute to the autism phenotype is still largely unknown. Investigating whether and how the neuronal structure and function are affected when these genes are mutated will provide insights toward developing effective interventions aimed at improving the lives of people with autism and their families. PMID:27909399

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

PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality

Directory of Open Access Journals (Sweden)

Rooman Marianne

2011-05-01

Full Text Available Abstract Background The rational design of modified proteins with controlled stability is of extreme importance in a whole range of applications, notably in the biotechnological and environmental areas, where proteins are used for their catalytic or other functional activities. Future breakthroughs in medical research may also be expected from an improved understanding of the effect of naturally occurring disease-causing mutations on the molecular level. Results PoPMuSiC-2.1 is a web server that predicts the thermodynamic stability changes caused by single site mutations in proteins, using a linear combination of statistical potentials whose coefficients depend on the solvent accessibility of the mutated residue. PoPMuSiC presents good prediction performances (correlation coefficient of 0.8 between predicted and measured stability changes, in cross validation, after exclusion of 10% outliers. It is moreover very fast, allowing the prediction of the stability changes resulting from all possible mutations in a medium size protein in less than a minute. This unique functionality is user-friendly implemented in PoPMuSiC and is particularly easy to exploit. Another new functionality of our server concerns the estimation of the optimality of each amino acid in the sequence, with respect to the stability of the structure. It may be used to detect structural weaknesses, i.e. clusters of non-optimal residues, which represent particularly interesting sites for introducing targeted mutations. This sequence optimality data is also expected to have significant implications in the prediction and the analysis of particular structural or functional protein regions. To illustrate the interest of this new functionality, we apply it to a dataset of known catalytic sites, and show that a much larger than average concentration of structural weaknesses is detected, quantifying how these sites have been optimized for function rather than stability. Conclusion The

Effects of protein phosphorylation on color stability of ground meat.

Science.gov (United States)

Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

2017-03-15

The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Influence of degree correlations on network structure and stability in protein-protein interaction networks

Directory of Open Access Journals (Sweden)

Zimmer Ralf

2007-08-01

Full Text Available Abstract Background The existence of negative correlations between degrees of interacting proteins is being discussed since such negative degree correlations were found for the large-scale yeast protein-protein interaction (PPI network of Ito et al. More recent studies observed no such negative correlations for high-confidence interaction sets. In this article, we analyzed a range of experimentally derived interaction networks to understand the role and prevalence of degree correlations in PPI networks. We investigated how degree correlations influence the structure of networks and their tolerance against perturbations such as the targeted deletion of hubs. Results For each PPI network, we simulated uncorrelated, positively and negatively correlated reference networks. Here, a simple model was developed which can create different types of degree correlations in a network without changing the degree distribution. Differences in static properties associated with degree correlations were compared by analyzing the network characteristics of the original PPI and reference networks. Dynamics were compared by simulating the effect of a selective deletion of hubs in all networks. Conclusion Considerable differences between the network types were found for the number of components in the original networks. Negatively correlated networks are fragmented into significantly less components than observed for positively correlated networks. On the other hand, the selective deletion of hubs showed an increased structural tolerance to these deletions for the positively correlated networks. This results in a lower rate of interaction loss in these networks compared to the negatively correlated networks and a decreased disintegration rate. Interestingly, real PPI networks are most similar to the randomly correlated references with respect to all properties analyzed. Thus, although structural properties of networks can be modified considerably by degree

ProteinSplit: splitting of multi-domain proteins using prediction of ordered and disordered regions in protein sequences for virtual structural genomics

International Nuclear Information System (INIS)

Wyrwicz, Lucjan S; Koczyk, Grzegorz; Rychlewski, Leszek; Plewczynski, Dariusz

2007-01-01

The annotation of protein folds within newly sequenced genomes is the main target for semi-automated protein structure prediction (virtual structural genomics). A large number of automated methods have been developed recently with very good results in the case of single-domain proteins. Unfortunately, most of these automated methods often fail to properly predict the distant homology between a given multi-domain protein query and structural templates. Therefore a multi-domain protein should be split into domains in order to overcome this limitation. ProteinSplit is designed to identify protein domain boundaries using a novel algorithm that predicts disordered regions in protein sequences. The software utilizes various sequence characteristics to assess the local propensity of a protein to be disordered or ordered in terms of local structure stability. These disordered parts of a protein are likely to create interdomain spacers. Because of its speed and portability, the method was successfully applied to several genome-wide fold annotation experiments. The user can run an automated analysis of sets of proteins or perform semi-automated multiple user projects (saving the results on the server). Additionally the sequences of predicted domains can be sent to the Bioinfo.PL Protein Structure Prediction Meta-Server for further protein three-dimensional structure and function prediction. The program is freely accessible as a web service at http://lucjan.bioinfo.pl/proteinsplit together with detailed benchmark results on the critical assessment of a fully automated structure prediction (CAFASP) set of sequences. The source code of the local version of protein domain boundary prediction is available upon request from the authors

Correlation of structural stability with functional remodeling of high-density lipoproteins: the importance of being disordered.

Science.gov (United States)

Guha, Madhumita; Gao, Xuan; Jayaraman, Shobini; Gursky, Olga

2008-11-04

High-density lipoproteins (HDLs) are protein-lipid assemblies that remove excess cell cholesterol and prevent atherosclerosis. HDLs are stabilized by kinetic barriers that decelerate protein dissociation and lipoprotein fusion. We propose that similar barriers modulate metabolic remodeling of plasma HDLs; hence, changes in particle composition that destabilize HDLs and accelerate their denaturation may accelerate their metabolic remodeling. To test this notion, we correlate existing reports on HDL-mediated cell cholesterol efflux and esterification, which are obligatory early steps in cholesterol removal, with our kinetic studies of HDL stability. The results support our hypothesis and show that factors accelerating cholesterol efflux and esterification in model discoidal lipoproteins (including reduced protein size, reduced fatty acyl chain length, and/or increased level of cis unsaturation) destabilize lipoproteins and accelerate their fusion and apolipoprotein dissociation. Oxidation studies of plasma spherical HDLs show a similar trend: mild oxidation by Cu(2+) or OCl(-) accelerates cell cholesterol efflux, protein dissociation, and HDL fusion, while extensive oxidation inhibits these reactions. Consequently, moderate destabilization may be beneficial for HDL functions by facilitating insertion of cholesterol and lipophilic enzymes, promoting dissociation of lipid-poor apolipoproteins, which are primary acceptors of cell cholesterol, and thereby accelerating HDL metabolism. Therefore, HDL stability must be delicately balanced to maintain the structural integrity of the lipoprotein assembly and ensure structural specificity necessary for interactions of HDL with its metabolic partners, while facilitating rapid HDL remodeling and turnover at key junctures of cholesterol transport. The inverse correlation between HDL stability and remodeling illustrates the functional importance of structural disorder in macromolecular assemblies stabilized by kinetic barriers.

Solution structure of the cold-shock-like protein from Rickettsia rickettsii

International Nuclear Information System (INIS)

Gerarden, Kyle P.; Fuchs, Andrew M.; Koch, Jonathan M.; Mueller, Melissa M.; Graupner, David R.; O’Rorke, Justin T.; Frost, Caleb D.; Heinen, Heather A.; Lackner, Emily R.; Schoeller, Scott J.; House, Paul G.; Peterson, Francis C.; Veldkamp, Christopher T.

2012-01-01

The solution structure of the cold-shock-like protein from R. rickettsii, the causative agent of Rocky Mountain spotted fever, is reported. Rocky Mountain spotted fever is caused by Rickettsia rickettsii infection. R. rickettsii can be transmitted to mammals, including humans, through the bite of an infected hard-bodied tick of the family Ixodidae. Since the R. rickettsii genome contains only one cold-shock-like protein and given the essential nature of cold-shock proteins in other bacteria, the structure of the cold-shock-like protein from R. rickettsii was investigated. With the exception of a short α-helix found between β-strands 3 and 4, the solution structure of the R. rickettsii cold-shock-like protein has the typical Greek-key five-stranded β-barrel structure found in most cold-shock domains. Additionally, the R. rickettsii cold-shock-like protein, with a ΔG of unfolding of 18.4 kJ mol −1 , has a similar stability when compared with other bacterial cold-shock proteins

Effects of inulin on the structure and emulsifying properties of protein components in dough.

Science.gov (United States)

Liu, Juan; Luo, Denglin; Li, Xuan; Xu, Baocheng; Zhang, Xiaoyu; Liu, Jianxue

2016-11-01

High-purity gliadin, glutenin and gluten fractions were extracted from wheat gluten flour. To investigate the effects of three types of inulin with different degrees of polymerization (DP) on the emulsifying properties, disulfide contents, secondary structures and microstructures of these fractions, Turbidimetry, spectrophotometer, Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) were used in this study. The results showed that the emulsifying activity of gliadin was higher than that of glutenin and gluten, but its emulsion stability was lower than that of glutenin. Adding inulin increased the emulsifying activity of the three protein fractions and emulsion stability of gliadin and gluten, but decreased the emulsion stability of glutenin and disulfide bond contents of glutenin and gluten. In the presence of inulin, the α-helical structure of the three proteins had no significant change, whereas the β-turn structure decreased and β-sheet structure increased. The SEM images showed that inulin had the most significant effect on the glutenin microstructure. In general, inulin with a higher DP had greater effects on the structure and emulsifying properties of protein components in dough. Copyright © 2016 Elsevier Ltd. All rights reserved.

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

International Nuclear Information System (INIS)

Shi, Jade; Schwantes, Christian; Bilsel, Osman

2017-01-01

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. We report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. The resulting prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. We then predict incisive single molecule FRET experiments, using these results, as a means of model validation. Our study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.

Influence of pH value on microstructure of oil-in-water emulsions stabilized by chickpea protein flour.

Science.gov (United States)

Felix, Manuel; Isurralde, Nadia; Romero, Alberto; Guerrero, Antonio

2018-01-01

Food industry is highly interested in the development of healthier formulations of oil-in-water emulsions, stabilized by plant proteins instead of egg or milk proteins. These emulsions would avoid allergic issues or animal fat. Among other plant proteins, legumes are a cost-competitive product. This work evaluates the influence of pH value (2.5, 5.0 and 7.5) on emulsions stabilized by chickpea-based emulsions at two different protein concentration (2.0 and 4.0 wt%). Microstructure of chickpea-based emulsions is assessed by means of backscattering, droplet size distributions and small amplitude oscillatory shear measurements. Visual appearances as well as confocal laser scanning microscopy images are obtained to provide useful information on the emulsions structure. Interestingly, results indicate that the pH value and protein concentration have a strong influence on emulsion microstructure and stability. Thus, the system which contains protein surfaces positively charged shows the highest viscoelastic properties, a good droplet size distribution profile and non-apparent destabilization phenomena. Interestingly, results also reveal the importance of rheological measurements in the prediction of protein interactions and emulsion stability since this technique is able to predict destabilization mechanisms sooner than other techniques such as backscattering or droplet size distribution measurements.

Protein Structure Prediction by Protein Threading

Science.gov (United States)

Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme.

Science.gov (United States)

Pelay-Gimeno, Marta; Bange, Tanja; Hennig, Sven; Grossmann, Tom N

2018-05-30

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. So far, macrocyclization approaches utilize a very limited structural diversity which complicates the design process. Here, we report an approach that enables cyclization via the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface exposed cysteines which are reacted with a triselectrophile resulting in the in situ cylization of the protein (INCYPRO). A bicyclic version of Sortase A was designed exhibiting increased tolerance towards thermal as well as chemical denaturation, and proved efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain resulting in up to 24 °C increased thermal stability. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stability of halophilic proteins: from dipeptide attributes to discrimination classifier.

Science.gov (United States)

Zhang, Guangya; Huihua, Ge; Yi, Lin

2013-02-01

To investigate the molecular features responsible for protein halophilicity is of great significance for understanding the structure basis of protein halo-stability and would help to develop a practical strategy for designing halophilic proteins. In this work, we have systematically analyzed the dipeptide composition of the halophilic and non-halophilic protein sequences. We observed the halophilic proteins contained more DA, RA, AD, RR, AP, DD, PD, EA, VG and DV at the expense of LK, IL, II, IA, KK, IS, KA, GK, RK and AI. We identified some macromolecular signatures of halo-adaptation, and thought the dipeptide composition might contain more information than amino acid composition. Based on the dipeptide composition, we have developed a machine learning method for classifying halophilic and non-halophilic proteins for the first time. The accuracy of our method for the training dataset was 100.0%, and for the 10-fold cross-validation was 93.1%. We also discussed the influence of some specific dipeptides on prediction accuracy. Copyright © 2012 Elsevier B.V. All rights reserved.

The construction of an amino acid network for understanding protein structure and function.

Science.gov (United States)

Yan, Wenying; Zhou, Jianhong; Sun, Maomin; Chen, Jiajia; Hu, Guang; Shen, Bairong

2014-06-01

Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein-protein interactions, and for understanding communication within and between proteins.

Testing the ability of non-methylamine osmolytes present in kidney cells to counteract the deleterious effects of urea on structure, stability and function of proteins.

Directory of Open Access Journals (Sweden)

Sheeza Khan

Full Text Available Human kidney cells are under constant urea stress due to its urine concentrating mechanism. It is believed that the deleterious effect of urea is counteracted by methylamine osmolytes (glycine betaine and glycerophosphocholine present in kidney cells. A question arises: Do the stabilizing osmolytes, non-methylamines (myo-inositol, sorbitol and taurine present in the kidney cells also counteract the deleterious effects of urea? To answer this question, we have measured structure, thermodynamic stability (ΔG D (o and functional activity parameters (K m and k cat of different model proteins in the presence of various concentrations of urea and each non-methylamine osmolyte alone and in combination. We observed that (i for each protein myo-inositol provides perfect counteraction at 1∶2 ([myo-inositol]:[urea] ratio, (ii any concentration of sorbitol fails to refold urea denatured proteins if it is six times less than that of urea, and (iii taurine regulates perfect counteraction in a protein specific manner; 1.5∶2.0, 1.2∶2.0 and 1.0∶2.0 ([taurine]:[urea] ratios for RNase-A, lysozyme and α-lactalbumin, respectively.

Relative Stabilities of Conserved and Non-Conserved Structures in the OB-Fold Superfamily

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Andrei T. Alexandrescu

2009-05-01

Full Text Available The OB-fold is a diverse structure superfamily based on a β-barrel motif that is often supplemented with additional non-conserved secondary structures. Previous deletion mutagenesis and NMR hydrogen exchange studies of three OB-fold proteins showed that the structural stabilities of sites within the conserved β-barrels were larger than sites in non-conserved segments. In this work we examined a database of 80 representative domain structures currently classified as OB-folds, to establish the basis of this effect. Residue-specific values were obtained for the number of Cα-Cα distance contacts, sequence hydrophobicities, crystallographic B-factors, and theoretical B-factors calculated from a Gaussian Network Model. All four parameters point to a larger average flexibility for the non-conserved structures compared to the conserved β-barrels. The theoretical B-factors and contact densities show the highest sensitivity.Our results suggest a model of protein structure evolution in which novel structural features develop at the periphery of conserved motifs. Core residues are more resistant to structural changes during evolution since their substitution would disrupt a larger number of interactions. Similar factors are likely to account for the differences in stability to unfolding between conserved and non-conserved structures.

Protein secondary structure: category assignment and predictability

DEFF Research Database (Denmark)

Andersen, Claus A.; Bohr, Henrik; Brunak, Søren

2001-01-01

In the last decade, the prediction of protein secondary structure has been optimized using essentially one and the same assignment scheme known as DSSP. We present here a different scheme, which is more predictable. This scheme predicts directly the hydrogen bonds, which stabilize the secondary......-forward neural network with one hidden layer on a data set identical to the one used in earlier work....

Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization

DEFF Research Database (Denmark)

Fonte, Pedro; Araújo, Francisca; Seabra, Vítor

2015-01-01

The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol...... formulations with externally added lyoprotectants, except trehalose, showed crystallinity. FTIR assessment showed that co-encapsulating lyoprotectants better preserved insulin structure upon lyophilization with a spectral area overlap of 82-87%, compared to only 72% in lyoprotectant absence. These results were...... confirmed by circular dichroism spectroscopy. Surprisingly, the simultaneous co-encapsulation and addition of lyoprotectants was detrimental to protein stabilization. The insulin in vitro release studies demonstrated that formulations with co-encapsulated trehalose, glucose, sucrose, fructose and sorbitol...

Structural deformation upon protein-protein interaction: a structural alphabet approach.

Science.gov (United States)

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-02-28

In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

Structural deformation upon protein-protein interaction: A structural alphabet approach

Directory of Open Access Journals (Sweden)

Lecornet Hélène

2008-02-01

Full Text Available Abstract Background In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. Results In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%. This proportion is even greater in the interface regions (41%. Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Conclusion Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

Science.gov (United States)

Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

2015-07-01

In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. © 2014 Wiley Periodicals, Inc.

Structural basis of thermal stability of the tungsten cofactor synthesis protein MoaB from Pyrococcus furiosus.

Directory of Open Access Journals (Sweden)

Nastassia Havarushka

Full Text Available Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15 °C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50 °C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.

A novel mechanism for antiglycative action of limonene through stabilization of protein conformation.

Science.gov (United States)

Joglekar, Madhav M; Panaskar, Shrimant N; Chougale, Ashok D; Kulkarni, Mahesh J; Arvindekar, Akalpita U

2013-10-01

Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 μM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to β-sheet transition wherein 18.5% β-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.

Effect of ionic liquid on activity, stability, and structure of enzymes: a review.

Science.gov (United States)

Naushad, Mu; Alothman, Zied Abdullah; Khan, Abbul Bashar; Ali, Maroof

2012-11-01

Ionic liquids have shown their potential as a solvent media for many enzymatic reactions as well as protein preservation, because of their unusual characteristics. It is also observed that change in cation or anion alters the physiochemical properties of the ionic liquids, which in turn influence the enzymatic reactions by altering the structure, activity, enatioselectivity, and stability of the enzymes. Thus, it is utmost need of the researchers to have full understanding of these influences created by ionic liquids before choosing or developing an ionic liquid to serve as solvent media for enzymatic reaction or protein preservation. So, in the present review, we try to shed light on effects of ionic liquids chemistry on structure, stability, and activity of enzymes, which will be helpful for the researchers in various biocatalytic applications. Copyright © 2012. Published by Elsevier B.V.

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

Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

Science.gov (United States)

Gorinstein, S; Zemser, M; Vargas-Albores, F; Ochoa, J L; Paredes-Lopez, O; Scheler, C; Aksu, S; Salnikow, J

1999-02-01

Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.

Tensegrity structures form, stability, and symmetry

CERN Document Server

Zhang, Jing Yao

2015-01-01

To facilitate a deeper understanding of tensegrity structures, this book focuses on their two key design problems: self-equilibrium analysis and stability investigation. In particular, high symmetry properties of the structures are extensively utilized. Conditions for self-equilibrium as well as super-stability of tensegrity structures are presented in detail. An analytical method and an efficient numerical method are given for self-equilibrium analysis of tensegrity structures: the analytical method deals with symmetric structures and the numerical method guarantees super-stability. Utilizing group representation theory, the text further provides analytical super-stability conditions for the structures that are of dihedral as well as tetrahedral symmetry. This book not only serves as a reference for engineers and scientists but is also a useful source for upper-level undergraduate and graduate students. Keeping this objective in mind, the presentation of the book is self-contained and detailed, with an abund...

Entropic formulation for the protein folding process: Hydrophobic stability correlates with folding rates

Science.gov (United States)

Dal Molin, J. P.; Caliri, A.

2018-01-01

possible connection between the hydrophobic component of protein stability and the native structural topology. We simulated those same 200 targets again with the Mq A, only. However, this time we evaluated the relative frequency {ϕq } in which each target visits its corresponding native structure along an appropriate simulation time. Due to the presence of the hydrophobic effect in our approach we obtained a strong correlation between the stability and the folding rate (R = 0 . 85). So, as faster a sequence found its target, as larger is the hydrophobic component of its stability. The strong correlation fulfills our last goal. This final finding suggests that the hydrophobic effect could not be a general stabilizing factor for proteins.

An Improved Methodology for Multidimensional High-Throughput Preformulation Characterization of Protein Conformational Stability

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Maddux, Nathaniel R.; Rosen, Ilan T.; Hu, Lei; Olsen, Christopher M.; Volkin, David B.; Middaugh, C. Russell

2013-01-01

The Empirical Phase Diagram (EPD) technique is a vector-based multidimensional analysis method for summarizing large data sets from a variety of biophysical techniques. It can be used to provide comprehensive preformulation characterization of a macromolecule’s higher-order structural integrity and conformational stability. In its most common mode, it represents a type of stimulus-response diagram using environmental variables such as temperature, pH, and ionic strength as the stimulus, with alterations in macromolecular structure being the response. Until now EPD analysis has not been available in a high throughput mode because of the large number of experimental techniques and environmental stressor/stabilizer variables typically employed. A new instrument has been developed that combines circular dichroism, UV-absorbance, fluorescence spectroscopy and light scattering in a single unit with a 6-position temperature controlled cuvette turret. Using this multifunctional instrument and a new software system we have generated EPDs for four model proteins. Results confirm the reproducibility of the apparent phase boundaries and protein behavior within the boundaries. This new approach permits two EPDs to be generated per day using only 0.5 mg of protein per EPD. Thus, the new methodology generates reproducible EPDs in high-throughput mode, and represents the next step in making such determinations more routine. PMID:22447621

A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis.

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Leslie Goo

2017-02-01

Full Text Available The structural flexibility or 'breathing' of the envelope (E protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F of the West Nile virus (WNV E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV, but not Zika virus (ZIKV, E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing.

Hemoglobin bioconjugates with surface-protected gold nanoparticles in aqueous media: The stability depends on solution pH and protein properties.

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Del Caño, Rafael; Mateus, Lucia; Sánchez-Obrero, Guadalupe; Sevilla, José Manuel; Madueño, Rafael; Blázquez, Manuel; Pineda, Teresa

2017-11-01

The identification of the factors that dictate the formation and physicochemical properties of protein-nanomaterial bioconjugates are important to understand their behavior in biological systems. The present work deals with the formation and characterization of bioconjugates made of the protein hemoglobin (Hb) and gold nanoparticles (AuNP) capped with three different molecular layers (citrate anions (c), 6-mercaptopurine (MP) and ω-mercaptoundecanoic acid (MUA)). The main focus is on the behavior of the bioconjugates in aqueous buffered solutions in a wide pH range. The stability of the bioconjugates have been studied by UV-visible spectroscopy by following the changes in the localized surface resonance plasmon band (LSRP), Dynamic light scattering (DLS) and zeta-potential pH titrations. It has been found that they are stable in neutral and alkaline solutions and, at pH lower than the protein isoelectric point, aggregation takes place. Although the surface chemical properties of the AuNPs confer different properties in respect to colloidal stability, once the bioconjugates are formed their properties are dictated by the Hb protein corona. The protein secondary structure, as analyzed by Attenuated total reflectance infrared (ATR-IR) spectroscopy, seems to be maintained under the conditions of colloidal stability but some small changes in protein conformation take place when the bioconjugates aggregate. These findings highlight the importance to keep the protein structure upon interaction with nanomaterials to drive the stability of the bioconjugates. Copyright © 2017 Elsevier Inc. All rights reserved.

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

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Vriend, Gert; Eijsink, Vincent

1993-08-01

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cercus. Several new techniques have been developed to improve the model-building procedures. Also a model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability

Structure of the Z Ring-associated Protein, ZapD, Bound to the C-terminal Domain of the Tubulin-like Protein, FtsZ, Suggests Mechanism of Z Ring Stabilization through FtsZ Cross-linking.

Science.gov (United States)

Schumacher, Maria A; Huang, Kuo-Hsiang; Zeng, Wenjie; Janakiraman, Anuradha

2017-03-03

Cell division in most bacteria is mediated by the tubulin-like FtsZ protein, which polymerizes in a GTP-dependent manner to form the cytokinetic Z ring. A diverse repertoire of FtsZ-binding proteins affects FtsZ localization and polymerization to ensure correct Z ring formation. Many of these proteins bind the C-terminal domain (CTD) of FtsZ, which serves as a hub for FtsZ regulation. FtsZ ring-associated proteins, ZapA-D (Zaps), are important FtsZ regulatory proteins that stabilize FtsZ assembly and enhance Z ring formation by increasing lateral assembly of FtsZ protofilaments, which then form the Z ring. There are no structures of a Zap protein bound to FtsZ; therefore, how these proteins affect FtsZ polymerization has been unclear. Recent data showed ZapD binds specifically to the FtsZ CTD. Thus, to obtain insight into the ZapD-CTD interaction and how it may mediate FtsZ protofilament assembly, we determined the Escherichia coli ZapD-FtsZ CTD structure to 2.67 Å resolution. The structure shows that the CTD docks within a hydrophobic cleft in the ZapD helical domain and adopts an unusual structure composed of two turns of helix separated by a proline kink. FtsZ CTD residue Phe-377 inserts into the ZapD pocket, anchoring the CTD in place and permitting hydrophobic contacts between FtsZ residues Ile-374, Pro-375, and Leu-378 with ZapD residues Leu-74, Trp-77, Leu-91, and Leu-174. The structural findings were supported by mutagenesis coupled with biochemical and in vivo studies. The combined data suggest that ZapD acts as a molecular cross-linking reagent between FtsZ protofilaments to enhance FtsZ assembly. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cross-linking proteins by laccase: Effects on the droplet size and rheology of emulsions stabilized by sodium caseinate.

Science.gov (United States)

Sato, A C K; Perrechil, F A; Costa, A A S; Santana, R C; Cunha, R L

2015-09-01

The aim of this work was to evaluate the influence of laccase and ferulic acid on the characteristics of oil-in-water emulsions stabilized by sodium caseinate at different pH (3, 5 and 7). Emulsions were prepared by high pressure homogenization of soybean oil with sodium caseinate solution containing varied concentrations of laccase (0, 1 and 5mg/mL) and ferulic acid (5 and 10mM). Laccase treatment and pH exerted a strong influence on the properties with a consequent effect on stability, structure and rheology of emulsions stabilized by Na-caseinate. At pH7, O/W emulsions were kinetically stable due to the negative protein charge which enabled electrostatic repulsion between oil droplets resulting in an emulsion with small droplet size, low viscosity, pseudoplasticity and viscoelastic properties. The laccase treatment led to emulsions showing shear-thinning behavior as a result of a more structured system. O/W emulsions at pH5 and 3 showed phase separation due to the proximity to protein pI, but the laccase treatment improved their stability of emulsions especially at pH3. At pH3, the addition of ferulic acid and laccase produced emulsions with larger droplet size but with narrower droplet size distribution, increased viscosity, pseudoplasticity and viscoelastic properties (gel-like behavior). Comparing laccase treatments, the combined addition of laccase and ferulic acid generally produced emulsions with lower stability (pH5), larger droplet size (pH3, 5 and 7) and higher pseudoplasticity (pH5 and 7) than emulsion with only ferulic acid. The results suggested that the cross-linking of proteins by laccase and ferulic acid improved protein emulsifying properties by changing functional mechanisms of the protein on emulsion structure and rheology, showing that sodium caseinate can be successfully used in acid products when treated with laccase. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study.

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Chi-Wen Lee

Full Text Available Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα-Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K-D49, E96R-D28, E96K-D28, S440K-E70, T231K-D388, and Q277E-D282 was detected, respectively. Reversing the polarity of T231K-D388 to T231D-D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K generated a melting temperature increase of 15.7°C. Thus, this study

Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

Science.gov (United States)

Sides, Cynthia R.; Liyanage, Rohana; Lay, Jackson O.; Philominathan, Sagaya Theresa Leena; Matsushita, Osamu; Sakon, Joshua

2012-03-01

Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.

Lipid nanotechnologies for structural studies of membrane-associated proteins.

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Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

2014-11-01

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

Science.gov (United States)

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

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Czaplicki Jerzy

2004-07-01

Full Text Available Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends.

Science.gov (United States)

Konishi, Akimitsu; Izumi, Takashi; Shimizu, Shigeomi

2016-09-23

Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends*

Science.gov (United States)

Izumi, Takashi; Shimizu, Shigeomi

2016-01-01

Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. PMID:27514743

Hydrolytic catalysis and structural stabilization in a designed metalloprotein

Science.gov (United States)

Zastrow, Melissa L.; Peacock, Anna F. A.; Stuckey, Jeanne A.; Pecoraro, Vincent L.

2011-01-01

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions – a Zn(II) ion which is important for catalytic activity and a Hg(II) ion which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate hydrolysis (pNPA) to within ~100-fold of the efficiency of human carbonic anhydrase (CA)II and is at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. While histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme uncovers necessary design features for future metalloenzymes containing one or more metals. PMID:22270627

Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies.

Science.gov (United States)

Scott, Daniel J; Kummer, Lutz; Egloff, Pascal; Bathgate, Ross A D; Plückthun, Andreas

2014-11-01

The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein adsorption at the electrified air-water interface: implications on foam stability.

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Engelhardt, Kathrin; Rumpel, Armin; Walter, Johannes; Dombrowski, Jannika; Kulozik, Ulrich; Braunschweig, Björn; Peukert, Wolfgang

2012-05-22

The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.

Getting to the core of protein pharmaceuticals – comprehensive structure analysis by mass spectrometry

DEFF Research Database (Denmark)

Leurs, Ulrike; Mistarz, Ulrik Hvid; Rand, Kasper Dyrberg

2015-01-01

. Mass spectrometry has evolved as a powerful tool for the characterization of both primary and higher order structures of protein pharmaceuticals. Furthermore, the chemical and physical stability of protein drugs, as well as their pharmacokinetics are nowadays routinely determined by mass spectrometry...

Why Is There a Glass Ceiling for Threading Based Protein Structure Prediction Methods?

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Skolnick, Jeffrey; Zhou, Hongyi

2017-04-20

Despite their different implementations, comparison of the best threading approaches to the prediction of evolutionary distant protein structures reveals that they tend to succeed or fail on the same protein targets. This is true despite the fact that the structural template library has good templates for all cases. Thus, a key question is why are certain protein structures threadable while others are not. Comparison with threading results on a set of artificial sequences selected for stability further argues that the failure of threading is due to the nature of the protein structures themselves. Using a new contact map based alignment algorithm, we demonstrate that certain folds are highly degenerate in that they can have very similar coarse grained fractions of native contacts aligned and yet differ significantly from the native structure. For threadable proteins, this is not the case. Thus, contemporary threading approaches appear to have reached a plateau, and new approaches to structure prediction are required.

Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization

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Ding Wei

2010-11-01

Full Text Available Abstract Background Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. Results In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. Conclusion Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples.

Effect of drying methods on the structure, thermo and functional properties of fenugreek (Trigonella foenum graecum) protein isolate.

Science.gov (United States)

Feyzi, Samira; Varidi, Mehdi; Zare, Fatemeh; Varidi, Mohammad Javad

2018-03-01

Different drying methods due to protein denaturation could alter the functional properties of proteins, as well as their structure. So, this study focused on the effect of different drying methods on amino acid content, thermo and functional properties, and protein structure of fenugreek protein isolate. Freeze and spray drying methods resulted in comparable protein solubility, dynamic surface and interfacial tensions, foaming and emulsifying properties except for emulsion stability. Vacuum oven drying promoted emulsion stability, surface hydrophobicity and viscosity of fenugreek protein isolate at the expanse of its protein solubility. Vacuum oven process caused a higher level of Maillard reaction followed by the spray drying process, which was confirmed by the lower amount of lysine content and less lightness, also more browning intensity. ΔH of fenugreek protein isolates was higher than soy protein isolate, which confirmed the presence of more ordered structures. Also, the bands which are attributed to the α-helix structures in the FTIR spectrum were in the shorter wave number region for freeze and spray dried fenugreek protein isolates that show more possibility of such structures. This research suggests that any drying method must be conducted in its gentle state in order to sustain native structure of proteins and promote their functionalities. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Stability of bulk metallic glass structure

Energy Technology Data Exchange (ETDEWEB)

Jain, H.; Williams, D.B.

2003-06-18

The fundamental origins of the stability of the (Pd-Ni){sub 80}P{sub 20} bulk metallic glasses (BMGs), a prototype for a whole class of BMG formers, were explored. While much of the properties of their BMGs have been characterized, their glass-stability have not been explained in terms of the atomic and electronic structure. The local structure around all three constituent atoms was obtained, in a complementary way, using extended X-ray absorption fine structure (EXAFS), to probe the nearest neighbor environment of the metals, and extended energy loss fine structure (EXELFS), to investigate the environment around P. The occupied electronic structure was investigated using X-ray photoelectron spectroscopy (XPS). The (Pd-Ni){sub 80}P{sub 20} BMGs receive their stability from cumulative, and interrelated, effects of both atomic and electronic origin. The stability of the (Pd-Ni){sub 80}P{sub 20} BMGs can be explained in terms of the stability of Pd{sub 60}Ni{sub 20}P{sub 20} and Pd{sub 30}Ni{sub 50}P{sub 20}, glasses at the end of BMG formation. The atomic structure in these alloys is very similar to those of the binary phosphide crystals near x=0 and x=80, which are trigonal prisms of Pd or Ni atoms surrounding P atoms. Such structures are known to exist in dense, randomly-packed systems. The structure of the best glass former in this series, Pd{sub 40}Ni{sub 40}P{sub 20} is further described by a weighted average of those of Pd{sub 30}Ni{sub 50}P{sub 20} and Pd{sub 60}Ni{sub 20}P{sub 20}. Bonding states present only in the ternary alloys were found and point to a further stabilization of the system through a negative heat of mixing between Pd and Ni atoms. The Nagel and Tauc criterion, correlating a decrease in the density of states at the Fermi level with an increase in the glass stability, was consistent with greater stability of the Pd{sub x}Ni{sub (80-x)}P{sub 20} glasses with respect to the binary alloys of P. A valence electron concentration of 1.8 e/a, which

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

Energy Technology Data Exchange (ETDEWEB)

Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

2007-01-01

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

Investigating homology between proteins using energetic profiles.

Science.gov (United States)

Wrabl, James O; Hilser, Vincent J

2010-03-26

Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may

Investigating homology between proteins using energetic profiles.

Directory of Open Access Journals (Sweden)

James O Wrabl

2010-03-01

Full Text Available Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved

Trade-off between positive and negative design of protein stability: from lattice models to real proteins.

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Orly Noivirt-Brik

2009-12-01

Full Text Available Two different strategies for stabilizing proteins are (i positive design in which the native state is stabilized and (ii negative design in which competing non-native conformations are destabilized. Here, the circumstances under which one strategy might be favored over the other are explored in the case of lattice models of proteins and then generalized and discussed with regard to real proteins. The balance between positive and negative design of proteins is found to be determined by their average "contact-frequency", a property that corresponds to the fraction of states in the conformational ensemble of the sequence in which a pair of residues is in contact. Lattice model proteins with a high average contact-frequency are found to use negative design more than model proteins with a low average contact-frequency. A mathematical derivation of this result indicates that it is general and likely to hold also for real proteins. Comparison of the results of correlated mutation analysis for real proteins with typical contact-frequencies to those of proteins likely to have high contact-frequencies (such as disordered proteins and proteins that are dependent on chaperonins for their folding indicates that the latter tend to have stronger interactions between residues that are not in contact in their native conformation. Hence, our work indicates that negative design is employed when insufficient stabilization is achieved via positive design owing to high contact-frequencies.

Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

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Kapoor, Abhijeet; Travesset, Alex

2014-03-01

We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

Stability Analysis and Stabilization of Miduk Heap Leaching Structure, Iran

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Mehdi Amini

2013-06-01

Full Text Available To construct copper heap leaching structures, a stepped heap of ore is placed over an isolated sloping surface and then washed with sulphuric acid. The isolated bed of such a heap consists of some natural and geosynthetic layers. Shear strength parameters between these layers are low, so they form the possible sliding surfaces of the heaps. Economic and environmental considerations call for studying such slides. In this study, firstly, results of the laboratory tests carried on the materials of the heap leaching structures bed are presented. Then, the instability mechanisms of such structures are investigated and proper approaches are summarized for their stabilization. Finally, stability of the Miduk copper heap is evaluated as a case history, and appropriate approaches and their effects are discussed for its stabilization.

Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding.

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Samanta, Sudipta; Mukherjee, Sanchita

2017-10-01

The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

Science.gov (United States)

Samanta, Sudipta; Mukherjee, Sanchita

2017-10-01

The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

Ultra-High Pressure Homogenization improves oxidative stability and interfacial properties of soy protein isolate-stabilized emulsions.

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Fernandez-Avila, C; Trujillo, A J

2016-10-15

Ultra-High Pressure Homogenization (100-300MPa) has great potential for technological, microbiological and nutritional aspects of fluid processing. Its effect on the oxidative stability and interfacial properties of oil-in-water emulsions prepared with 4% (w/v) of soy protein isolate and soybean oil (10 and 20%, v/v) were studied and compared to emulsions treated by conventional homogenization (15MPa). Emulsions were characterized by particle size, emulsifying activity index, surface protein concentration at the interface and by transmission electron microscopy. Primary and secondary lipid oxidation products were evaluated in emulsions upon storage. Emulsions with 20% oil treated at 100 and 200MPa exhibited the most oxidative stability due to higher amount of oil and protein surface load at the interface. This manuscript addresses the improvement in oxidative stability in emulsions treated by UHPH when compared to conventional emulsions. Copyright © 2016 Elsevier Ltd. All rights reserved.

DNA nanotubes for NMR structure determination of membrane proteins.

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

2013-04-01

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

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

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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.

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

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Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2016-03-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

Trimethylamine N-oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine

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Liao, Yi-Ting; Manson, Anthony C.; DeLyser, Michael R.; Noid, William G.; Cremer, Paul S.

2017-01-01

We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air–water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer–water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins. PMID:28228526

Functional structural motifs for protein-ligand, protein-protein, and protein-nucleic acid interactions and their connection to supersecondary structures.

Science.gov (United States)

Kinjo, Akira R; Nakamura, Haruki

2013-01-01

Protein functions are mediated by interactions between proteins and other molecules. One useful approach to analyze protein functions is to compare and classify the structures of interaction interfaces of proteins. Here, we describe the procedures for compiling a database of interface structures and efficiently comparing the interface structures. To do so requires a good understanding of the data structures of the Protein Data Bank (PDB). Therefore, we also provide a detailed account of the PDB exchange dictionary necessary for extracting data that are relevant for analyzing interaction interfaces and secondary structures. We identify recurring structural motifs by classifying similar interface structures, and we define a coarse-grained representation of supersecondary structures (SSS) which represents a sequence of two or three secondary structure elements including their relative orientations as a string of four to seven letters. By examining the correspondence between structural motifs and SSS strings, we show that no SSS string has particularly high propensity to be found interaction interfaces in general, indicating any SSS can be used as a binding interface. When individual structural motifs are examined, there are some SSS strings that have high propensity for particular groups of structural motifs. In addition, it is shown that while the SSS strings found in particular structural motifs for nonpolymer and protein interfaces are as abundant as in other structural motifs that belong to the same subunit, structural motifs for nucleic acid interfaces exhibit somewhat stronger preference for SSS strings. In regard to protein folds, many motif-specific SSS strings were found across many folds, suggesting that SSS may be a useful description to investigate the universality of ligand binding modes.

Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

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Miller, Craig R; Lee, Kuo Hao; Wichman, Holly A; Ytreberg, F Marty

2014-01-01

Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.

A specific role for the ZipA protein in cell division: stabilization of the FtsZ protein.

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Pazos, Manuel; Natale, Paolo; Vicente, Miguel

2013-02-01

In Escherichia coli, the cell division protein FtsZ is anchored to the cytoplasmic membrane by the action of the bitopic membrane protein ZipA and the cytoplasmic protein FtsA. Although the presence of both ZipA and FtsA is strictly indispensable for cell division, an FtsA gain-of-function mutant FtsA* (R286W) can bypass the ZipA requirement for cell division. This observation casts doubts on the role of ZipA and its need for cell division. Maxicells are nucleoid-free bacterial cells used as a whole cell in vitro system to probe protein-protein interactions without the need of protein purification. We show that ZipA protects FtsZ from the ClpXP-directed degradation observed in E. coli maxicells and that ZipA-stabilized FtsZ forms membrane-attached spiral-like structures in the bacterial cytoplasm. The overproduction of the FtsZ-binding ZipA domain is sufficient to protect FtsZ from degradation, whereas other C-terminal ZipA partial deletions lacking it are not. Individual overproduction of the proto-ring component FtsA or its gain-of-function mutant FtsA* does not result in FtsZ protection. Overproduction of FtsA or FtsA* together with ZipA does not interfere with the FtsZ protection. Moreover, neither FtsA nor FtsA* protects FtsZ when overproduced together with ZipA mutants lacking the FZB domain. We propose that ZipA protects FtsZ from degradation by ClpP by making the FtsZ site of interaction unavailable to the ClpX moiety of the ClpXP protease. This role cannot be replaced by either FtsA or FtsA*, suggesting a unique function for ZipA in proto-ring stability.

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

Science.gov (United States)

De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

2018-01-01

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

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DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J. (Tennessee-HSC); (SJCH)

2012-12-10

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

Directory of Open Access Journals (Sweden)

Barna Dey

2009-05-01

Full Text Available The human immunodeficiency virus type 1 (HIV-1 exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

Science.gov (United States)

Zavodszky, M; Chen, C W; Huang, J K; Zolkiewski, M; Wen, L; Krishnamoorthi, R

2001-01-01

bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.

Sensitivity of system stability to model structure

Science.gov (United States)

Hosack, G.R.; Li, H.W.; Rossignol, P.A.

2009-01-01

A community is stable, and resilient, if the levels of all community variables can return to the original steady state following a perturbation. The stability properties of a community depend on its structure, which is the network of direct effects (interactions) among the variables within the community. These direct effects form feedback cycles (loops) that determine community stability. Although feedback cycles have an intuitive interpretation, identifying how they form the feedback properties of a particular community can be intractable. Furthermore, determining the role that any specific direct effect plays in the stability of a system is even more daunting. Such information, however, would identify important direct effects for targeted experimental and management manipulation even in complex communities for which quantitative information is lacking. We therefore provide a method that determines the sensitivity of community stability to model structure, and identifies the relative role of particular direct effects, indirect effects, and feedback cycles in determining stability. Structural sensitivities summarize the degree to which each direct effect contributes to stabilizing feedback or destabilizing feedback or both. Structural sensitivities prove useful in identifying ecologically important feedback cycles within the community structure and for detecting direct effects that have strong, or weak, influences on community stability. The approach may guide the development of management intervention and research design. We demonstrate its value with two theoretical models and two empirical examples of different levels of complexity. ?? 2009 Elsevier B.V. All rights reserved.

Laccases stabilization with phosphatidylcholine liposomes

OpenAIRE

Martí, M.; Zille, Andrea; Paulo, Artur Cavaco; Parra, J. L.; Coderch, L.

2012-01-01

In recent years, there has been an upsurge of interest in enzyme treatment of textile fibres. Enzymes are globular proteins whose catalytic function is due to their three dimensional structure. For this reason, stability strategies make use of compounds that avoid dismantling or distorting protein 3D structures. This study is concerned with the use of microencapsulation techniques to optimize enzyme stabilization. Laccases were embedded in phophatidylcholine liposomes and their encaps...

Structure and stability of complexes of agmatine with some functional receptor residues of proteins

Science.gov (United States)

Remko, Milan; Broer, Ria; Remková, Anna; Van Duijnen, Piet Th.

2017-04-01

The paper reports the results of a theoretical study of the conformational behavior and basicity of biogenic amine agmatine. The complexes modelling of agmatine - protein interaction are also under scrutiny of our investigation using the Becke3LYP and B97D levels of the density functional theory. The relative stabilities (Gibbs energies) of individual complexes are by both DFT methods described equally. Hydration has a dramatic effect on the hydrogen bonded complexes studied. The pairing acidic carboxylate group with different agmatine species resulted in charged hydrogen bond complexes containing negatively charged acetate species acting as proton acceptors.

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

Science.gov (United States)

Sinha, Rajeshwari; Khare, Sunil K.

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants. PMID:24782853

Structural stability of nonlinear population dynamics.

Science.gov (United States)

Cenci, Simone; Saavedra, Serguei

2018-01-01

In population dynamics, the concept of structural stability has been used to quantify the tolerance of a system to environmental perturbations. Yet, measuring the structural stability of nonlinear dynamical systems remains a challenging task. Focusing on the classic Lotka-Volterra dynamics, because of the linearity of the functional response, it has been possible to measure the conditions compatible with a structurally stable system. However, the functional response of biological communities is not always well approximated by deterministic linear functions. Thus, it is unclear the extent to which this linear approach can be generalized to other population dynamics models. Here, we show that the same approach used to investigate the classic Lotka-Volterra dynamics, which is called the structural approach, can be applied to a much larger class of nonlinear models. This class covers a large number of nonlinear functional responses that have been intensively investigated both theoretically and experimentally. We also investigate the applicability of the structural approach to stochastic dynamical systems and we provide a measure of structural stability for finite populations. Overall, we show that the structural approach can provide reliable and tractable information about the qualitative behavior of many nonlinear dynamical systems.

Structural stability of nonlinear population dynamics

Science.gov (United States)

Cenci, Simone; Saavedra, Serguei

2018-01-01

In population dynamics, the concept of structural stability has been used to quantify the tolerance of a system to environmental perturbations. Yet, measuring the structural stability of nonlinear dynamical systems remains a challenging task. Focusing on the classic Lotka-Volterra dynamics, because of the linearity of the functional response, it has been possible to measure the conditions compatible with a structurally stable system. However, the functional response of biological communities is not always well approximated by deterministic linear functions. Thus, it is unclear the extent to which this linear approach can be generalized to other population dynamics models. Here, we show that the same approach used to investigate the classic Lotka-Volterra dynamics, which is called the structural approach, can be applied to a much larger class of nonlinear models. This class covers a large number of nonlinear functional responses that have been intensively investigated both theoretically and experimentally. We also investigate the applicability of the structural approach to stochastic dynamical systems and we provide a measure of structural stability for finite populations. Overall, we show that the structural approach can provide reliable and tractable information about the qualitative behavior of many nonlinear dynamical systems.

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14.

Science.gov (United States)

Schwaighofer, Andreas; Pechlaner, Maria; Oostenbrink, Chris; Kotlowski, Caroline; Araman, Can; Mastrogiacomo, Rosa; Pelosi, Paolo; Knoll, Wolfgang; Nowak, Christoph; Larisika, Melanie

2014-04-18

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant. Copyright © 2014 Elsevier Inc. All rights reserved.

Stability of integral membrane proteins under high hydrostatic pressure: the LH2 and LH3 antenna pigment-protein complexes from photosynthetic bacteria.

Science.gov (United States)

Kangur, Liina; Timpmann, Kõu; Freiberg, Arvi

2008-07-03

The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure

The Escherichia coli antiterminator protein BglG stabilizes the 5 ...

Indian Academy of Sciences (India)

Unknown

Keywords. Antitermination; mRNA stability; RNA binding protein ... factor, Rho, and the pBR322 copy number protein, Rop, have been .... Transcription analysis using the oligo- ..... Retarded RNA turnover in Escherichia coli a means of main-.

Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

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Chi-Ho Chan

Full Text Available Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p, leading to the up-shifting and broadening of the protein stability curves.

Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization

DEFF Research Database (Denmark)

Fonte, Pedro; Andrade, Fernanda; Azevedo, Cláudia

2016-01-01

, sucrose and sorbitol as cryoprotectants was evaluated. METHODS: Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at -80°C, in liquid nitrogen, or ramped cooling at -40°C. Upon lyophilization, the stability of protein structure...

The role of milk proteins in the structure formation of dairy products

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Olga Rybak

2015-04-01

Full Text Available Introduction. The structure of dairy products is a complex of proteins, fat, minerals and water that determines the texture and sensory properties of the product. Material and methods. The fermented milks (using the example of yogurt, cheese, ice cream, aerated milk and frozen fruit desserts have been researched. Scientific articles, published during 2000 and 2014 years, as well as theses and monographs of dairy science have been analysed too. Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis. Results and discussion. The scientific understanding of the milk proteins’ role in the structure formation of dairy product has been summarized. Negligible changes of structure as a result of compositional or technological changes can lead to shifts in the stability, texture and rheology of products, which are closely related to each other. The allowance of these properties has significant influence on the manufacturing. Acid coagulation is a major functional property of milk proteins, which used in the structure formation of cheese and fermented dairy products. However, the form and properties of milk curd depend on the heat treatment of milk before fermentation. Milk proteins exhibit other functional properties (emulsification and partial coalescence of fat globules, aeration and foam stability during a churning, viscosity increasing of external phase in the development of structure in the ice cream, aerated milk and frozen fruit desserts. Conclusions. It is expedient to use results into a further study of the structure formation mechanism of dairy products and the development of recommendations in order to an efficient production.

The role of milk proteins in the structure formation of dairy products

Directory of Open Access Journals (Sweden)

Olga Rybak

2014-09-01

Full Text Available Introduction. The structure of dairy products is a complex of proteins, fat, minerals and water that determines the texture and sensory properties of the product. Material and methods. The fermented milks (using the example of yogurt, cheese, ice cream, aerated milk and frozen fruit desserts have been researched. Scientific articles, published during 2000 and 2014 years, as well as theses and monographs of dairy science have been analysed too. Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis. Results and discussion. The scientific understanding of the milk proteins’ role in the structure formation of dairy product has been summarized. Negligible changes of structure as a result of compositional or technological changes can lead to shifts in the stability, texture and rheology of products, which are closely related to each other. The allowance of these properties has significant influence on the manufacturing. Acid coagulation is a major functional property of milk proteins, which used in the structure formation of cheese and fermented dairy products. However, the form and properties of milk curd depend on the heat treatment of milk before fermentation. Milk proteins exhibit other functional properties (emulsification and partial coalescence of fat globules, aeration and foam stability during a churning, viscosity increasing of external phase in the development of structure in the ice cream, aerated milk and frozen fruit desserts. Conclusions. It is expedient to use results into a further study of the structure formation mechanism of dairy products and the development of recommendations in order to an efficient production.

The role of milk proteins in the structure formation of dairy products

Directory of Open Access Journals (Sweden)

O. Rybak

2015-05-01

Full Text Available Introduction. The structure of dairy products is a complex of proteins, fat, minerals and water that determines the texture and sensory properties of the product. Material and methods. The fermented milks (using the example of yogurt, cheese, ice cream, aerated milk and frozen fruit desserts have been researched. Scientific articles, published during 2000 and 2014 years, as well as theses and monographs of dairy science have been analysed too. Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis. Results and discussion. The scientific understanding of the milk proteins’ role in the structure formation of dairy product has been summarized. Negligible changes of structure as a result of compositional or technological changes can lead to shifts in the stability, texture and rheology of products, which are closely related to each other. The allowance of these properties has significant influence on the manufacturing. Acid coagulation is a major functional property of milk proteins, which used in the structure formation of cheese and fermented dairy products. However, the form and properties of milk curd depend on the heat treatment of milk before fermentation. Milk proteins exhibit other functional properties (emulsification and partial coalescence o f fatglobules, aeration and foam stability during a churning, viscosity increasing of external phase in the development of structure in the ice cream, aerated milk and frozen fruit desserts. Conclusions.It is expedient to use results into a further study of the structure formation mechanism of dairy products and the development of recommendations in order to an efficient production.

Stability of the octameric structure affects plasminogen-binding capacity of streptococcal enolase.

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Amanda J Cork

Full Text Available Group A Streptococcus (GAS is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen. Streptococcal surface enolase (SEN is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen to its binding sites, leading to more efficient plasmin(ogen binding and activation.

Modulation of the Extent of Cooperative Structural Change During Protein Folding by Chemical Denaturant.

Science.gov (United States)

Jethva, Prashant N; Udgaonkar, Jayant B

2017-09-07

Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N ↔ I N ↔ I 2 ↔ U model, in which structural changes occur noncooperatively during the N ↔ I N and I N ↔ I 2 transitions, and occur cooperatively during the I 2 ↔ U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.

Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala.

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Timothy J Jarome

Full Text Available Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.

Metal stabilization of collagen and de novo designed mimetic peptides

OpenAIRE

Parmar, Avanish S.; Xu, Fei; Pike, Douglas H.; Belure, Sandeep V.; Hasan, Nida F.; Drzewiecki, Kathryn E.; Shreiber, David I.; Nanda, Vikas

2015-01-01

We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacen...

The Use of Polymerized Genipin for the Stabilization of the Collagen Structure of Animal Hides

Science.gov (United States)

Animal hides are the major byproduct of meat industry and the collagen fibers is the main constituent. Crosslinkers play a key role in stabilizing the collagen structure for useful applications. Genipin is widely used as an ideal biological protein crosslinking agent due to its low toxicity compare...

Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability

International Nuclear Information System (INIS)

Bhuiya, Mohammad Wadud; Suryadi, Jimmy; Zhou, Zholi; Brown, Bernard Andrew II

2013-01-01

The crystal structure of A. pernix L7Ae is reported, providing insight into the extreme thermostability of this protein. Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that directs post-transcriptional modification of archaeal RNAs. The L7Ae protein from Aeropyrum pernix (Ap L7Ae), a member of the Crenarchaea, was found to have an extremely high melting temperature (>383 K). The crystal structure of Ap L7Ae has been determined to a resolution of 1.56 Å. The structure of Ap L7Ae was compared with the structures of two homologs: hyperthermophilic Methanocaldococcus jannaschii L7Ae and the mesophilic counterpart mammalian 15.5 kD protein. The primary stabilizing feature in the Ap L7Ae protein appears to be the large number of ion pairs and extensive ion-pair network that connects secondary-structural elements. To our knowledge, Ap L7Ae is among the most thermostable single-domain monomeric proteins presently observed

Effects of monohydric alcohols and polyols on the thermal stability of a protein

Energy Technology Data Exchange (ETDEWEB)

Murakami, Shota [Graduate School of Energy Science, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kinoshita, Masahiro, E-mail: kinoshit@iae.kyoto-u.ac.jp [Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011 (Japan)

2016-03-28

The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the

Graphene oxide as a protein matrix: influence on protein biophysical properties.

Science.gov (United States)

Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

2015-10-19

This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

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David J Leibly

Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

Conformational energy calculations on polypeptides and proteins: use of a statistical mechanical procedure for evaluating structure and properties.

Science.gov (United States)

Scheraga, H A; Paine, G H

1986-01-01

We are using a variety of theoretical and computational techniques to study protein structure, protein folding, and higher-order structures. Our earlier work involved treatments of liquid water and aqueous solutions of nonpolar and polar solutes, computations of the stabilities of the fundamental structures of proteins and their packing arrangements, conformations of small cyclic and open-chain peptides, structures of fibrous proteins (collagen), structures of homologous globular proteins, introduction of special procedures as constraints during energy minimization of globular proteins, and structures of enzyme-substrate complexes. Recently, we presented a new methodology for predicting polypeptide structure (described here); the method is based on the calculation of the probable and average conformation of a polypeptide chain by the application of equilibrium statistical mechanics in conjunction with an adaptive, importance sampling Monte Carlo algorithm. As a test, it was applied to Met-enkephalin.

Truncation of the C-terminal region of Toscana Virus NSs protein is critical for interferon-β antagonism and protein stability.

Science.gov (United States)

Gori Savellini, Gianni; Gandolfo, Claudia; Cusi, Maria Grazia

2015-12-01

Toscana Virus (TOSV) is a Phlebovirus responsible for central nervous system (CNS) injury in humans. The TOSV non-structural protein (NSs), which interacting with RIG-I leads to its degradation, was analysed in the C terminus fragment in order to identify its functional domains. To this aim, two C-terminal truncated NSs proteins, Δ1C-NSs (aa 1-284) and Δ2C-NSs (aa 1-287) were tested. Only Δ1C-NSs did not present any inhibitory effect on RIG-I and it showed a greater stability than the whole NSs protein. Moreover, the deletion of the TLQ aa sequence interposed between the two ΔC constructs caused a greater accumulation of the protein with a weak inhibitory effect on RIG-I, indicating some involvement of these amino acids in the NSs activity. Nevertheless, all the truncated proteins were still able to interact with RIG-I, suggesting that the domains responsible for RIG-I signaling and RIG-I interaction are mapped on different regions of the protein. Copyright © 2015 Elsevier Inc. All rights reserved.

Modularity in protein structures: study on all-alpha proteins.

Science.gov (United States)

Khan, Taushif; Ghosh, Indira

2015-01-01

Modularity is known as one of the most important features of protein's robust and efficient design. The architecture and topology of proteins play a vital role by providing necessary robust scaffolds to support organism's growth and survival in constant evolutionary pressure. These complex biomolecules can be represented by several layers of modular architecture, but it is pivotal to understand and explore the smallest biologically relevant structural component. In the present study, we have developed a component-based method, using protein's secondary structures and their arrangements (i.e. patterns) in order to investigate its structural space. Our result on all-alpha protein shows that the known structural space is highly populated with limited set of structural patterns. We have also noticed that these frequently observed structural patterns are present as modules or "building blocks" in large proteins (i.e. higher secondary structure content). From structural descriptor analysis, observed patterns are found to be within similar deviation; however, frequent patterns are found to be distinctly occurring in diverse functions e.g. in enzymatic classes and reactions. In this study, we are introducing a simple approach to explore protein structural space using combinatorial- and graph-based geometry methods, which can be used to describe modularity in protein structures. Moreover, analysis indicates that protein function seems to be the driving force that shapes the known structure space.

Mapping monomeric threading to protein-protein structure prediction.

Science.gov (United States)

Guerler, Aysam; Govindarajoo, Brandon; Zhang, Yang

2013-03-25

The key step of template-based protein-protein structure prediction is the recognition of complexes from experimental structure libraries that have similar quaternary fold. Maintaining two monomer and dimer structure libraries is however laborious, and inappropriate library construction can degrade template recognition coverage. We propose a novel strategy SPRING to identify complexes by mapping monomeric threading alignments to protein-protein interactions based on the original oligomer entries in the PDB, which does not rely on library construction and increases the efficiency and quality of complex template recognitions. SPRING is tested on 1838 nonhomologous protein complexes which can recognize correct quaternary template structures with a TM score >0.5 in 1115 cases after excluding homologous proteins. The average TM score of the first model is 60% and 17% higher than that by HHsearch and COTH, respectively, while the number of targets with an interface RMSD benchmark proteins. Although the relative performance of SPRING and ZDOCK depends on the level of homology filters, a combination of the two methods can result in a significantly higher model quality than ZDOCK at all homology thresholds. These data demonstrate a new efficient approach to quaternary structure recognition that is ready to use for genome-scale modeling of protein-protein interactions due to the high speed and accuracy.

Computational analysis of histidine mutations on the structural stability of human tyrosinases leading to albinism insurgence.

Science.gov (United States)

Hassan, Mubashir; Abbas, Qamar; Raza, Hussain; Moustafa, Ahmed A; Seo, Sung-Yum

2017-07-25

Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N 180 , R 202 , Q 202 , R 211 , Y 363 , R 367 , Y 367 and D 390 ) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr 367 (-0.079) and Q/R 202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln 202 , Arg 202 , Tyr 367 and D 390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R 202 and Y/R 363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R 202 and Y/R 363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.

Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

Science.gov (United States)

Keefe, Andrew J.; Jiang, Shaoyi

2012-01-01

Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

Design of sweet protein based sweeteners: hints from structure-function relationships.

Science.gov (United States)

Rega, Michele Fortunato; Di Monaco, Rossella; Leone, Serena; Donnarumma, Federica; Spadaccini, Roberta; Cavella, Silvana; Picone, Delia

2015-04-15

Sweet proteins represent a class of natural molecules, which are extremely interesting regarding their potential use as safe low-calories sweeteners for individuals who need to control sugar intake, such as obese or diabetic subjects. Punctual mutations of amino acid residues of MNEI, a single chain derivative of the natural sweet protein monellin, allow the modulation of its taste. In this study we present a structural and functional comparison between MNEI and a sweeter mutant Y65R, containing an extra positive charge on the protein surface, in conditions mimicking those of typical beverages. Y65R exhibits superior sweetness in all the experimental conditions tested, has a better solubility at mild acidic pH and preserves a significant thermal stability in a wide range of pH conditions, although slightly lower than MNEI. Our findings confirm the advantages of structure-guided protein engineering to design improved low-calorie sweeteners and excipients for food and pharmaceutical preparations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Heat shock protein 90 positively regulates Chikungunya virus replication by stabilizing viral non-structural protein nsP2 during infection.

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Indrani Das

Full Text Available BACKGROUND: The high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV emphasize the need to understand the biology of the virus for developing effective antiviral therapies. METHODS AND FINDINGS: In this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90 mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27 and Indian outbreak strain of 2006 (DRDE-06. Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production. CONCLUSION: Hsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06

Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

International Nuclear Information System (INIS)

Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

2008-01-01

A water network stabilizes the structure of cellular retionic acid binding protein II. The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the α2 helix at its entrance. This chain of interactions acts as a ‘pillar’ that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the α2 helix adopting an altered conformation compared with wild-type CRABPII

Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

Directory of Open Access Journals (Sweden)

Kanika Bajaj

2007-12-01

Full Text Available When incorporated into a polypeptide chain, proline (Pro differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

Science.gov (United States)

Bajaj, Kanika; Madhusudhan, M S; Adkar, Bharat V; Chakrabarti, Purbani; Ramakrishnan, C; Sali, Andrej; Varadarajan, Raghavan

2007-12-01

When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

Stability patterns for a size-structured population model and its stage-structured counterpart

DEFF Research Database (Denmark)

Zhang, Lai; Pedersen, Michael; Lin, Zhigui

2015-01-01

In this paper we compare a general size-structured population model, where a size-structured consumer feeds upon an unstructured resource, to its simplified stage-structured counterpart in terms of equilibrium stability. Stability of the size-structured model is understood in terms of an equivale...... to the population level....

Protein capped nanosilver free radical oxidation: role of biomolecule capping on nanoparticle colloidal stability and protein oxidation.

Science.gov (United States)

Ahumada, Manuel; Bohne, Cornelia; Oake, Jessy; Alarcon, Emilio I

2018-05-03

We studied the effect of human serum albumin protein capped spherical nanosilver on the nanoparticle stability upon peroxyl radical oxidation. The nanoparticle-protein composite is less prone to oxidation compared to the individual components. However, higher concentrations of hydrogen peroxide were formed in the nanoparticle-protein system.

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

Science.gov (United States)

Guinn, Emily J.; Pegram, Laurel M.; Capp, Michael W.; Pollock, Michelle N.; Record, M. Thomas

2011-01-01

To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values. PMID:21930943

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

DEFF Research Database (Denmark)

Mi, Li-Zhi; Grey, Michael J; Nishida, Noritaka

2008-01-01

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase...... differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane...

Protein enriched pasta: structure and digestibility of its protein network.

Science.gov (United States)

Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

2016-02-01

Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

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Bedouelle, Hugues

2016-02-01

The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Structure-based barcoding of proteins.

Science.gov (United States)

Metri, Rahul; Jerath, Gaurav; Kailas, Govind; Gacche, Nitin; Pal, Adityabarna; Ramakrishnan, Vibin

2014-01-01

A reduced representation in the format of a barcode has been developed to provide an overview of the topological nature of a given protein structure from 3D coordinate file. The molecular structure of a protein coordinate file from Protein Data Bank is first expressed in terms of an alpha-numero code and further converted to a barcode image. The barcode representation can be used to compare and contrast different proteins based on their structure. The utility of this method has been exemplified by comparing structural barcodes of proteins that belong to same fold family, and across different folds. In addition to this, we have attempted to provide an illustration to (i) the structural changes often seen in a given protein molecule upon interaction with ligands and (ii) Modifications in overall topology of a given protein during evolution. The program is fully downloadable from the website http://www.iitg.ac.in/probar/. © 2013 The Protein Society.

Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes.

Science.gov (United States)

Martin, Juliette; Regad, Leslie; Etchebest, Catherine; Camproux, Anne-Claude

2008-11-15

Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Voronoï tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions.

CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction

KAUST Repository

Cui, Xuefeng

2016-06-15

Motivation: Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment, threading and alignment-free methods, protein homology detection remains a challenging open problem. Recently, network methods that try to find transitive paths in the protein structure space demonstrate the importance of incorporating network information of the structure space. Yet, current methods merge the sequence space and the structure space into a single space, and thus introduce inconsistency in combining different sources of information. Method: We present a novel network-based protein homology detection method, CMsearch, based on cross-modal learning. Instead of exploring a single network built from the mixture of sequence and structure space information, CMsearch builds two separate networks to represent the sequence space and the structure space. It then learns sequence–structure correlation by simultaneously taking sequence information, structure information, sequence space information and structure space information into consideration. Results: We tested CMsearch on two challenging tasks, protein homology detection and protein structure prediction, by querying all 8332 PDB40 proteins. Our results demonstrate that CMsearch is insensitive to the similarity metrics used to define the sequence and the structure spaces. By using HMM–HMM alignment as the sequence similarity metric, CMsearch clearly outperforms state-of-the-art homology detection methods and the CASP-winning template-based protein structure prediction methods.

Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

OpenAIRE

Strub, Caroline; Alies, Carole; Lougarre, Andrée; Ladurantie, Caroline; Czaplicki, Jerzy; Fournier, Didier

2004-01-01

Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful...

Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

Science.gov (United States)

Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

2013-06-01

The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

Contribution of simple saccharides to the stabilization of amyloid structure

International Nuclear Information System (INIS)

Fung, Justin; Darabie, Audrey A.; McLaurin, JoAnne

2005-01-01

The use of osmolytes or chaperones to stabilize proteins/peptides that misfold in neurodegenerative diseases is an attractive concept for drug development. We have investigated the role of a series of small carbohydrates for protection of the natively structured Alzheimer's amyloid-β peptides (Aβ). Using circular dichroism spectroscopy to follow the β-structural transitions and electron microscopy to examine tertiary structural characteristics, we demonstrate that the hydrogen bonding capacity of the carbohydrate determines the inhibition or promotion of fibrillogenesis. Three sugar molecules that vary only in their distribution of potential H-bonding partners promote various structural changes in Aβ. Two of these sugar molecules are excluded from Aβ during aggregation and promote mature fibre growth, while the other binds Aβ promoting nucleation and the accumulation of protofibrils. Our studies suggest that utilization of a combinatorial strategy to alter H-bonding capacity across a simple carbohydrate molecule may represent a novel drug design strategy

Structural and energetic study of cation-π-cation interactions in proteins.

Science.gov (United States)

Pinheiro, Silvana; Soteras, Ignacio; Gelpí, Josep Lluis; Dehez, François; Chipot, Christophe; Luque, F Javier; Curutchet, Carles

2017-04-12

Cation-π interactions of aromatic rings and positively charged groups are among the most important interactions in structural biology. The role and energetic characteristics of these interactions are well established. However, the occurrence of cation-π-cation interactions is an unexpected motif, which raises intriguing questions about its functional role in proteins. We present a statistical analysis of the occurrence, composition and geometrical preferences of cation-π-cation interactions identified in a set of non-redundant protein structures taken from the Protein Data Bank. Our results demonstrate that this structural motif is observed at a small, albeit non-negligible frequency in proteins, and suggest a preference to establish cation-π-cation motifs with Trp, followed by Tyr and Phe. Furthermore, we have found that cation-π-cation interactions tend to be highly conserved, which supports their structural or functional role. Finally, we have performed an energetic analysis of a representative subset of cation-π-cation complexes combining quantum-chemical and continuum solvation calculations. Our results point out that the protein environment can strongly screen the cation-cation repulsion, leading to an attractive interaction in 64% of the complexes analyzed. Together with the high degree of conservation observed, these results suggest a potential stabilizing role in the protein fold, as demonstrated recently for a miniature protein (Craven et al., J. Am. Chem. Soc. 2016, 138, 1543). From a computational point of view, the significant contribution of non-additive three-body terms challenges the suitability of standard additive force fields for describing cation-π-cation motifs in molecular simulations.

Crystal structure of the Japanese encephalitis virus envelope protein.

Science.gov (United States)

Luca, Vincent C; AbiMansour, Jad; Nelson, Christopher A; Fremont, Daved H

2012-02-01

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

Directory of Open Access Journals (Sweden)

Yaiza Fernández-García

2016-06-01

Full Text Available Andes virus (ANDV is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

Src protein-tyrosine kinase structure and regulation

International Nuclear Information System (INIS)

Roskoski, Robert

2004-01-01

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

Chromatin structure and dynamics in hot environments: architectural proteins and DNA topoisomerases of thermophilic archaea.

Science.gov (United States)

Visone, Valeria; Vettone, Antonella; Serpe, Mario; Valenti, Anna; Perugino, Giuseppe; Rossi, Mosè; Ciaramella, Maria

2014-09-25

In all organisms of the three living domains (Bacteria, Archaea, Eucarya) chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair). Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C), chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity.

Chromatin Structure and Dynamics in Hot Environments: Architectural Proteins and DNA Topoisomerases of Thermophilic Archaea

Directory of Open Access Journals (Sweden)

Valeria Visone

2014-09-01

Full Text Available In all organisms of the three living domains (Bacteria, Archaea, Eucarya chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair. Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C, chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity.

Stability of Bulk Metallic Glass Structure. Final Report

Energy Technology Data Exchange (ETDEWEB)

Jain, H.; Williams, D. B.

2003-06-01

The fundamental origins of the stability of the (Pd-Ni){sub 80}P{sub 20} bulk metallic glasses (BMGs), a prototype for a whole class of BMG formers, were explored. While much of the properties of their BMGs have been characterized, their glass-stability have not been explained in terms of the atomic and electronic structure. The local structure around all three constituent atoms was obtained, in a complementary way, using extended X-ray absorption fine structure (EXAFS), to probe the nearest neighbor environment of the metals, and extended energy loss fine structure (EXELFS), to investigate the environment around P. The occupied electronic structure was investigated using X-ray photoelectron spectroscopy (XPS). The (Pd-Ni){sub 80}P{sub 20} BMGs receive their stability from cumulative, and interrelated, effects of both atomic and electronic origin. The stability of the (Pd-Ni){sub 80}P{sub 20} BMGs can be explained in terms of the stability of Pd{sub 60}Ni{sub 20}P{sub 20} and Pd{sub 30}Ni{sub 50}P{sub 20}, glasses at the end of BMG formation. The atomic structure in these alloys is very similar to those of the binary phosphide crystals near x=0 and x=80, which are trigonal prisms of Pd or Ni atoms surrounding P atoms. Such structures are known to exist in dense, randomly-packed systems. The structure of the best glass former in this series, Pd{sub 40}Ni{sub 40}P{sub 20} is further described by a weighted average of those of Pd{sub 30}Ni{sub 50}P{sub 20} and Pd{sub 60}Ni{sub 20}P{sub 20}. Bonding states present only in the ternary alloys were found and point to a further stabilization of the system through a negative heat of mixing between Pd and Ni atoms. The Nagel and Tauc criterion, correlating a decrease in the density of states at the Fermi level with an increase in the glass stability, was consistent with greater stability of the Pd{sub x}Ni{sub 80-x}P{sub 20} glasses with respect to the binary alloys of P. A valence electron concentration of 1.8 e/a, which

The structure at 1.7 Å resolution of the protein product of the At2g17340 gene from Arabidopsis thaliana

International Nuclear Information System (INIS)

Bitto, Eduard; Bingman, Craig A.; Allard, Simon T. M.; Wesenberg, Gary E.; Phillips, George N. Jr

2005-01-01

The crystal structure of the 40.8 kDa At2g17340 protein from A. thaliana was determined at 1.7 Å resolution. The structure provides the first insight into the structural organization of the Pfam01937.11 family and establishes that the proteins of this family coordinate a metal in its putative active site. The crystal structure of the At2g17340 protein from A. thaliana was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 16.9% (R free = 22.1%) at 1.7 Å resolution. At2g17340 is a member of the Pfam01937.11 protein family and its structure provides the first insight into the structural organization of this family. A number of fully and highly conserved residues defined by multiple sequence alignment of members of the Pfam01937.11 family were mapped onto the structure of At2g17340. The fully conserved residues are involved in the coordination of a metal ion and in the stabilization of loops surrounding the metal site. Several additional highly conserved residues also map into the vicinity of the metal-binding site, while others are clearly involved in stabilizing the hydrophobic core of the protein. The structure of At2g17340 represents a new fold in protein conformational space

Protein structural transition at negatively charged electrode surfaces. Effects of temperature and current density

Czech Academy of Sciences Publication Activity Database

Černocká, Hana; Ostatná, Veronika; Paleček, Emil

2015-01-01

Roč. 174, AUG 2015 (2015), s. 356-360 ISSN 0013-4686 R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR(CZ) GA15-15479S; GA ČR(CZ) GA13-00956S Institutional support: RVO:68081707 Keywords : Bovine serum albumin * sensing of surface-attached protein stability * protein structural transition at Hg Subject RIV: BO - Biophysics Impact factor: 4.803, year: 2015

MOLECULAR MODELING INDICATES THAT HOMOCYSTEINE INDUCES CONFORMATIONAL CHANGES IN THE STRUCTURE OF PUTATIVE TARGET PROTEINS

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Yumnam Silla

2015-09-01

Full Text Available An elevated level of homocysteine, a reactive thiol containing amino acid is associated with a multitude of complex diseases. A majority (>80% of homocysteine in circulation is bound to protein cysteine residues. Although, till date only 21 proteins have been experimentally shown to bind with homocysteine, using an insilico approach we had earlier identified several potential target proteins that could bind with homocysteine. Shomocysteinylation of proteins could potentially alter the structure and/or function of the protein. Earlier studies have shown that binding of homocysteine to protein alters its function. However, the effect of homocysteine on the target protein structure has not yet been documented. In the present work, we assess conformational or structural changes if any due to protein homocysteinylation using two proteins, granzyme B (GRAB and junctional adhesion molecule 1 (JAM1, which could potentially bind to homocysteine. We, for the first time, constructed computational models of homocysteine bound to target proteins and monitored their structural changes using explicit solvent molecular dynamic (MD simulation. Analysis of homocysteine bound trajectories revealed higher flexibility of the active site residues and local structural perturbations compared to the unbound native structure’s simulation, which could affect the stability of the protein. In addition, secondary structure analysis of homocysteine bound trajectories also revealed disappearance of â-helix within the G-helix and linker region that connects between the domain regions (as defined in the crystal structure. Our study thus captures the conformational transitions induced by homocysteine and we suggest these structural alterations might have implications for hyperhomocysteinemia induced pathologies.

X-ray structure of the mammalian GIRK2-βγ G-protein complex

Energy Technology Data Exchange (ETDEWEB)

Whorton, Matthew R.; MacKinnon, Roderick [Rockefeller

2013-07-30

G-protein-gated inward rectifier K⁺ (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K⁺ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K⁺ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP₂) and intracellular Na⁺ ions participate in multi-ligand regulation of GIRK channels.

Effective stabilization of CLA by microencapsulation in pea protein.

Science.gov (United States)

Costa, A M M; Nunes, J C; Lima, B N B; Pedrosa, C; Calado, V; Torres, A G; Pierucci, A P T R

2015-02-01

CLA was microencapsulated by spray drying in ten varied wall systems (WS) consisting of pea protein isolate or pea protein concentrate (PPC) alone at varied core:WS ratios (1:2; 1:3 and 1:4), or blended with maltodextrin (M) and carboxymethylcellulose at a pea protein:carbohydrate ratio of 3:1. The physical-chemical properties of the CLA microparticles were characterised by core retention, microencapsulation efficiency (ME), particle size and moisture. CLA:M:PPC (1:1:3) showed the most promising results, thus we evaluated the effect of M addition in the WS on other physical-chemical characteristics and oxidative stability (CLA isomer profile, quantification of CLA and volatile compounds by SPME coupled with CG-MS) during two months of storage at room temperature, CLA:PPC (1:4) was selected for comparisons. CLA:M:PPC (1:1:3) microparticles demonstrated better morphology, solubility, dispersibility and higher glass-transition temperature values. M addition did not influence the oxidative stability of CLA, however its presence improved physical-chemical characteristics necessary for food applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Immobilization-stabilization of proteins on nanofibrillated cellulose derivatives and their bioactive film formation.

Science.gov (United States)

Arola, Suvi; Tammelin, Tekla; Setälä, Harri; Tullila, Antti; Linder, Markus B

2012-03-12

In a number of different applications for enzymes and specific binding proteins a key technology is the immobilization of these proteins to different types of supports. In this work we describe a concept for protein immobilization that is based on nanofibrillated cellulose (NFC). NFC is a form of cellulose where fibers have been disintegrated into fibrils that are only a few nanometers in diameter and have a very large aspect ratio. Proteins were conjugated through three different strategies using amine, epoxy, and carboxylic acid functionalized NFC. The conjugation chemistries were chosen according to the reactive groups on the NFC derivatives; epoxy amination, heterobifunctional modification of amino groups, and EDC/s-NHS activation of carboxylic acid groups. The conjugation reactions were performed in solution and immobilization was performed by spin coating the protein-NCF conjugates. The structure of NFC was shown to be advantageous for both protein performance and stability. The use of NFC allows all covalent chemistry to be performed in solution, while the immobilization is achieved by a simple spin coating or spreading of the protein-NFC conjugates on a support. This allows more scalable methods and better control of conditions compared to the traditional methods that depend on surface reactions.

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Science.gov (United States)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

SDSL-ESR-based protein structure characterization.

Science.gov (United States)

Strancar, Janez; Kavalenka, Aleh; Urbancic, Iztok; Ljubetic, Ajasja; Hemminga, Marcus A

2010-03-01

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation.

Science.gov (United States)

Schlimpert, Susan; Wasserstrom, Sebastian; Chandra, Govind; Bibb, Maureen J; Findlay, Kim C; Flärdh, Klas; Buttner, Mark J

2017-07-25

During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

Structure and non-structure of centrosomal proteins.

Science.gov (United States)

Dos Santos, Helena G; Abia, David; Janowski, Robert; Mortuza, Gulnahar; Bertero, Michela G; Boutin, Maïlys; Guarín, Nayibe; Méndez-Giraldez, Raúl; Nuñez, Alfonso; Pedrero, Juan G; Redondo, Pilar; Sanz, María; Speroni, Silvia; Teichert, Florian; Bruix, Marta; Carazo, José M; Gonzalez, Cayetano; Reina, José; Valpuesta, José M; Vernos, Isabelle; Zabala, Juan C; Montoya, Guillermo; Coll, Miquel; Bastolla, Ugo; Serrano, Luis

2013-01-01

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php.

Microencapsulation structures based on protein-coated liposomes obtained through electrospraying for the stabilization and improved bioaccessibility of curcumin.

Science.gov (United States)

Gómez-Mascaraque, Laura G; Casagrande Sipoli, Caroline; de La Torre, Lucimara Gaziola; López-Rubio, Amparo

2017-10-15

Novel food-grade hybrid encapsulation structures based on the entrapment of phosphatidylcholine liposomes, within a WPC matrix through electrospraying, were developed and used as delivery vehicles for curcumin. The loading capacity and encapsulation efficiency of the proposed system was studied, and the suitability of the approach to stabilize curcumin and increase its bioaccessibility was assessed. Results showed that the maximum loading capacity of the liposomes was around 1.5% of curcumin, although the loading capacity of the hybrid microencapsulation structures increased with the curcumin content by incorporation of curcumin microcrystals upon electrospraying. Microencapsulation of curcumin within the proposed hybrid structures significantly increased its bioaccessibility (∼1.7-fold) compared to the free compound, and could successfully stabilize it against degradation in PBS (pH=7.4). The proposed approach thus proved to be a promising alternative to produce powder-like functional ingredients. Copyright © 2017 Elsevier Ltd. All rights reserved.

Positively selected sites in cetacean myoglobins contribute to protein stability

DEFF Research Database (Denmark)

Dasmeh, Pouria; Serohijos, Adrian W R; Kepp, Kasper P

2013-01-01

Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity...... between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected...

Alkaline transition of pseudoazurin Met16X mutant proteins: protein stability influenced by the substitution of Met16 in the second sphere coordination.

Science.gov (United States)

Abdelhamid, Rehab F; Obara, Yuji; Kohzuma, Takamitsu

2008-01-01

Several blue copper proteins are known to change the active site structure at alkaline pH (alkaline transition). Spectroscopic studies of Met16Phe, Met16Tyr, Met16Trp, and Met16Val pseudoazurin variants were performed to investigate the second sphere role through alkaline transition. The visible electronic absorption and resonance Raman spectra of Met16Phe, Met16Tyr, and Met16Trp variants showed the increasing of axial component at pH approximately 11 like wild-type PAz. The visible electronic absorption and far-UV CD spectra of Met16Val demonstrated that the destabilization of the protein structure was triggered at pH>11. Resonance Raman (RR) spectra of PAz showed that the intensity-weighted averaged Cu-S(Cys) stretching frequency was shifted to higher frequency region at pH approximately 11. The higher frequency shift of Cu-S(Cys) bond is implied the stronger Cu-S(Cys) bond at alkaline transition pH approximately 11. The visible electronic absorption and far-UV CD spectra of Met16X PAz revealed that the Met16Val variant is denatured at pH>11, but Met16Phe, Met16Tyr, and Met16Trp mutant proteins are not denatured even at pH>11. These observations suggest that Met16 is important to maintain the protein structure through the possible weak interaction between methionine -SCH3 part and coordinated histidine imidazole moiety. The introduction of pi-pi interaction in the second coordination sphere may be contributed to the enhancement of protein structure stability.

Comparison of the colloidal stability, bioaccessibility and antioxidant activity of corn protein hydrolysate and sodium caseinate stabilized curcumin nanoparticles.

Science.gov (United States)

Wang, Yong-Hui; Yuan, Yang; Yang, Xiao-Quan; Wang, Jin-Mei; Guo, Jian; Lin, Yuan

2016-07-01

The aims of this work were to construct corn protein hydrolysate (CPH)-based curcumin nanoparticles (Cur NPs) and to compare the colloidal stability, bioaccessibility and antioxidant activity of the Cur NPs stabilized CPH and sodium caseinate (NaCas) respectively. The results indicated that Cur solubility could be considerably improved after the Cur NPs fabrication. The spectroscopy results demonstrated that the solubilization of Cur should be attributed to its complexation with CPH or NaCas. The Cur NPs exhibited good colloidal stability after 1 week's storage but showed smaller (40 nm) size in CPH than in NaCas (100 nm). After lyophilization, the Cur NPs powders showed good rehydration properties and chemical stability, and compared with NaCas, the size of Cur NPs stabilized by CPH was still smaller. Additionally, the Cur NPs exhibited higher chemical stability against the temperature compared with free Cur, and the CPH could protect Cur from degradation more efficiently. Comparing with NaCas, the Cur NPs stabilized by CPH exhibited better bioaccessibility and antioxidant activity. This study demonstrated that CPH may be better than NaCas in Cur NPs fabrication and it opens up the possibility of using hydrophobic protein hydrolysate to construct the NPs delivery system.

Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

Science.gov (United States)

Bian, Liujiao; Ji, Xu

2014-01-01

Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

Thermodynamic database for proteins: features and applications.

Science.gov (United States)

Gromiha, M Michael; Sarai, Akinori

2010-01-01

We have developed a thermodynamic database for proteins and mutants, ProTherm, which is a collection of a large number of thermodynamic data on protein stability along with the sequence and structure information, experimental methods and conditions, and literature information. This is a valuable resource for understanding/predicting the stability of proteins, and it can be accessible at http://www.gibk26.bse.kyutech.ac.jp/jouhou/Protherm/protherm.html . ProTherm has several features including various search, display, and sorting options and visualization tools. We have analyzed the data in ProTherm to examine the relationship among thermodynamics, structure, and function of proteins. We describe the progress on the development of methods for understanding/predicting protein stability, such as (i) relationship between the stability of protein mutants and amino acid properties, (ii) average assignment method, (iii) empirical energy functions, (iv) torsion, distance, and contact potentials, and (v) machine learning techniques. The list of online resources for predicting protein stability has also been provided.

Influence of protic ionic liquids on the structure and stability of succinylated Con A.

Science.gov (United States)

Attri, Pankaj; Venkatesu, Pannuru

2012-01-01

We report the synthesis of a series of ionic liquids (ILs) from various ions having different kosmotropicity including dihydrogen phosphate (H(2)PO(4)(-)), hydrogen sulfate (HSO(4)(-)) and acetate (CH(3)COO(-)) as anions and chaotropic cation such as trialkylammonium cation. To characterize the biomolecular interactions of ILs with protein, we have explored the stability of succinylated Con A (S Con A) in the presence of these aqueous ILs, which are varied combinations of kosmotropic anion with chaotropic cation such as triethylammonium dihydrogen phosphate [(CH(3)CH(2))(3)NH][H(2)PO(4)] (TEAP), trimethylammonium acetate [(CH(3))(3)NH][CH(3)COO] (TMAA), trimethylammonium dihydrogen phosphate [(CH(3))(3)NH][H(2)PO(4)] (TMAP) and trimethylammonium hydrogen sulfate [(CH(3))(3)NH][HSO(4)] (TMAS). Circular dichroism (CD) and fluorescence experiments have been used to characterize the stability of S Con A by ILs. Our data distinctly demonstrate that the long alkyl chain IL TEAP is a strong stabilizer for S Con A. Further, our experimental results reveal that TEAP is an effective refolding enhancer for S Con A from a thermally denatured protein structure. Copyright © 2012 Elsevier B.V. All rights reserved.

Structural and thermodynamic characterization of the Escherichia coli RelBE toxin-antitoxin system: indication for a functional role of differential stability

DEFF Research Database (Denmark)

Cherny, Izhack; Overgaard, Martin; Borch, Jonas

2007-01-01

on the folding and stability of the protein pair in solution. Here we structurally and thermodynamically characterize the RelBE system components from E. coli in solution, both separately and in their complexed state. The RelB antitoxin, an alpha-helical protein according to circular dichroism and infrared.......5 degrees C, and exhibits exceptional sensitivity to heat. Complex formation, accompanied by a structural transition, leads to a 12 degrees C increase in the TM and substantial heat resistance. Moreover, in vivo interaction and protein footprint experiments indicate that the C-terminal part of Rel...

TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

Science.gov (United States)

Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

2014-01-01

In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. PMID:25287913

A structural basis for cellular uptake of GST-fold proteins.

Directory of Open Access Journals (Sweden)

Melanie J Morris

Full Text Available It has recently emerged that glutathione transferase enzymes (GSTs and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.

BLAST-based structural annotation of protein residues using Protein Data Bank.

Science.gov (United States)

Singh, Harinder; Raghava, Gajendra P S

2016-01-25

In the era of next-generation sequencing where thousands of genomes have been already sequenced; size of protein databases is growing with exponential rate. Structural annotation of these proteins is one of the biggest challenges for the computational biologist. Although, it is easy to perform BLAST search against Protein Data Bank (PDB) but it is difficult for a biologist to annotate protein residues from BLAST search. A web-server StarPDB has been developed for structural annotation of a protein based on its similarity with known protein structures. It uses standard BLAST software for performing similarity search of a query protein against protein structures in PDB. This server integrates wide range modules for assigning different types of annotation that includes, Secondary-structure, Accessible surface area, Tight-turns, DNA-RNA and Ligand modules. Secondary structure module allows users to predict regular secondary structure states to each residue in a protein. Accessible surface area predict the exposed or buried residues in a protein. Tight-turns module is designed to predict tight turns like beta-turns in a protein. DNA-RNA module developed for predicting DNA and RNA interacting residues in a protein. Similarly, Ligand module of server allows one to predicted ligands, metal and nucleotides ligand interacting residues in a protein. In summary, this manuscript presents a web server for comprehensive annotation of a protein based on similarity search. It integrates number of visualization tools that facilitate users to understand structure and function of protein residues. This web server is available freely for scientific community from URL http://crdd.osdd.net/raghava/starpdb .

New protein structures provide an updated understanding of phenylketonuria.

Science.gov (United States)

Jaffe, Eileen K

2017-08-01

Phenylketonuria (PKU) and less severe hyperphenylalaninemia (HPA) constitute the most common inborn error of amino acid metabolism, and is most often caused by defects in phenylalanine hydroxylase (PAH) function resulting in accumulation of Phe to neurotoxic levels. Despite the success of dietary intervention in preventing permanent neurological damage, individuals living with PKU clamor for additional non-dietary therapies. The bulk of disease-associated mutations are PAH missense variants, which occur throughout the entire 452 amino acid human PAH protein. While some disease-associated mutations affect protein structure (e.g. truncations) and others encode catalytically dead variants, most have been viewed as defective in protein folding/stability. Here we refine this view to address how PKU-associated missense variants can perturb the equilibrium among alternate native PAH structures (resting-state PAH and activated PAH), thus shifting the tipping point of this equilibrium to a neurotoxic Phe concentration. This refined view of PKU introduces opportunities for the design or discovery of therapeutic pharmacological chaperones that can help restore the tipping point to healthy Phe levels and how such a therapeutic might work with or without the inhibitory pharmacological chaperone BH 4 . Dysregulation of an equilibrium of architecturally distinct native PAH structures departs from the concept of "misfolding", provides an updated understanding of PKU, and presents an enhanced foundation for understanding genotype/phenotype relationships. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterisation of protein stability in rod-insert vaginal rings.

Science.gov (United States)

Pattani, Aditya; Lowry, Deborah; Curran, Rhonda M; McGrath, Stephanie; Kett, Vicky L; Andrews, Gavin P; Malcolm, R Karl

2012-07-01

A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 °C, but not when stored at 40 °C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 °C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40 °C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general. Copyright

Effect of the geometry of confining media on the stability and folding rate of α -helix proteins

Science.gov (United States)

Wang, Congyue; Piroozan, Nariman; Javidpour, Leili; Sahimi, Muhammad

2018-05-01

Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding

Biochemical and Structural Analysis of Hormone-sensitive Lipase Homolog EstE7: Insight into the Stabilized Dimerization of HSL-Homolog Proteins

International Nuclear Information System (INIS)

Nam, Ki Hyun; Park, Sung Ha; Lee, Won Ho; Hwang, Kwang Yeon

2010-01-01

Hormone sensitive lipase (HSL) plays a major role in energy homeostasis and lipid metabolism. Several crystal structures of HSL-homolog proteins have been identified, which has led to a better understanding of its molecular function. HSLhomolog proteins exit as both monomer and dimer, but the biochemical and structural basis for such oligomeric states has not been successfully elucidated. Therefore, we determined the crystal structure of HSL-homolog protein EstE7 from a metagenome library at 2.2 A resolution and characterized the oligomeric states of EstE7 both structurally and biochemically. EstE7 protein prefers the dimeric state in solution, which is supported by its higher enzymatic activity in the dimeric state. In the crystal form, EstE7 protein shows two-types of dimeric interface. Specifically, dimerization via the external β8-strand occurred through tight association between two pseudosymmetric folds via salt bridges, hydrogen bonds and van der Waals interactions. This dimer formation was similar to that of other HSL-homolog protein structures such as AFEST, BEFA, and EstE1. We anticipate that our results will provide insight into the oligomeric state of HSLhomolog proteins

Carotenoid-protein complexes and their stability towards oxygen and radiation

International Nuclear Information System (INIS)

Ramakrishnan, T.V.; Francis, F.J.

1980-01-01

Carotenoid-protein complexes isolated from fresh mangoes were found to be more stable to oxygen and radiation when dissolved in water as compared with β-carotene in petroleum ether. Part of the pigment could be released from the complex by gamma irradiation. Observations on the stability of the carotenoid (98% β-carotene) in the complex indicated that the pigment is either associated with the lipid prosthetic group of the protein or loosely attached to the protein by weak hydrophobic bonds. (author)

Cysteine residue is not essential for CPM protein thermal-stability assay.

Science.gov (United States)

Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

2015-05-01

A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

Effect of pasteurization on the protein composition and oxidative stability of beer during storage.

Science.gov (United States)

Lund, Marianne N; Hoff, Signe; Berner, Torben S; Lametsch, René; Andersen, Mogens L

2012-12-19

The impacts of pasteurization of a lager beer on protein composition and the oxidative stability were studied during storage at 22 °C for 426 days in the dark. Pasteurization clearly improved the oxidative stability of beer determined by ESR spectroscopy, whereas it had a minor negative effect on the volatile profile by increasing volatile compounds that is generally associated with heat treatment and a loss of fruity ester aroma. A faster rate of radical formation in unpasteurized beer was consistent with a faster consumption of sulfite. Beer proteins in the unpasteurized beer were more degraded, most likely due to proteolytic enzyme activity of yeast remnants and more precipitation of proteins was also observed. The differences in soluble protein content and composition are suggested to result in differences in the contents of prooxidative metals as a consequence of the proteins ability to bind metals. This also contributes to the differences in oxidative stabilities of the beers.

Structural evidence for solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations.

Science.gov (United States)

Lenton, Samuel; Walsh, Danielle L; Rhys, Natasha H; Soper, Alan K; Dougan, Lorna

2016-07-21

Halophilic organisms have adapted to survive in high salt environments, where mesophilic organisms would perish. One of the biggest challenges faced by halophilic proteins is the ability to maintain both the structure and function at molar concentrations of salt. A distinct adaptation of halophilic proteins, compared to mesophilic homologues, is the abundance of aspartic acid on the protein surface. Mutagenesis and crystallographic studies of halophilic proteins suggest an important role for solvent interactions with the surface aspartic acid residues. This interaction, between the regions of the acidic protein surface and the solvent, is thought to maintain a hydration layer around the protein at molar salt concentrations thereby allowing halophilic proteins to retain their functional state. Here we present neutron diffraction data of the monomeric zwitterionic form of aspartic acid solutions at physiological pH in 0.25 M and 2.5 M concentration of potassium chloride, to mimic mesophilic and halophilic-like environmental conditions. We have used isotopic substitution in combination with empirical potential structure refinement to extract atomic-scale information from the data. Our study provides structural insights that support the hypothesis that carboxyl groups on acidic residues bind water more tightly under high salt conditions, in support of the residue-ion interaction model of halophilic protein stabilisation. Furthermore our data show that in the presence of high salt the self-association between the zwitterionic form of aspartic acid molecules is reduced, suggesting a possible mechanism through which protein aggregation is prevented.

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

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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

The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

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Anna C. Maurer

2018-05-01

Full Text Available Summary: The adeno-associated virus (AAV vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. : Maurer et al. describe a phenotype-to-phylogeny mapping strategy correlating phenotypic variation in AAVs to a reconstructed phylogeny, revealing capsid structure-function relationships relevant to that phenotype. Dependence on the viral co-factor AAP for capsid assembly is examined, and capsid functional motifs, in addition to mechanistic roles of AAP, are elucidated. Keywords: AAV, AAP, adeno-associated virus, capsid assembly, manufacturing, capsid, vector engineering, structure-function, gene therapy

Direct measurements of protein-stabilized gold nanoparticle interactions.

Science.gov (United States)

Eichmann, Shannon L; Bevan, Michael A

2010-09-21

We report integrated video and total internal reflection microscopy measurements of protein stabilized 110 nm Au nanoparticles confined in 280 nm gaps in physiological media. Measured potential energy profiles display quantitative agreement with Brownian dynamic simulations that include hydrodynamic interactions and camera exposure time and noise effects. Our results demonstrate agreement between measured nonspecific van der Waals and adsorbed protein interactions with theoretical potentials. Confined, lateral nanoparticle diffusivity measurements also display excellent agreement with predictions. These findings provide a basis to interrogate specific biomacromolecular interactions in similar experimental configurations and to design future improved measurement methods.

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

Directory of Open Access Journals (Sweden)

Chen Wang

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

NAPS: Network Analysis of Protein Structures

Science.gov (United States)

Chakrabarty, Broto; Parekh, Nita

2016-01-01

Traditionally, protein structures have been analysed by the secondary structure architecture and fold arrangement. An alternative approach that has shown promise is modelling proteins as a network of non-covalent interactions between amino acid residues. The network representation of proteins provide a systems approach to topological analysis of complex three-dimensional structures irrespective of secondary structure and fold type and provide insights into structure-function relationship. We have developed a web server for network based analysis of protein structures, NAPS, that facilitates quantitative and qualitative (visual) analysis of residue–residue interactions in: single chains, protein complex, modelled protein structures and trajectories (e.g. from molecular dynamics simulations). The user can specify atom type for network construction, distance range (in Å) and minimal amino acid separation along the sequence. NAPS provides users selection of node(s) and its neighbourhood based on centrality measures, physicochemical properties of amino acids or cluster of well-connected residues (k-cliques) for further analysis. Visual analysis of interacting domains and protein chains, and shortest path lengths between pair of residues are additional features that aid in functional analysis. NAPS support various analyses and visualization views for identifying functional residues, provide insight into mechanisms of protein folding, domain-domain and protein–protein interactions for understanding communication within and between proteins. URL:http://bioinf.iiit.ac.in/NAPS/. PMID:27151201

Stability and the structure of continuous-time economic models

NARCIS (Netherlands)

Nieuwenhuis, H.J.; Schoonbeek, L.

In this paper we investigate the relationship between the stability of macroeconomic, or macroeconometric, continuous-time models and the structure of the matrices appearing in these models. In particular, we concentrate on dominant-diagonal structures. We derive general stability results for models

Constraint Network Analysis (CNA): a Python software package for efficiently linking biomacromolecular structure, flexibility, (thermo-)stability, and function.

Science.gov (United States)

Pfleger, Christopher; Rathi, Prakash Chandra; Klein, Doris L; Radestock, Sebastian; Gohlke, Holger

2013-04-22

For deriving maximal advantage from information on biomacromolecular flexibility and rigidity, results from rigidity analyses must be linked to biologically relevant characteristics of a structure. Here, we describe the Python-based software package Constraint Network Analysis (CNA) developed for this task. CNA functions as a front- and backend to the graph-based rigidity analysis software FIRST. CNA goes beyond the mere identification of flexible and rigid regions in a biomacromolecule in that it (I) provides a refined modeling of thermal unfolding simulations that also considers the temperature-dependence of hydrophobic tethers, (II) allows performing rigidity analyses on ensembles of network topologies, either generated from structural ensembles or by using the concept of fuzzy noncovalent constraints, and (III) computes a set of global and local indices for quantifying biomacromolecular stability. This leads to more robust results from rigidity analyses and extends the application domain of rigidity analyses in that phase transition points ("melting points") and unfolding nuclei ("structural weak spots") are determined automatically. Furthermore, CNA robustly handles small-molecule ligands in general. Such advancements are important for applying rigidity analysis to data-driven protein engineering and for estimating the influence of ligand molecules on biomacromolecular stability. CNA maintains the efficiency of FIRST such that the analysis of a single protein structure takes a few seconds for systems of several hundred residues on a single core. These features make CNA an interesting tool for linking biomacromolecular structure, flexibility, (thermo-)stability, and function. CNA is available from http://cpclab.uni-duesseldorf.de/software for nonprofit organizations.

Correlation between mechanical behavior of protein films at the air/water interface and intrinsic stability of protein molecules

NARCIS (Netherlands)

Martin, A.H.; Cohen Stuart, M.A.; Bos, M.A.; Vliet, T. van

2005-01-01

The relation between mechanical film properties of various adsorbed protein layers at the air/water interface and intrinsic stability of the corresponding proteins is discussed. Mechanical film properties were determined by surface deformation in shear and dilation. In shear, fracture stress, σf,

Stability of buffer-free freeze-dried formulations: A feasibility study of a monoclonal antibody at high protein concentrations.

Science.gov (United States)

Garidel, Patrick; Pevestorf, Benjamin; Bahrenburg, Sven

2015-11-01

We studied the stability of freeze-dried therapeutic protein formulations over a range of initial concentrations (from 40 to 160 mg/mL) and employed a variety of formulation strategies (including buffer-free freeze dried formulations, or BF-FDF). Highly concentrated, buffer-free liquid formulations of therapeutic monoclonal antibodies (mAbs) have been shown to be a viable alternative to conventionally buffered preparations. We considered whether it is feasible to use the buffer-free strategy in freeze-dried formulations, as an answer to some of the known drawbacks of conventional buffers. We therefore conducted an accelerated stability study (24 weeks at 40 °C) to assess the feasibility of stabilizing freeze-dried formulations without "classical" buffer components. Factors monitored included pH stability, protein integrity, and protein aggregation. Because the protein solutions are inherently self-buffering, and the system's buffer capacity scales with protein concentration, we included highly concentrated buffer-free freeze-dried formulations in the study. The tested formulations ranged from "fully formulated" (containing both conventional buffer and disaccharide stabilizers) to "buffer-free" (including formulations with only disaccharide lyoprotectant stabilizers) to "excipient-free" (with neither added buffers nor stabilizers). We evaluated the impacts of varying concentrations, buffering schemes, pHs, and lyoprotectant additives. At the end of 24 weeks, no change in pH was observed in any of the buffer-free formulations. Unbuffered formulations were found to have shorter reconstitution times and lower opalescence than buffered formulations. Protein stability was assessed by visual inspection, sub-visible particle analysis, protein monomer content, charge variants analysis, and hydrophobic interaction chromatography. All of these measures found the stability of buffer-free formulations that included a disaccharide stabilizer comparable to buffer

Structure of Dynamic, Taxol-Stabilized, and GMPPCP-Stabilized Microtubule.

Science.gov (United States)

Ginsburg, Avi; Shemesh, Asaf; Millgram, Abigail; Dharan, Raviv; Levi-Kalisman, Yael; Ringel, Israel; Raviv, Uri

2017-09-14

Microtubule (MT) is made of αβ-tubulin heterodimers that dynamically assemble into a hollow nanotube composed of straight protofilaments. MT dynamics is facilitated by hydrolysis of guanosine-5'-triphosphate (GTP) and can be inhibited by either anticancer agents like taxol or the nonhydrolyzable GTP analogues like GMPPCP. Using high-resolution synchrotron X-ray scattering, we have measured and analyzed the scattering curves from solutions of dynamic MT (in other words, in the presence of excess GTP and free of dynamic-inhibiting agents) and examined the effect of two MT stabilizers: taxol and GMPPCP. Previously, we have analyzed the structure of dynamic MT by docking the atomic model of tubulin dimer onto a 3-start left handed helical lattice, derived from the PDB ID 3J6F . 3J6F corresponds to a MT with 14 protofilaments. In this paper, we took into account the possibility of having MT structures containing between 12 and 15 protofilaments. MTs with 12 protofilaments were never observed. We determined the radii, the pitch, and the distribution of protofilament number that best fit the scattering data from dynamic MT or stabilized MT by taxol or GMPPCP. We found that the protofilament number distribution shifted when the MT was stabilized. Taxol increased the mass fraction of MT with 13 protofilaments and decreased the mass fraction of MT with 14 protofilaments. GMPPCP reduced the mass fraction of MT with 15 protofilaments and increased the mass fraction of MT with 14 protofilaments. The pitch, however, remained unchanged regardless of whether the MT was dynamic or stabilized. Higher tubulin concentrations increased the fraction of dynamic MT with 14 protofilaments.

Insulin structure and stability.

Science.gov (United States)

Brange, J; Langkjoer, L

1993-01-01

Insulin is composed of 51 amino acids in two peptide chains (A and B) linked by two disulfide bonds. The three-dimensional structure of the insulin molecule (insulin monomer), essentially the same in solution and in solid phase, exists in two main conformations. These differ in the extent of helix in the B chain which is governed by the presence of phenol or its derivatives. In acid and neutral solutions, in concentrations relevant for pharmaceutical formulation, the insulin monomer assembles to dimers and at neutral pH, in the presence of zinc ions, further to hexamers. Many crystalline modifications of insulin have been identified but only those with the hexamer as the basic unit are utilized in preparations for therapy. The insulin hexamer forms a relatively stable unit but some flexibility remains within the individual molecules. The intrinsic flexibility at the ends of the B chain plays an important role in governing the physical and chemical stability of insulin. A variety of chemical changes of the primary structure (yielding insulin derivatives), and physical modifications of the secondary to quaternary structures (resulting in "denaturation," aggregation, and precipitation) are known to affect insulin and insulin preparations during storage and use (Fig. 8). The tendency of insulin to undergo structural transformation resulting in aggregation and formation of insoluble insulin fibrils has been one of the most intriguing and widely studied phenomena in relation to insulin stability. Although the exact mechanism of fibril formation is still obscure, it is now clear that the initial step is an exposure of certain hydrophobic residues, normally buried in the three-dimensional structure, to the surface of the insulin monomer. This requires displacement of the COOH-terminal B-chain residues from their normal position which can only be accomplished via monomerization of the insulin. Therefore, most methods stabilizing insulin against fibrillation share the

Phase stability and electronic structure of transition-metal aluminides

International Nuclear Information System (INIS)

Carlsson, A.E.

1992-01-01

This paper will describe the interplay between die electronic structure and structural energetics in simple, complex, and quasicrystalline Al-transition metal (T) intermetallics. The first example is the Ll 2 -DO 22 competition in Al 3 T compounds. Ab-initio electronic total-energy calculations reveal surprisingly large structural-energy differences, and show that the phase stability of both stoichiometric and ternary-substituted compounds correlates closely with a quasigap in the electronic density of states (DOS). Secondly, ab-initio calculations for the structural stability of the icosahedrally based Al 12 W structure reveal similar quasigap effects, and provide a simple physical explanation for the stability of the complex aluminide structures. Finally, parametrized tight-binding model calculations for the Al-Mn quasicrystal reveal a large spread in the local Mn DOS behavior, and support a two-site model for the quasicrystal's magnetic behavior

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

International Nuclear Information System (INIS)

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

2006-01-01

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

Structure of a periplasmic glucose-binding protein from Thermotoga maritima

International Nuclear Information System (INIS)

Palani, Kandavelu; Kumaran, Desigan; Burley, Stephen K.; Swaminathan, Subramanyam

2012-01-01

The periplasmic glucose-binding protein from T. maritima consists of two domains with the ligand β-d-glucose buried between them. The two domains adopt a closed conformation. ABC transport systems have been characterized in organisms ranging from bacteria to humans. In most bacterial systems, the periplasmic component is the primary determinant of specificity of the transport complex as a whole. Here, the X-ray crystal structure of a periplasmic glucose-binding protein (GBP) from Thermotoga maritima determined at 2.4 Å resolution is reported. The molecule consists of two similar α/β domains connected by a three-stranded hinge region. In the current structure, a ligand (β-d-glucose) is buried between the two domains, which have adopted a closed conformation. Details of the substrate-binding sites revealed features that determine substrate specificity. In toto, ten residues from both domains form eight hydrogen bonds to the bound sugar and four aromatic residues (two from each domain) stabilize the substrate through stacking interactions

Salinity induced changes in cell membrane stability, protein and ...

African Journals Online (AJOL)

control), 4.7, 9.4 and 14.1 dS m-1 to determine the effect of salt on vegetative growth, relative water content, cell membrane stability, protein and RNA contents in sand culture experiment. Fresh and dry weights of plants, shoots and roots decreased ...

The Effect of Protein PEGylation on Physical Stability in Liquid Formulation

DEFF Research Database (Denmark)

Holm, Louise Stenstrup; Mcumber, Aaron; Rasmussen, Jakob Ewald

2014-01-01

The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg...... approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation...

A Comparison of Protein Stability in Prefillable Syringes Made of Glass and Plastic.

Science.gov (United States)

Waxman, Lloyd; Vilivalam, Vinod D

2017-01-01

The development of protein therapeutics requires stabilization of these labile molecules during shipment and storage. Biologics, particularly monoclonal antibodies, are frequently packaged at high concentration in prefillable syringes traditionally made of glass. However, some biologics are unstable in glass due to sensitivity to silicone oil, tungsten, glue, or metal ions. Syringes made from the plastic cyclic olefin polymer, Daikyo Crystal Zenith® (CZ), with a Flurotec-laminated piston, have none of these issues. This study compared the stability of several proteins including biotherapeutics when stored up to 14 months at 5 °C and 25 °C in prefillable siliconized syringes made of glass or silicone oil-free CZ syringes, and when subjected to mild agitation by end-over-end rotation at room temperature. At each time point, proteins were analyzed by several techniques including turbidity, size exclusion high-performance liquid chromatography, reversed phase high-performance liquid chromatography, ion-exchange chromatography, electrophoresis, and light scattering to monitor changes in aggregation and degradation. The results show that proteins have comparable stability when stored in glass syringes or in syringes made of CZ sterilized by E-beam or autoclave. In addition, proteins stressed by agitation were generally more stable and aggregated less in syringes made of CZ than in ones made of glass. LAY ABSTRACT: Biotherapeutic protein drugs such as monoclonal antibodies are frequently packaged at high concentration in prefillable syringes, which allows the drug to be directly administered by the patient or caregiver. Protein drugs, or biologics, can be unstable, and may aggregate, particularly when shaken. These aggregates can be immunogenic, stimulating the body's immune system to produce antibodies that can reduce the drug's efficacy. Although prefillable syringes are traditionally made of glass, some biologics are unstable in glass syringes due to the presence of

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

Science.gov (United States)

Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

USING BIOPOLYMERS TO STABILIZE THE PROTEIN OXYGEN FOAM

Directory of Open Access Journals (Sweden)

N. V. Nepovinnyh

2013-01-01

Full Text Available The cottage cheese whey as an oxygen cocktail foaming base and natural juices as a flavoring ingredient are analyzed. The lifetime of foam generated by the serum proteins is not long: foam falls off rapidly; because from the foam liquid is released (syneresis. The effects of plant polysaccharides on the stabilization of the protein foam oxygen cocktail is studied. It was shown that the use of plant polysaccharides (guar gum, high methoxyl citrus pectin, locust been gum prolong the life of the foam up to 20 times, compared with conventional blowing agents. It was found that oxygen foam properties depend on the molecular weight of guar gum.

CD-loop Extension in Zika Virus Envelope Protein Key for Stability and Pathogenesis.

Science.gov (United States)

Gallichotte, Emily N; Dinnon, Kenneth H; Lim, Xin-Ni; Ng, Thiam-Seng; Lim, Elisa X Y; Menachery, Vineet D; Lok, Shee-Mei; Baric, Ralph S

2017-12-05

With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barré syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A, and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type-dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus, opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

International Nuclear Information System (INIS)

Różycki, Bartosz; Cieplak, Marek

2014-01-01

We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme

Prediction of protein–protein interactions: unifying evolution and structure at protein interfaces

International Nuclear Information System (INIS)

Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

2011-01-01

The vast majority of the chores in the living cell involve protein–protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein–protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations

Microstructure and in vitro cellular response to novel soy protein-based porous structures for tissue regeneration applications.

Science.gov (United States)

Olami, Hilla; Zilberman, Meital

2016-02-01

Interest in the development of new bioresorbable structures for various tissue engineering applications is on the rise. In the current study, we developed and studied novel soy protein-based porous blends as potential new scaffolds for such applications. Soy protein has several advantages over the various types of natural proteins employed for biomedical applications due to its low price, non-animal origin and relatively long storage time and stability. In the present study, blends of soy protein with other polymers (gelatin, pectin and alginate) were added and chemically cross-linked using the cross-linking agents carbodiimide or glyoxal, and the porous structure was obtained through lyophilization. The resulting blend porous structures were characterized using environmental scanning microscopy, and the cytotoxicity of these scaffolds was examined in vitro. The biocompatibility of the scaffolds was also evaluated in vitro by seeding and culturing human fibroblasts on these scaffolds. Cell growth morphology and adhesion were examined histologically. The results show that these blends can be assembled into porous three-dimensional structures by combining chemical cross-linking with freeze-drying. The achieved blend structures combine suitable porosity with a large pore size (100-300 µm). The pore structure in the soy-alginate scaffolds possesses adequate interconnectivity compared to that of the soy-gelatin scaffolds. However, porous structure was not observed for the soy-pectin blend, which presented a different structure with significantly lower porosities than all other groups. The in vitro evaluation of these porous soy blends demonstrated that soy-alginate blends are advantageous over soy-gelatin blends and exhibited adequate cytocompatibility along with better cell infiltration and stability. These soy protein scaffolds may be potentially useful as a cellular/acellular platform for skin regeneration applications. © The Author(s) 2015.

Structural anatomy of telomere OB proteins.

Science.gov (United States)

Horvath, Martin P

2011-10-01

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.

Protein Structure and the Sequential Structure of mRNA

DEFF Research Database (Denmark)

Brunak, Søren; Engelbrecht, Jacob

1996-01-01

entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein

Directory of Open Access Journals (Sweden)

Deeksha Vishwamitra

2015-09-01

Full Text Available Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+ T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5(p23;q35 that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

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

Unifying dynamical and structural stability of equilibria

Science.gov (United States)

Arnoldi, Jean-François; Haegeman, Bart

2016-09-01

We exhibit a fundamental relationship between measures of dynamical and structural stability of linear dynamical systems-e.g. linearized models in the vicinity of equilibria. We show that dynamical stability, quantified via the response to external perturbations (i.e. perturbation of dynamical variables), coincides with the minimal internal perturbation (i.e. perturbations of interactions between variables) able to render the system unstable. First, by reformulating a result of control theory, we explain that harmonic external perturbations reflect the spectral sensitivity of the Jacobian matrix at the equilibrium, with respect to constant changes of its coefficients. However, for this equivalence to hold, imaginary changes of the Jacobian's coefficients have to be allowed. The connection with dynamical stability is thus lost for real dynamical systems. We show that this issue can be avoided, thus recovering the fundamental link between dynamical and structural stability, by considering stochastic noise as external and internal perturbations. More precisely, we demonstrate that a linear system's response to white-noise perturbations directly reflects the intensity of internal white-noise disturbance that it can accommodate before becoming stochastically unstable.

Automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal.

Science.gov (United States)

Bordoli, Lorenza; Schwede, Torsten

2012-01-01

Comparative protein structure modeling is a computational approach to build three-dimensional structural models for proteins using experimental structures of related protein family members as templates. Regular blind assessments of modeling accuracy have demonstrated that comparative protein structure modeling is currently the most reliable technique to model protein structures. Homology models are often sufficiently accurate to substitute for experimental structures in a wide variety of applications. Since the usefulness of a model for specific application is determined by its accuracy, model quality estimation is an essential component of protein structure prediction. Comparative protein modeling has become a routine approach in many areas of life science research since fully automated modeling systems allow also nonexperts to build reliable models. In this chapter, we describe practical approaches for automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal.

Automated Protein Structure Modeling with SWISS-MODEL Workspace and the Protein Model Portal

OpenAIRE

Bordoli, Lorenza; Schwede, Torsten

2012-01-01

Comparative protein structure modeling is a computational approach to build three-dimensional structural models for proteins using experimental structures of related protein family members as templates. Regular blind assessments of modeling accuracy have demonstrated that comparative protein structure modeling is currently the most reliable technique to model protein structures. Homology models are often sufficiently accurate to substitute for experimental structures in a wide variety of appl...

Micropore Structure of Cement-Stabilized Gold Mine Tailings

Directory of Open Access Journals (Sweden)

Joon Kyu Lee

2018-03-01

Full Text Available Mine tailings have often to be stabilized by mixing them with cementing agents. In this study, the pore structure of gold tailings stabilized with Portland cement was evaluated by means of mercury intrusion porosimetry. The investigation was conducted on samples prepared with different fractions of tailings and cement as well as on samples activated with elevated temperature curing and chemical (CaCl2 addition. It was observed that all mixed samples exhibit a mono-modal pore size distribution, indicating that the cement-stabilized tailings are characterized by a single-porosity structure. The results also showed that the higher fraction of tailings and cement leads to a dense and finer pore structure. The total porosity of mixture samples decreases with increasing curing temperature and CaCl2 concentration due to the acceleration of hydration reaction.

In Search of Functional Advantages of Knots in Proteins.

OpenAIRE

Dabrowski-Tumanski, P.; Stasiak, A.; Sulkowska, J.I.

2016-01-01

We analysed the structure of deeply knotted proteins representing three unrelated families of knotted proteins. We looked at the correlation between positions of knotted cores in these proteins and such local structural characteristics as the number of intra-chain contacts, structural stability and solvent accessibility. We observed that the knotted cores and especially their borders showed strong enrichment in the number of contacts. These regions showed also increased thermal stability, whe...

Beta-structures in fibrous proteins.

Science.gov (United States)

Kajava, Andrey V; Squire, John M; Parry, David A D

2006-01-01

The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

Synergistic and antagonistic effects of plant and dairy protein blends on the physicochemical stability of lycopene-loaded emulsions

NARCIS (Netherlands)

Ho, Kacie K.H.Y.; Schroën, Karin; San Martín-González, M.F.; Berton-Carabin, Claire C.

2018-01-01

Whey-plant protein-based emulsions had high physicochemical stability. Whey and plant protein blend-based interfaces were viscoelastic while casein-based interfaces were relatively viscous. Whey-plant and plant-plant protein blends behaved synergistically leading to enhanced emulsion stability.

Structural basis for the aminoacid composition of proteins from halophilic archea.

Directory of Open Access Journals (Sweden)

Xavier Tadeo

2009-12-01

Full Text Available Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.

Ultra-High Pressure Homogenization enhances physicochemical properties of soy protein isolate-stabilized emulsions.

Science.gov (United States)

Fernández-Ávila, C; Escriu, R; Trujillo, A J

2015-09-01

The effect of Ultra-High Pressure Homogenization (UHPH, 100-300MPa) on the physicochemical properties of oil-in-water emulsions prepared with 4.0% (w/v) of soy protein isolate (SPI) and soybean oil (10 and 20%, v/v) was studied and compared to emulsions treated by conventional homogenization (CH, 15MPa). CH emulsions were prepared with non-heated and heated (95°C for 15min) SPI dispersions. Emulsions were characterized by particle size determination with laser diffraction, rheological properties using a rotational rheometer by applying measurements of flow curve and by transmission electron microscopy. The variation on particle size and creaming was assessed by Turbiscan® analysis, and visual observation of the emulsions was also carried out. UHPH emulsions showed much smaller d 3.2 values and greater physical stability than CH emulsions. The thermal treatment of SPI prior CH process did not improve physical stability properties. In addition, emulsions containing 20% of oil exhibited greater physical stability compared to emulsions containing 10% of oil. Particularly, UHPH emulsions treated at 100 and 200MPa with 20% of oil were the most stable due to low particle size values (d 3.2 and Span), greater viscosity and partial protein denaturation. These results address the physical stability improvement of protein isolate-stabilized emulsions by using the emerging UHPH technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

The interface of protein structure, protein biophysics, and molecular evolution

Science.gov (United States)

Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

2012-01-01

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

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.

Influence of Tableting on the Conformation and Thermal Stability of Trypsin as a Model Protein

DEFF Research Database (Denmark)

Klukkert, Marten; Van De Weert, Marco; Fanø, Mathias

2015-01-01

was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially...... reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied...... compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs....

In Search of Functional Advantages of Knots in Proteins.

Science.gov (United States)

Dabrowski-Tumanski, Pawel; Stasiak, Andrzej; Sulkowska, Joanna I

2016-01-01

We analysed the structure of deeply knotted proteins representing three unrelated families of knotted proteins. We looked at the correlation between positions of knotted cores in these proteins and such local structural characteristics as the number of intra-chain contacts, structural stability and solvent accessibility. We observed that the knotted cores and especially their borders showed strong enrichment in the number of contacts. These regions showed also increased thermal stability, whereas their solvent accessibility was decreased. Interestingly, the active sites within these knotted proteins preferentially located in the regions with increased number of contacts that also have increased thermal stability and decreased solvent accessibility. Our results suggest that knotting of polypeptide chains provides a favourable environment for the active sites observed in knotted proteins. Some knotted proteins have homologues without a knot. Interestingly, these unknotted homologues form local entanglements that retain structural characteristics of the knotted cores.

Effect of calcium ions on structure and stability of the C1q-like domain of otolin-1 from human and zebrafish.

Science.gov (United States)

Hołubowicz, Rafał; Wojtas, Magdalena; Taube, Michał; Kozak, Maciej; Ożyhar, Andrzej; Dobryszycki, Piotr

2017-12-01

Otolin-1 is a collagen-like protein expressed in the inner ear of vertebrates. It provides an organic scaffold for otoliths in fish and otoconia in land vertebrates. In this study, the expression and purification procedure of C1q-like domain of otolin-1 from human and zebrafish was developed. The structure and stability of the proteins were investigated. The results of sedimentation velocity analytical ultracentrifugation and small-angle X-ray scattering indicated that the C1q-like domain of otolin-1 forms stable trimers in solution in the presence of calcium ions. It was also observed that calcium ions influenced the secondary structure of the proteins. C1q-like domains were stabilized by the calcium ions. The human variant was especially affected by the calcium ions. The results indicate the importance of the C1q-like domain for the assembly of the organic matrix of otoliths and otoconia. © 2017 Federation of European Biochemical Societies.

Neural Networks for protein Structure Prediction

DEFF Research Database (Denmark)

Bohr, Henrik

1998-01-01

This is a review about neural network applications in bioinformatics. Especially the applications to protein structure prediction, e.g. prediction of secondary structures, prediction of surface structure, fold class recognition and prediction of the 3-dimensional structure of protein backbones...

Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

International Nuclear Information System (INIS)

Yu, P.

2007-01-01

The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

Effects of mannose, fructose, and fucose on the structure, stability, and hydration of lysozyme in aqueous solution

DEFF Research Database (Denmark)

Rahim, Abdoul; Peters, Günther H.J.; Jalkanen, Karl J.

2013-01-01

The bio-protective properties of monosaccharaides, namely mannose, fructose and fucose, on the stability and dynamical properties of the NMR determined hen egg-white lysozyme structure have been investigated by means of molecular dynamics simulations at room temperature in aqueous solution and in...... of the solvent and sugar distributions around lysozyme was used to investigate the interfacial solvent and sugar structure near the protein surface.......The bio-protective properties of monosaccharaides, namely mannose, fructose and fucose, on the stability and dynamical properties of the NMR determined hen egg-white lysozyme structure have been investigated by means of molecular dynamics simulations at room temperature in aqueous solution and in 7...... and 13 wt % concentrations of the three sugars. Results are discussed in the framework of the bio-protective phenomena. The three sugars show similar bio-protective behaviours at room temperature (300 K) in the concentration range studied as shown by the small RMSDs of the resulting MD structures from...

Computing Stability Effects of Mutations in Human Superoxide Dismutase 1

DEFF Research Database (Denmark)

Kepp, Kasper Planeta

2014-01-01

Protein stability is affected in several diseases and is of substantial interest in efforts to correlate genotypes to phenotypes. Superoxide dismutase 1 (SOD1) is a suitable test case for such correlations due to its abundance, stability, available crystal structures and thermochemical data......, and physiological importance. In this work, stability changes of SOD1 mutations were computed with five methods, CUPSAT, I-Mutant2.0, I-Mutant3.0, PoPMuSiC, and SDM, with emphasis on structural sensitivity as a potential issue in structure-based protein calculation. The large correlation between experimental...... literature data of SOD1 dimers and monomers (r = 0.82) suggests that mutations in separate protein monomers are mostly additive. PoPMuSiC was most accurate (typical MAE ∼ 1 kcal/mol, r ∼ 0.5). The relative performance of the methods was not very structure-dependent, and the more accurate methods also...

In vivo stability and inertness of various direct labelled and chelate-tagged protein

International Nuclear Information System (INIS)

Janoki, A.; Korosi, L.; Klivenyi, G.; Spett, B.

1987-01-01

There were looking for methods giving precise information about composition and activity distribution of protein components, both in the initial samples and serum samples after intravenous administration. It was tested the applicability of electroimmunoassay, polyacrilamide gel electrophoresis and high performance liquid chromatography for the assessment of in vivo stability and labelled proteins. The model compound was human serum albumin (HSA) labelled with 99m Tc and 125 I, respectively. Bifunctional chelate labelling was done with desferrioxamine, in this case protein was labelled with 67 Ga. Biodistribution of the labelled compounds and their elimination from the blood were studied in rabbits. Experience with various labelling proteins, especially with Tc-Sn-HSA system indicate that in vivo stability of this compounds are generally low. Following intravenous injection of proteins labelled with metal isotopes, due to dilution and to the presence of considerable amount of compatitive protein in the serum, part of the label is being detached from the carrier protein. Distribution of the detached metal is different from the original distribution of the protein. This problem arises also with radiopharmaceuticals based on monoclonal antibodies. (M.E.L.) [es

Biophysics of protein evolution and evolutionary protein biophysics

Science.gov (United States)

Sikosek, Tobias; Chan, Hue Sun

2014-01-01

The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

Structure based alignment and clustering of proteins (STRALCP)

Science.gov (United States)

Zemla, Adam T.; Zhou, Carol E.; Smith, Jason R.; Lam, Marisa W.

2013-06-18

Disclosed are computational methods of clustering a set of protein structures based on local and pair-wise global similarity values. Pair-wise local and global similarity values are generated based on pair-wise structural alignments for each protein in the set of protein structures. Initially, the protein structures are clustered based on pair-wise local similarity values. The protein structures are then clustered based on pair-wise global similarity values. For each given cluster both a representative structure and spans of conserved residues are identified. The representative protein structure is used to assign newly-solved protein structures to a group. The spans are used to characterize conservation and assign a "structural footprint" to the cluster.

Validation-driven protein-structure improvement

NARCIS (Netherlands)

Touw, W.G.

2016-01-01

High-quality protein structure models are essential for many Life Science applications, such as protein engineering, molecular dynamics, drug design, and homology modelling. The WHAT_CHECK model validation project and the PDB_REDO model optimisation project have shown that many structure models in

Understanding Protein-Protein Interactions Using Local Structural Features

DEFF Research Database (Denmark)

Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier

2013-01-01

Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... interacting and non-interacting protein pairs to classify the structural features that sustain the binding (or non-binding) behavior. Our study indicates that not only the interacting region but also the rest of the protein surface are important for the interaction fate. The interpretation...... to score the likelihood of the interaction between two proteins and to develop a method for the prediction of PPIs. We have tested our method on several sets with unbalanced ratios of interactions and non-interactions to simulate real conditions, obtaining accuracies higher than 25% in the most unfavorable...

The effect of charge-introduction mutations on E. coli thioredoxin stability.

Science.gov (United States)

Perez-Jimenez, Raul; Godoy-Ruiz, Raquel; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

2005-04-01

Technological applications of proteins are often hampered by their low-stability and, consequently, the development of procedures for protein stabilization is of considerable biotechnological interest. Here, we use simple electrostatics to determine positions in E. coli thioredoxin at which mutations that introduce new charged residues are expected to lead to stability enhancement. We also obtain the corresponding mutants and characterize their stability using differential scanning calorimetry. The results are interpreted in terms of the accessibility in the native structure of the mutated residues and the potential effect of the mutations on the residual structure of the denatured state.

Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

Directory of Open Access Journals (Sweden)

Kevin A James

Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

Effect of high-intensity ultrasound on the technofunctional properties and structure of jackfruit (Artocarpus heterophyllus) seed protein isolate.

Science.gov (United States)

Resendiz-Vazquez, J A; Ulloa, J A; Urías-Silvas, J E; Bautista-Rosales, P U; Ramírez-Ramírez, J C; Rosas-Ulloa, P; González-Torres, L

2017-07-01

The influence of high-intensity ultrasound (HIU) on the technofunctional properties and structure of jackfruit seed protein isolate (JSPI) was investigated. Protein solutions (10%, w/v) were sonicated for 15min at 20kHz to the following levels of power output: 200, 400, and 600W (pulse duration: on-time, 5s; off-time 1s). Compared with untreated JSPI, HIU at 200W and 400W improved the oil holding capacity (OHC) and emulsifying capacity (EC), but the emulsifying activity (EA) and emulsion stability (ES) increased at 400W and 600W. The foaming capacity (FC) increased after all HIU treatments, as opposed to the water holding capacity (WHC), least gelation concentration (LGC), and foaming stability (FS), which all decreased except at pH 4 for FS. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) showed changes in the molecular weight of protein fractions after HIU treatment. Scanning electron microscopy (SEM) demonstrated that HIU disrupted the microstructure of JSPI, exhibiting larger aggregates. Surface hydrophobicity and protein solubility of the JSPI dispersions were enhanced after ultrasonication, which increased the destruction of internal hydrophobic interactions of protein molecules and accelerated the molecular motion of proteins to cause protein aggregation. These changes in the technofunctional and structural properties of JSPI could meet the complex needs of manufactured food products. Copyright © 2017 Elsevier B.V. All rights reserved.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

Directory of Open Access Journals (Sweden)

Yushen Du

2016-11-01

Full Text Available Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp, we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.

Spray dried microparticles of chia oil using emulsion stabilized by whey protein concentrate and pectin by electrostatic deposition.

Science.gov (United States)

Noello, C; Carvalho, A G S; Silva, V M; Hubinger, M D

2016-11-01

Chia seed oil has a high content of α-linolenic acid (60%) and linoleic acid (20%). Use of this oil in different products is limited due to its liquid state, and the presence of insaturation is a trigger for oxidation. In this context, to facilitate the incorporation of chia oil in food products and increase its protection against oxidation, the aim of this work was to produce chia oil microparticles by spray drying using emulsions stabilized by whey protein concentrate (ζ-potential +13.4 at pH3.8) and pectin (ζ-potential -40.4 at pH3.8) through the electrostatic layer-by-layer deposition technique and emulsions prepared with only whey protein concentrate. Emulsions stabilized by whey protein concentrate and stabilized by whey protein concentrate-pectin were prepared using maltodextrin (10 DE) and modified starch (Hi-Cap® 100). They were characterized in relation to stability, droplet size, ζ-Potential and optical microscopy. The microparticles were characterized in relation to moisture content, water activity, particle size, microstructure and oxidative stability by the Rancimat method. Emulsions stabilized by whey protein concentrate-pectin with added maltodextrin 10 DE and emulsions stabilized by whey protein concentrate with added modified starch (Hi-Cap® 100) were stable after 24h. Emulsions stabilized by whey protein concentrate and by whey protein concentrate-pectin showed droplets with mean diameter ranging from 0.80 to 1.31μm, respectively and ζ-potential varying from -6.9 to -27.43mV, respectively. After spray drying, the microparticles showed an mean diameter ranging from 7.00 to 9.00μm. All samples presented high encapsulation efficiency values, above 99%. Microparticles produced with modified starch showed a smoother spherical surface than particles with maltodextrin 10 DE, which presented a wrinkled surface. All microparticles exhibited higher oxidative stability than chia oil in pure form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efficient protein structure search using indexing methods.

Science.gov (United States)

Kim, Sungchul; Sael, Lee; Yu, Hwanjo

2013-01-01

Understanding functions of proteins is one of the most important challenges in many studies of biological processes. The function of a protein can be predicted by analyzing the functions of structurally similar proteins, thus finding structurally similar proteins accurately and efficiently from a large set of proteins is crucial. A protein structure can be represented as a vector by 3D-Zernike Descriptor (3DZD) which compactly represents the surface shape of the protein tertiary structure. This simplified representation accelerates the searching process. However, computing the similarity of two protein structures is still computationally expensive, thus it is hard to efficiently process many simultaneous requests of structurally similar protein search. This paper proposes indexing techniques which substantially reduce the search time to find structurally similar proteins. In particular, we first exploit two indexing techniques, i.e., iDistance and iKernel, on the 3DZDs. After that, we extend the techniques to further improve the search speed for protein structures. The extended indexing techniques build and utilize an reduced index constructed from the first few attributes of 3DZDs of protein structures. To retrieve top-k similar structures, top-10 × k similar structures are first found using the reduced index, and top-k structures are selected among them. We also modify the indexing techniques to support θ-based nearest neighbor search, which returns data points less than θ to the query point. The results show that both iDistance and iKernel significantly enhance the searching speed. In top-k nearest neighbor search, the searching time is reduced 69.6%, 77%, 77.4% and 87.9%, respectively using iDistance, iKernel, the extended iDistance, and the extended iKernel. In θ-based nearest neighbor serach, the searching time is reduced 80%, 81%, 95.6% and 95.6% using iDistance, iKernel, the extended iDistance, and the extended iKernel, respectively.

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.

A 'periodic table' for protein structures.

Science.gov (United States)

Taylor, William R

2002-04-11

Current structural genomics programs aim systematically to determine the structures of all proteins coded in both human and other genomes, providing a complete picture of the number and variety of protein structures that exist. In the past, estimates have been made on the basis of the incomplete sample of structures currently known. These estimates have varied greatly (between 1,000 and 10,000; see for example refs 1 and 2), partly because of limited sample size but also owing to the difficulties of distinguishing one structure from another. This distinction is usually topological, based on the fold of the protein; however, in strict topological terms (neglecting to consider intra-chain cross-links), protein chains are open strings and hence are all identical. To avoid this trivial result, topologies are determined by considering secondary links in the form of intra-chain hydrogen bonds (secondary structure) and tertiary links formed by the packing of secondary structures. However, small additions to or loss of structure can make large changes to these perceived topologies and such subjective solutions are neither robust nor amenable to automation. Here I formalize both secondary and tertiary links to allow the rigorous and automatic definition of protein topology.

Atomistic mechanisms governing structural stability change of zinc antimony thermoelectrics

Energy Technology Data Exchange (ETDEWEB)

Yang, Xiaolong [Frontier Institute of Science and Technology, Xi' an Jiaotong University, Xi' an 710054 (China); Lin, Jianping, E-mail: jaredlin@163.com [School of Materials Science and Engineering, Xiamen University of Technology, Xiamen 361024 (China); Qiao, Guanjun [State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China); Wang, Zhao, E-mail: zwangzhao@gmail.com [Frontier Institute of Science and Technology, Xi' an Jiaotong University, Xi' an 710054 (China); State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China)

2015-01-05

The structural stability of thermoelectric materials is a subject of growing importance for their energy harvesting applications. Here, we study the microscopic mechanisms governing the structural stability change of zinc antimony at its working temperature, using molecular dynamics combined with experimental measurements of the electrical and thermal conductivity. Our results show that the temperature-dependence of the thermal and electrical transport coefficients is strongly correlated with a structural transition. This is found to be associated with a relaxation process, in which a group of Zn atoms migrates between interstitial sites. This atom migration gradually leads to a stabilizing structural transition of the entire crystal framework, and then results in a more stable crystal structure of β–Zn{sub 4}Sb{sub 3} at high temperature.

Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions.

Science.gov (United States)

Sutariya, Suresh; Patel, Hasmukh

2017-05-15

Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H 2 O 2 in the range of 0-0.144 H 2 O 2 to protein ratios (HTPR) by the addition of the required quantity of H 2 O 2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H 2 O 2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H 2 O 2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H 2 O 2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein structure recognition: From eigenvector analysis to structural threading method

Science.gov (United States)

Cao, Haibo

In this work, we try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. We found a strong correlation between amino acid sequence and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, we give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part include discussions of interactions among amino acids residues, lattice HP model, and the designablity principle. In the second part, we try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in our eigenvector study of protein contact matrix. We believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, we discuss a threading method based on the correlation between amino acid sequence and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, we list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches.

Protein Structure Recognition: From Eigenvector Analysis to Structural Threading Method

Energy Technology Data Exchange (ETDEWEB)

Cao, Haibo [Iowa State Univ., Ames, IA (United States)

2003-01-01

In this work, they try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. They found a strong correlation between amino acid sequences and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, they give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part includes discussions of interactions among amino acids residues, lattice HP model, and the design ability principle. In the second part, they try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in the eigenvector study of protein contact matrix. They believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, they discuss a threading method based on the correlation between amino acid sequences and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, they list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches.

Protein Structure Recognition: From Eigenvector Analysis to Structural Threading Method

International Nuclear Information System (INIS)

Haibo Cao

2003-01-01

In this work, they try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. They found a strong correlation between amino acid sequences and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, they give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part includes discussions of interactions among amino acids residues, lattice HP model, and the design ability principle. In the second part, they try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in the eigenvector study of protein contact matrix. They believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, they discuss a threading method based on the correlation between amino acid sequences and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, they list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches

Innovative Ingredients and Emerging Technologies for Controlling Ice Recrystallization, Texture, and Structure Stability in Frozen Dairy Desserts: A Review.

Science.gov (United States)

Soukoulis, Christos; Fisk, Ian

2016-11-17

Over the past decade, ice cream manufacturers have developed a strong understanding of the functionality of key ingredients and processing, developing effective explanations for the link between structure forming agents, stability mechanisms, and perceived quality. Increasing demand for products perceived as healthier/more natural with minimal processing has identified a number of new tools to improve quality and storage stability of frozen dairy desserts. Ingredients such as dietary fiber, polysaccharides, prebiotics, alternate sweeteners, fat sources rich in unsaturated fatty acids and ice strucsturing proteins (ISP) have been successfully applied as cryoprotective, texturizing, and structuring agents. Emerging minimal processing technologies including hydrostatic pressure processing, ultrasonic or high pressure assisted freezing, low temperature extrusion and enzymatically induced biopolymers crosslinking have been evaluated for their ability to improve colloidal stability, texture and sensory quality. It is therefore timely for a comprehensive review.

Force spectroscopy studies on protein-ligand interactions: a single protein mechanics perspective.

Science.gov (United States)

Hu, Xiaotang; Li, Hongbin

2014-10-01

Protein-ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein-ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein-ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein-ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Structures composing protein domains.

Science.gov (United States)

Kubrycht, Jaroslav; Sigler, Karel; Souček, Pavel; Hudeček, Jiří

2013-08-01

This review summarizes available data concerning intradomain structures (IS) such as functionally important amino acid residues, short linear motifs, conserved or disordered regions, peptide repeats, broadly occurring secondary structures or folds, etc. IS form structural features (units or elements) necessary for interactions with proteins or non-peptidic ligands, enzyme reactions and some structural properties of proteins. These features have often been related to a single structural level (e.g. primary structure) mostly requiring certain structural context of other levels (e.g. secondary structures or supersecondary folds) as follows also from some examples reported or demonstrated here. In addition, we deal with some functionally important dynamic properties of IS (e.g. flexibility and different forms of accessibility), and more special dynamic changes of IS during enzyme reactions and allosteric regulation. Selected notes concern also some experimental methods, still more necessary tools of bioinformatic processing and clinically interesting relationships. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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).

Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking

NARCIS (Netherlands)

Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

2015-01-01

A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80. °C for 15. min. During heating of w/o emulsions containing 10% (w/v) WPI

Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy.

Science.gov (United States)

McHaourab, Hassane S; Steed, P Ryan; Kazmier, Kelli

2011-11-09

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 Å-80 Å) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

Protein structure similarity from principle component correlation analysis

Directory of Open Access Journals (Sweden)

Chou James

2006-01-01

Full Text Available Abstract Background Owing to rapid expansion of protein structure databases in recent years, methods of structure comparison are becoming increasingly effective and important in revealing novel information on functional properties of proteins and their roles in the grand scheme of evolutionary biology. Currently, the structural similarity between two proteins is measured by the root-mean-square-deviation (RMSD in their best-superimposed atomic coordinates. RMSD is the golden rule of measuring structural similarity when the structures are nearly identical; it, however, fails to detect the higher order topological similarities in proteins evolved into different shapes. We propose new algorithms for extracting geometrical invariants of proteins that can be effectively used to identify homologous protein structures or topologies in order to quantify both close and remote structural similarities. Results We measure structural similarity between proteins by correlating the principle components of their secondary structure interaction matrix. In our approach, the Principle Component Correlation (PCC analysis, a symmetric interaction matrix for a protein structure is constructed with relationship parameters between secondary elements that can take the form of distance, orientation, or other relevant structural invariants. When using a distance-based construction in the presence or absence of encoded N to C terminal sense, there are strong correlations between the principle components of interaction matrices of structurally or topologically similar proteins. Conclusion The PCC method is extensively tested for protein structures that belong to the same topological class but are significantly different by RMSD measure. The PCC analysis can also differentiate proteins having similar shapes but different topological arrangements. Additionally, we demonstrate that when using two independently defined interaction matrices, comparison of their maximum

Advances in Computational Stability Analysis of Composite Aerospace Structures

International Nuclear Information System (INIS)

Degenhardt, R.; Araujo, F. C. de

2010-01-01

European aircraft industry demands for reduced development and operating costs. Structural weight reduction by exploitation of structural reserves in composite aerospace structures contributes to this aim, however, it requires accurate and experimentally validated stability analysis of real structures under realistic loading conditions. This paper presents different advances from the area of computational stability analysis of composite aerospace structures which contribute to that field. For stringer stiffened panels main results of the finished EU project COCOMAT are given. It investigated the exploitation of reserves in primary fibre composite fuselage structures through an accurate and reliable simulation of postbuckling and collapse. For unstiffened cylindrical composite shells a proposal for a new design method is presented.

Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition.

Science.gov (United States)

Schlundt, Andreas; Tants, Jan-Niklas; Sattler, Michael

2017-04-15

Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain

Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).

Science.gov (United States)

Yong, K J; Scott, D J

2015-03-01

Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS. © 2014 Wiley Periodicals, Inc.

Stability of globular proteins in H2O and D2O

NARCIS (Netherlands)

Efimova, Y. M.; Haemers, S.; Wierczinski, B.; Norde, W.; van Well, A. A.

2007-01-01

In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the

Recent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applications.

Science.gov (United States)

Sivapragasam, Magaret; Moniruzzaman, Muhammad; Goto, Masahiro

2016-08-01

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) - potentially attractive "green" and "designer" solvents - rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule-based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein-, enzyme-, and DNA-based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule-friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL-based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule-based applications. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SDSL-ESR-based protein structure characterization

NARCIS (Netherlands)

Strancar, J.; Kavalenka, A.A.; Urbancic, I.; Ljubetic, A.; Hemminga, M.A.

2010-01-01

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

Science.gov (United States)

Oulhen, Nathalie; Wessel, Gary M

2016-10-01

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. Copyright © 2016 Elsevier Inc. All rights reserved.

Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

DEFF Research Database (Denmark)

Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta

2018-01-01

In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest....... The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously...... been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool....

Metal Stabilization of Collagen and de Novo Designed Mimetic Peptides.

Science.gov (United States)

Parmar, Avanish S; Xu, Fei; Pike, Douglas H; Belure, Sandeep V; Hasan, Nida F; Drzewiecki, Kathryn E; Shreiber, David I; Nanda, Vikas

2015-08-18

We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo- and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal-specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.

Effects of Ecohydraulic Bank Stabilization Structures on Bank Stability and Macroinvertebrate Community in Surabaya River

Directory of Open Access Journals (Sweden)

Daru Setyo Rini

2018-01-01

Full Text Available There were 18 accelerated erosion sites identified along 7 km of Surabaya River Fishery Sanctuary Area. A model of ecohydraulic bank stabilization was applied to reduce bank erosion in Surabaya River at Gresik Regency Indonesia. The model is combination of reprofiled and revegetated bank with rock toe reinforcement and  addition of log groynes. Various native plant species were planted and naturally grown to establish multi-strata littoral vegetation structure. This study assessed effects of ecohydraulic bank stabilization on bank morphology, near bank velocity and littoral macroinvertebrate community during September 2014 to August 2016. The study found that rock toe enforcement, log groynes and reprofiled bank slope could stabilized the eroded bank, and littoral vegetation formation reduced near bank velocity at restored sites. There were 31 families of macroinvertebrate found in Surabaya River with high abundance of moderately pollution sensitive taxa Atyidae and pollution tolerant taxa Corixidae, Chironomidae and Tubificidae. The taxa richness, diversity index and abundance of sensitive and moderately sensitive macroinvertebrate group were increased after application of ecohydraulic bank stabilization at restored area. The results shown that ecohydraulic bank stabilization structure provides multi-benefits in improving bank stabilization against erosion and providing new micro-habitats for biotic community. Keywords:  ecohydraulic bank stabilization, macroinvertebrates, riparian restoration

Structure stability index allocation theory and measurement of laser prototype facility

International Nuclear Information System (INIS)

Zhang Junwei; China Academy of Engineering Physics, Mianyang; Zhou Hai; Feng Bin; Lin Donghui; Jing Feng; Zhou Yi; Wang Shilong

2008-01-01

Structure stability is an important design index of ICF driver. Based on laser prototype facility(TIL) design characteristic of multi-pass amplifier and frame structure, the optical matrix is used to analyze the single optical element influence on the beam drift and get the mathematic model. Considering all the optical elements influence on the beam drift, the mathematic model of the optical element stability index allocation is built, the parameter relation of the mathematic model is defined according to the structure characteristic of TIL, the stability index of each optical element is got as the support structure design index. Charge-coupled device(CCD) detect technology is used to measure the general beam stability of TIL. The root mean square beam drift in x and y direction are 2.78 μm, the difference between peak and valley values are 14.4 μm and 15.60 μm, respectively. The result indicates that the stability drift of the prototype facility can satisfy the design requirement, the way of the stability allocation is reasonable. (authors)

Do we see what we should see? Describing non-covalent interactions in protein structures including precision

Directory of Open Access Journals (Sweden)

Manickam Gurusaran

2014-01-01

Full Text Available The power of X-ray crystal structure analysis as a technique is to `see where the atoms are'. The results are extensively used by a wide variety of research communities. However, this `seeing where the atoms are' can give a false sense of security unless the precision of the placement of the atoms has been taken into account. Indeed, the presentation of bond distances and angles to a false precision (i.e. to too many decimal places is commonplace. This article has three themes. Firstly, a basis for a proper representation of protein crystal structure results is detailed and demonstrated with respect to analyses of Protein Data Bank entries. The basis for establishing the precision of placement of each atom in a protein crystal structure is non-trivial. Secondly, a knowledge base harnessing such a descriptor of precision is presented. It is applied here to the case of salt bridges, i.e. ion pairs, in protein structures; this is the most fundamental place to start with such structure-precision representations since salt bridges are one of the tenets of protein structure stability. Ion pairs also play a central role in protein oligomerization, molecular recognition of ligands and substrates, allosteric regulation, domain motion and α-helix capping. A new knowledge base, SBPS (Salt Bridges in Protein Structures, takes these structural precisions into account and is the first of its kind. The third theme of the article is to indicate natural extensions of the need for such a description of precision, such as those involving metalloproteins and the determination of the protonation states of ionizable amino acids. Overall, it is also noted that this work and these examples are also relevant to protein three-dimensional structure molecular graphics software.

Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study.

Science.gov (United States)

Németh, Attila; Kamondi, Szilárd; Szilágyi, András; Magyar, Csaba; Kovári, Zoltán; Závodszky, Péter

2002-05-02

Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.

Stabilization and structural adjustment in Mozambique