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Nucleic Acids Research Jul 2018Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a...
Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a limited number of relevant experimental tools. Widely-used experimental techniques, such as calorimetry or spectroscopy, critically depend on a proper data analysis. Currently, there are only separate data analysis tools available for each type of experiment with a limited model selection. To address this problem, we have developed the CalFitter web server to be a unified platform for comprehensive data fitting and analysis of protein thermal denaturation data. The server allows simultaneous global data fitting using any combination of input data types and offers 12 protein unfolding pathway models for selection, including irreversible transitions often missing from other tools. The data fitting produces optimal parameter values, their confidence intervals, and statistical information to define unfolding pathways. The server provides an interactive and easy-to-use interface that allows users to directly analyse input datasets and simulate modelled output based on the model parameters. CalFitter web server is available free at https://loschmidt.chemi.muni.cz/calfitter/.
Topics: Computational Biology; Internet; Models, Molecular; Protein Denaturation; Protein Folding; Protein Unfolding; Software
PubMed: 29762722
DOI: 10.1093/nar/gky358 -
Post-transcriptional expression regulation in the yeast Saccharomyces cerevisiae on a genomic scale.Molecular & Cellular Proteomics : MCP Nov 2004Based on large-scale data for the yeast Saccharomyces cerevisiae (protein and mRNA abundance, translational status, transcript length), we investigate the relation of...
Based on large-scale data for the yeast Saccharomyces cerevisiae (protein and mRNA abundance, translational status, transcript length), we investigate the relation of transcription, translation, and protein turnover on a genome-wide scale. We elucidate variations between different spatial cell compartments and functional modules by comparing protein-to-mRNA ratios, translational activity, and a novel descriptor for protein-specific degradation (protein half-life descriptor). This analysis helps to understand the cell's strategy to use transcriptional and post-transcriptional regulation mechanisms for managing protein levels. For instance, it is possible to identify modules that are subject to suppressed translation under normal conditions ("translation on demand"). In order to reduce inconsistencies between the datasets, we compiled a new reference mRNA abundance dataset and we present a novel approach to correct large microarray signals for a saturation bias. Accounting for ribosome density based on transcript length rather than ORF length improves the correlation of observed protein levels to translational activity. We discuss potential causes for the deviations of these correlations. Finally, we introduce a quantitative descriptor for protein degradation (protein half-life descriptor) and compare it to measured half-lives. The study demonstrates significant post-transcriptional control of protein levels for a number of different compartments and functional modules, which is missed when exclusively focusing on transcript levels.
Topics: Cell Compartmentation; Computational Biology; Gene Expression Regulation, Fungal; Genome, Fungal; Protein Biosynthesis; Protein Denaturation; Proteome; RNA Processing, Post-Transcriptional; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 15326222
DOI: 10.1074/mcp.M400099-MCP200 -
International Journal of Molecular... Sep 2022Thermophilic proteins have various practical applications in theoretical research and in industry. In recent years, the demand for thermophilic proteins on an industrial...
Thermophilic proteins have various practical applications in theoretical research and in industry. In recent years, the demand for thermophilic proteins on an industrial scale has been increasing; therefore, the engineering of thermophilic proteins has become a hot direction in the field of protein engineering. However, the exact mechanism of thermostability of proteins is not yet known, for engineering thermophilic proteins knowing the basis of thermostability is necessary. In order to understand the basis of the thermostability in proteins, we have made a statistical analysis of the sequences, secondary structures, hydrogen bonds, salt bridges, DHA (Donor-Hydrogen-Accepter) angles, and bond lengths of ten pairs of thermophilic proteins and their non-thermophilic orthologous. Our findings suggest that polar amino acids contribute to thermostability in proteins by forming hydrogen bonds and salt bridges which provide resistance against protein denaturation. Short bond length and a wider DHA angle provide greater bond stability in thermophilic proteins. Moreover, the increased frequency of aromatic amino acids in thermophilic proteins contributes to thermal stability by forming more aromatic interactions. Additionally, the coil, helix, and loop in the secondary structure also contribute to thermostability.
Topics: Amino Acids; Hydrogen Bonding; Protein Denaturation; Protein Engineering; Proteins; Temperature
PubMed: 36077513
DOI: 10.3390/ijms231710116 -
Physical Chemistry Chemical Physics :... Jan 2018Solution pH plays an important role in protein dynamics, stability, and folding; however, detailed mechanisms remain poorly understood. Here we use continuous constant...
Solution pH plays an important role in protein dynamics, stability, and folding; however, detailed mechanisms remain poorly understood. Here we use continuous constant pH molecular dynamics in explicit solvent with pH replica exchange to describe the pH profile of the folding cooperativity of a miniprotein BBL, which has drawn intense debate in the past. Our data reconciled the two opposing hypotheses (downhill vs. two-state) and uncovered a sparsely populated unfolding intermediate. As pH is lowered from 7 to 5, the folding barrier vanishes. As pH continues to decrease, the unfolding barrier lowers and denaturation is triggered by the protonation of Asp162, consistent with experimental evidence. Interestingly, unfolding proceeded via an intermediate, with intact secondary structure and a compact, unlocked hydrophobic core shielded from solvent, lending support to the recent hypothesis of a universal dry molten globule in protein folding. Our work demonstrates that constant pH molecular dynamics is a unique tool for testing this and other hypotheses to advance the knowledge in protein dynamics, stability, and folding.
Topics: Hydrogen-Ion Concentration; Nuclear Magnetic Resonance, Biomolecular; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Thermodynamics
PubMed: 29336449
DOI: 10.1039/c7cp08296g -
Cellular and Molecular Life Sciences :... Mar 1997The stabilization of proteins is discussed from the theoretical and practical points of view. Methods are described for kinetic stabilization and protection from... (Review)
Review
The stabilization of proteins is discussed from the theoretical and practical points of view. Methods are described for kinetic stabilization and protection from deterioration, as well as the thermodynamic stabilization of proteins.
Topics: Animals; Humans; Hydrogen-Ion Concentration; Protein Denaturation; Proteins; Thermodynamics
PubMed: 9104483
DOI: 10.1007/pl00000593 -
Journal of Chemical Theory and... Nov 2015Chemical denaturants are the most commonly used perturbation applied to study protein stability and folding kinetics as well as the properties of unfolded polypeptides....
Chemical denaturants are the most commonly used perturbation applied to study protein stability and folding kinetics as well as the properties of unfolded polypeptides. We build on recent work balancing the interactions of proteins and water, and accurate models for the solution properties of urea and guanidinium chloride, to develop a combined force field that is able to capture the strength of interactions between proteins and denaturants. We use solubility data for a model tetraglycine peptide in each denaturant to tune the protein-denaturant interaction by a novel simulation methodology. We validate the results against data for more complex sequences: single-molecule Förster resonance energy transfer data for a 34-residue fragment of the globular protein CspTm and photoinduced electron transfer quenching data for the disordered peptides C(AGQ)nW in denaturant solution as well as the chemical denaturation of the mini-protein Trp cage. The combined force field model should aid our understanding of denaturation mechanisms and the interpretation of experiment.
Topics: Models, Molecular; Molecular Dynamics Simulation; Peptides; Protein Denaturation; Proteins; Urea
PubMed: 26574341
DOI: 10.1021/acs.jctc.5b00778 -
Biophysical Journal Dec 2015The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length...
The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining (3)JNC' couplings transmitted through H-bonds, the temperature and urea-concentration dependence of (1)HN and (15)N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and (3)JNC' H-bond couplings, are identified with an accuracy of 90% by (1)HN temperature coefficients. The accuracy is improved to 95% when (15)N temperature coefficients are also included. In contrast, the urea dependence of (1)HN and (15)N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility.
Topics: Bacteriophage P22; Capsid Proteins; Hydrogen Bonding; Models, Molecular; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Solvents; Temperature; Urea
PubMed: 26682823
DOI: 10.1016/j.bpj.2015.11.010 -
Protein Science : a Publication of the... Mar 2016Determining the energetics of the unfolded state of a protein is essential for understanding the folding mechanics of ordered proteins and the structure-function...
Determining the energetics of the unfolded state of a protein is essential for understanding the folding mechanics of ordered proteins and the structure-function relation of intrinsically disordered proteins. Here, we adopt a coil-globule transition theory to develop a general scheme to extract interaction and free energy information from single-molecule fluorescence resonance energy transfer spectroscopy. By combining protein stability data, we have determined the free energy difference between the native state and the maximally collapsed denatured state in a number of systems, providing insight on the specific/nonspecific interactions in protein folding. Both the transfer and binding models of the denaturant effects are demonstrated to account for the revealed linear dependence of inter-residue interactions on the denaturant concentration, and are thus compatible under the coil-globule transition theory to further determine the dimension and free energy of the conformational ensemble of the unfolded state. The scaling behaviors and the effective θ-state are also discussed.
Topics: Fluorescence Resonance Energy Transfer; Protein Conformation; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Thermodynamics
PubMed: 26683260
DOI: 10.1002/pro.2865 -
Journal of Computational Chemistry Sep 2012Accounting for target flexibility and selecting "hot spots" most likely to be able to bind an inhibitor continue to be challenges in the field of structure-based drug...
Accounting for target flexibility and selecting "hot spots" most likely to be able to bind an inhibitor continue to be challenges in the field of structure-based drug design, especially in the case of protein-protein interactions. Computational fragment-based approaches using molecular dynamics (MD) simulations are a promising emerging technology having the potential to address both of these challenges. However, the optimal MD conditions permitting sufficient target flexibility while also avoiding fragment-induced target denaturation remain ambiguous. Using one such technology (Site Identification by Ligand Competitive Saturation, SILCS), conditions were identified to either prevent denaturation or identify and exclude trajectories in which subtle but important denaturation was occurring. The target system used was the well-characterized protein cytokine IL-2, which is involved in a protein-protein interface and, in its unliganded crystallographic form, lacks surface pockets that can serve as small-molecule binding sites. Nonetheless, small-molecule inhibitors have previously been discovered that bind to two "cryptic" binding sites that emerge only in the presence of ligand binding, highlighting the important role of IL-2 flexibility. Using the above conditions, SILCS with hydrophobic fragments was able to identify both sites based on favorable fragment binding while avoiding IL-2 denaturation. An important additional finding was that acetonitrile, a water-miscible fragment, fails to identify either site yet can induce target denaturation, highlighting the importance of fragment choice.
Topics: Binding Sites; Crystallography, X-Ray; Drug Discovery; Interleukin-2; Ligands; Models, Molecular; Molecular Dynamics Simulation; Protein Denaturation; Software; Structure-Activity Relationship
PubMed: 22641475
DOI: 10.1002/jcc.23026 -
Protein Science : a Publication of the... Dec 2003A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and...
A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use (17)O and (2)H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein. Whereas CD shows an apparently two-state transition, MRD reveals a more complex process involving at least two intermediates. At least one water molecule binds persistently (with residence time >10 nsec) to the protein even in 7.5 M urea, where the large internal binding cavity is disrupted and CD indicates a fully denatured protein. This may be the water molecule buried near the small hydrophobic folding core at the D-E turn in the native protein. The MRD data also provide insights about transient (residence time <1 nsec) interactions of urea and water with the native and denatured protein. In the denatured state, both water and urea rotation is much more retarded than for a fully solvated polypeptide. The MRD results support a picture of the denatured state where solvent penetrates relatively compact clusters of polypeptide segments.
Topics: Carrier Proteins; Fatty Acid-Binding Proteins; Models, Molecular; Protein Binding; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Solutions; Solvents; Urea; Water
PubMed: 14627737
DOI: 10.1110/ps.03262603