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The Journal of Physical Chemistry. B Oct 2023Chemical unfolding with guanidineHCl or urea is a common method to study the conformational stability of proteins. The analysis of unfolding isotherms is usually...
Chemical unfolding with guanidineHCl or urea is a common method to study the conformational stability of proteins. The analysis of unfolding isotherms is usually performed with an empirical linear extrapolation method (LEM). A large positive free energy is assigned to the native protein, which is usually considered to be a minimum of the free energy. The method thus contradicts common expectations. Here, we present a multistate cooperative model that addresses specifically the binding of the denaturant to the protein and the cooperativity of the protein unfolding equilibrium. The model is based on a molecular statistical-mechanical partition function of the ensemble, but simple solutions for the calculation of the binding isotherm and the associated free energy are presented. The model is applied to 23 published unfolding isotherms of small and large proteins. For a given denaturant, the binding constant depends on temperature and pH but shows little protein specificity. Chemical unfolding is less cooperative than thermal unfolding. The cooperativity parameter σ is at least 2 orders of magnitude larger than that of thermal unfolding. The multistate cooperative model predicts zero free energy for the native protein, which becomes strongly negative beyond the midpoint concentration of unfolding. The free energy to unfold a cooperative unit corresponds exactly to the diffusive energy of the denaturant concentration gradient necessary for unfolding. The temperature dependence of unfolding isotherms yields the denaturant-induced unfolding entropy and, in turn, the unfolding enthalpy. The multistate cooperative model provides molecular insight and is as simple to apply as the LEM but avoids the conceptual difficulties of the latter.
PubMed: 37735883
DOI: 10.1021/acs.jpcb.3c03558 -
Biomolecules Feb 2020Most of the human diseases related to various proteopathies are confined to the brain, which leads to the development of various forms of neurological disorders. The...
Most of the human diseases related to various proteopathies are confined to the brain, which leads to the development of various forms of neurological disorders. The human brain consists of several osmolytic compounds, such as N-Acetylaspartate (NAA), myo-inositol (mI), glutamate (Glu), glutamine (Gln), creatine (Cr), and choline-containing compounds (Cho). Among these osmolytes, the level of NAA drastically decreases under neurological conditions, and, hence, NAA is considered to be one of the most widely accepted neuronal biomarkers in several human brain disorders. To date, no data are available regarding the effect of NAA on protein stability, and, therefore, the possible effect of NAA under proteopathic conditions has not been fully uncovered. To gain an insight into the effect of NAA on protein stability, thermal denaturation and structural measurements were carried out using two model proteins at different pH values. The results indicate that NAA increases the protein stability with an enhancement of structure formation. We also observed that the stabilizing ability of NAA decreases in a pH-dependent manner. Our study indicates that NAA is an efficient protein stabilizer at a physiological pH.
Topics: Aspartic Acid; Biomarkers; Brain Chemistry; Choline; Creatine; Glutamic Acid; Glutamine; Humans; Hydrogen-Ion Concentration; Inositol; Muramidase; Neurons; Osmosis; Protein Unfolding; Temperature; Thermodynamics
PubMed: 32059525
DOI: 10.3390/biom10020286 -
FEBS Letters Sep 2020Molecular chaperones are highly conserved proteins that promote proper folding of other proteins in vivo. Diverse chaperone systems assist de novo protein folding and... (Review)
Review
Molecular chaperones are highly conserved proteins that promote proper folding of other proteins in vivo. Diverse chaperone systems assist de novo protein folding and trafficking, the assembly of oligomeric complexes, and recovery from stress-induced unfolding. A fundamental function of molecular chaperones is to inhibit unproductive protein interactions by recognizing and protecting hydrophobic surfaces that are exposed during folding or following proteotoxic stress. Beyond this basic principle, it is now clear that chaperones can also actively and specifically accelerate folding reactions in an ATP-dependent manner. We focus on the bacterial Hsp70 and chaperonin systems as paradigms, and review recent work that has advanced our understanding of how these chaperones act as catalysts of protein folding.
Topics: Adenosine Triphosphate; Biocatalysis; Chaperonin 60; Chaperonins; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; HSP70 Heat-Shock Proteins; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Chaperones; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Interaction Domains and Motifs; Protein Unfolding
PubMed: 32446288
DOI: 10.1002/1873-3468.13844 -
Molecules (Basel, Switzerland) Oct 2022The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be... (Review)
Review
The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.
Topics: Protein Denaturation; Protein Conformation; Deuterium Oxide; Aryldialkylphosphatase; Electrophoresis, Polyacrylamide Gel; Cholinesterases; Biological Products; Thermodynamics; Protein Folding
PubMed: 36296453
DOI: 10.3390/molecules27206861 -
Cold Spring Harbor Perspectives in... Apr 2020The functional health of the proteome is determined by properties of the proteostasis network (PN) that regulates protein synthesis, folding, macromolecular assembly,... (Review)
Review
The functional health of the proteome is determined by properties of the proteostasis network (PN) that regulates protein synthesis, folding, macromolecular assembly, translocation, and degradation. In eukaryotes, the PN also integrates protein biogenesis across compartments within the cell and between tissues of metazoans for organismal health and longevity. Additionally, in metazoans, proteome stability and the functional health of proteins is optimized for development and yet declines throughout aging, accelerating the risk for misfolding, aggregation, and cellular dysfunction. Here, I describe the cell-nonautonomous regulation of organismal PN by tissue communication and cell stress-response pathways. These systems are robust from development through reproductive maturity and are genetically programmed to decline abruptly in early adulthood by repression of the heat shock response and other cell-protective stress responses, thus compromising the ability of cells and tissues to properly buffer against the cumulative stress of protein damage during aging. While the failure of multiple protein quality control processes during aging challenges cellular function and tissue health, genetic studies, and the identification of small-molecule proteostasis regulators suggests strategies that can be employed to reset the PN with potential benefit on cellular health and organismal longevity.
Topics: Aging; Animals; Caenorhabditis elegans; Fibroblasts; Gene Expression Regulation; Heat Shock Transcription Factors; Heat-Shock Proteins; Heat-Shock Response; Humans; Longevity; Protein Denaturation; Protein Folding; Proteins; Proteome; Proteostasis; Quality Control; Risk; Signal Transduction; Stress, Physiological
PubMed: 30962274
DOI: 10.1101/cshperspect.a034074 -
Biomolecules Jan 2021The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate... (Review)
Review
The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). Here, we summarise recent insights into the structure, assembly and protein secretion mechanisms of the T3SS with a focus on energy transduction and protein transport across the cytoplasmic membrane.
Topics: Adenosine Triphosphatases; Bacterial Proteins; Cell Membrane; Cytoplasm; Flagella; Gram-Negative Bacteria; Hydrolysis; Membrane Proteins; Protein Denaturation; Protein Transport; Substrate Specificity; Type III Secretion Systems
PubMed: 33572887
DOI: 10.3390/biom11020186 -
Advanced Science (Weinheim,... Feb 2022Protein-based hydrogels have attracted great attention due to their excellent biocompatible properties, but often suffer from weak mechanical strength. Conventional...
Protein-based hydrogels have attracted great attention due to their excellent biocompatible properties, but often suffer from weak mechanical strength. Conventional strengthening strategies for protein-based hydrogels are to introduce nanoparticles or synthetic polymers for improving their mechanical strength, but often compromise their biocompatibility. Here, a new, general, protein unfolding-chemical coupling (PNC) strategy is developed to fabricate pure protein hydrogels without any additives to achieve both high mechanical strength and excellent cell biocompatibility. This PNC strategy combines thermal-induced protein unfolding/gelation to form a physically-crosslinked network and a -NH2/-COOH coupling reaction to generate a chemicallycrosslinked network. Using bovine serum albumin (BSA) as a globular protein, PNC-BSA hydrogels show macroscopic transparency, high stability, high mechanical properties (compressive/tensile strength of 115/0.43 MPa), fast stiffness/toughness recovery of 85%/91% at room temperature, good fatigue resistance, and low cell cytotoxicity and red blood cell hemolysis. More importantly, the PNC strategy can be not only generally applied to silk fibroin, ovalbumin, and milk albumin protein to form different, high strength protein hydrogels, but also modified with PEDOT/PSS nanoparticles as strain sensors and fluorescent fillers as color sensors. This work demonstrates a new, universal, PNC method to prepare high strength, multi-functional, pure protein hydrogels beyond a few available today.
Topics: Fibroins; Hydrogels; Polymers; Protein Unfolding; Serum Albumin, Bovine
PubMed: 34939355
DOI: 10.1002/advs.202102557 -
ELife Sep 2022Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained...
Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.
Topics: Bayes Theorem; Lipid Bilayers; Membrane Proteins; Microscopy, Atomic Force; Protein Unfolding; Proteomics
PubMed: 36094473
DOI: 10.7554/eLife.77427 -
Communications Biology Jul 2021Proteins as force-sensors respond to mechanical cues and regulate signaling in physiology. Proteins commonly connect the source and response points of mechanical cues in...
Proteins as force-sensors respond to mechanical cues and regulate signaling in physiology. Proteins commonly connect the source and response points of mechanical cues in two conformations, independent proteins in end-to-end geometry and protein complexes in handshake geometry. The force-responsive property of independent proteins in end-to-end geometry is studied extensively using single-molecule force spectroscopy (SMFS). The physiological significance of the complex conformations in force-sensing is often disregarded as mere surge protectors. However, with the potential of force-steering, protein complexes possess a distinct mechano-responsive property over individual force-sensors. To decipher, we choose a force-sensing protein, cadherin-23, from tip-link complex and perform SMFS using end-to-end geometry and handshake complex geometry. We measure higher force-resilience of cadherin-23 with preferential shorter extensions in handshake mode of pulling over the direct mode. The handshake geometry drives the force-response of cadherin-23 through different potential-energy landscapes than direct pulling. Analysis of the dynamic network structure of cadherin-23 under tension indicates narrow force-distributions among residues in cadherin-23 in direct pulling, resulting in low force-dissipation paths and low resilience to force. Overall, the distinct and superior mechanical responses of cadherin-23 in handshake geometry than single protein geometry highlight a probable evolutionary drive of protein-protein complexes as force-conveyors over independent ones.
Topics: Anisotropy; Biomechanical Phenomena; Cadherins; Protein Unfolding
PubMed: 34326473
DOI: 10.1038/s42003-021-02445-y -
Protein Science : a Publication of the... Jan 2021Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited...
Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature T , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations.
Topics: Crystallography, X-Ray; Hot Temperature; Membrane Proteins; Multiprotein Complexes; Protein Folding; Protein Unfolding; Software
PubMed: 33140490
DOI: 10.1002/pro.3986