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Journal of the American Chemical Society Apr 2022Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the...
Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the freezing point of water. Understanding the mechanisms that determine cold denaturation and the rules that permit its observation is an important challenge. A way to approach them is to be able to induce cold denaturation in an otherwise stable protein by means of mutations. Here, we studied CyaY, a relatively stable bacterial protein with no detectable cold denaturation and a high melting temperature of 54 °C. We have characterized for years the yeast orthologue of CyaY, Yfh1, a protein that undergoes cold and heat denaturation at 5 and 35 °C, respectively. We demonstrate that, by transferring to CyaY the lessons learnt from Yfh1, we can induce cold denaturation by introducing a restricted number of carefully designed mutations aimed at destabilizing the overall fold and inducing electrostatic frustration. We used molecular dynamics simulations to rationalize our findings and demonstrate the individual effects observed experimentally with the various mutants. Our results constitute the first example of rationally designed cold denaturation and demonstrate the importance of electrostatic frustration on the mechanism of cold denaturation.
Topics: Cold Temperature; Hot Temperature; Molecular Dynamics Simulation; Protein Denaturation; Proteins; Thermodynamics
PubMed: 35427450
DOI: 10.1021/jacs.1c13355 -
International Journal of Hyperthermia :... Dec 2005Changes in growth temperature induce both activating and inactivating responses from cells, with the magnitude of the temperature change being among the factors that... (Review)
Review
Changes in growth temperature induce both activating and inactivating responses from cells, with the magnitude of the temperature change being among the factors that influence which type of response dominates. Aside from upregulated enzyme activity, induction of thermotolerance is the most widely studied and best understood activating response that cells exhibit following heat shock. Inactivating responses to heat shock that are of biomedical interest include heat radiosensitization and cytotoxicity. Interestingly, the activation energy for inducing thermotolerance, heat cytotoxicity, and radiosensitization all fall within a similar range of 120-146 kcal per mole. The relatively high activation energy for each of these responses suggests that they all involve a heat-induced molecular transition as a trigger, and several lines of research suggest strongly that protein denaturation is the common transition that triggers all three responses. Low levels of protein denaturation are sufficient to attract the 90 kDa heat shock protein (HSP90) such that it frees up heat shock factor 1, which then trimerizes to form an active transcription factor that upregulates expression of heat shock proteins. Upregulation of heat shock proteins and other heat-induced events result in the development of thermotolerance, which protects cells from subsequent exposure to heat shock and other stresses. A more severe heat shock increases protein denaturation proportionately and leads to aggregation of both denatured and native proteins. This results in inactivation of protein synthesis, cell cycle progression, and DNA repair processes such that cells either die or are sensitized to radiation and other cytotoxic events. The ultimate fate of cells following a heat shock depends upon the summation of the activation and inactivation events that are induced, which appears to be governed by the resultant magnitude of protein denaturation and aggregation. Treatments that stabilize cellular proteins against denaturation and aggregation reduce the magnitude of inactivating responses while increasing that of activating responses for a given heat shock (time at temperature), while treatments that sensitize proteins to denaturation and aggreation have the converse effect. These findings support the conclusion that the determinant of the cellular response to heat shock is the amount of heat-induced protein denaturation and aggregation and not the time at temperature.
Topics: Animals; Cell Physiological Phenomena; DNA-Binding Proteins; Environment; Fever; HSP90 Heat-Shock Proteins; Heat Shock Transcription Factors; Hot Temperature; Protein Denaturation; Transcription Factors
PubMed: 16338849
DOI: 10.1080/02656730500307298 -
International Journal of Hyperthermia :... Mar 2004Hyperthermia at temperatures above 41 degrees C denatures a set of thermolabile cellular proteins located in all parts of the cell. Non-histone nuclear proteins,... (Review)
Review
Hyperthermia at temperatures above 41 degrees C denatures a set of thermolabile cellular proteins located in all parts of the cell. Non-histone nuclear proteins, including those comprising the nuclear matrix, appear to be particularly thermolabile. Heating isolated nuclear matrices of Chinese hamster lung (CHL) V79 cells to 46 degrees C at 1 degree C/min results in approximately 15% denaturation. Protein unfolding during denaturation exposes buried hydrophobic residues, which increases protein-protein interactions and results in the co-aggregation of denatured thermolabile proteins and native, aggregative-sensitive nuclear proteins. This aggregated protein, the majority of which is native, is insoluble and resistant to extraction during isolation of nuclei and is responsible for the increased protein content, usually expressed as an increased protein:DNA ratio, of nuclei isolated from heated cells. A large fraction of the aggregated protein is found to be associated with the nuclear matrix, distributed throughout the fibre network and nucleolus. Three general consequences of nuclear protein denaturation and aggregation of relevance to cellular damage are: (1) protein (enzyme) inactivation, both direct inactivation of thermolabile proteins and indirect inactivation due to co-aggregation; (2) reduced accessibility and altered physical properties of DNA due to masking by aggregated protein; and (3) protein redistribution into and out of the nucleus. Functional impairment of the nucleus appears to be due to one or a combination of these basic mechanisms.
Topics: Animals; Humans; Hyperthermia, Induced; Nuclear Proteins; Protein Denaturation; Radiation Tolerance
PubMed: 15195506
DOI: 10.1080/02656730310001637334 -
ACS Applied Materials & Interfaces Oct 2023Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a...
Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a liquid-based atomic force microscope and graphene liquid-cell-based scanning transmission electron microscope make it possible to observe chemically induced protein unfolding. To illustrate this capability, ferritin proteins were deposited on a graphene surface, and the concentration-dependent urea- or guanidinium-induced changes of morphology were monitored for holo-ferritin with its ferrihydrite core as well as apo-ferritin without this core. Depending on the chaotropic agent the liquid-based imaging setup captured an unexpected transformation of natively folded holo-ferritin proteins into rings after urea treatment but not after guanidinium treatment. Urea treatment of apo-ferritin did not result in nanorings, confirming that nanorings are a specific signature of denaturation of holo-ferritins after exposture to sufficiently high urea concentrations. Mapping the images with molecular dynamics simulations of ferritin subunits in urea solutions suggests that electrostatic destabilization triggers denaturation of ferritin as urea makes direct contact with the protein and also disrupts the water H-bonding network in the ferritin solvation shell. Our findings deepen the understanding of protein denaturation studied using label-free techniques operating at the solid-liquid interface.
Topics: Guanidine; Protein Denaturation; Graphite; Ferritins; Urea
PubMed: 37797325
DOI: 10.1021/acsami.3c10510 -
IUBMB Life Jun 2009Protein research is generally recognized as experimental science and knowledge of protein science is not constructed axiomatically. In this article, we show that much of... (Review)
Review
Protein research is generally recognized as experimental science and knowledge of protein science is not constructed axiomatically. In this article, we show that much of our present knowledge of protein science is explainable by principles of protein thermodynamic structure theory. A deductive system for protein knowledge has been developed and several fundamental questions of protein science can be theoretically resolved.
Topics: Protein Conformation; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Receptors, Cell Surface; Receptors, Drug; Thermodynamics
PubMed: 19472177
DOI: 10.1002/iub.214 -
Biophysical Journal Aug 2021The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger...
The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.
Topics: Entropy; Plant Proteins; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics
PubMed: 34087209
DOI: 10.1016/j.bpj.2021.05.019 -
Biochemistry May 2019Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the...
Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the degree of structure and the compactness of denatured states populated in these experiments are still open questions. Here, building on previous works, we show that thermally and chemically denatured protein states are distinct from the viewpoint of far-ultraviolet circular dichroism experiments that report on the local conformational status of peptide bonds. The differences identified are independent of protein length, structural class, or experimental conditions, highlighting the presence of two sub-ensembles within the denatured states. The sub-ensembles, U and U for thermally induced and denaturant-induced unfolded states, respectively, can exclusively exchange populations as a function of temperature at high chemical denaturant concentrations. Empirical analysis suggests that chemically denatured states are ∼50% more expanded than the thermally denatured chains of the same protein. Our observations hint that the strength of protein backbone-backbone interactions and identity versus backbone-solvent/co-solvent interactions determine the conformational distributions. We discuss the implications for protein folding mechanisms, the heterogeneity in relaxation rates, and folding diffusion coefficients.
Topics: Circular Dichroism; DNA-Binding Proteins; Escherichia coli Proteins; Hot Temperature; Kinetics; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Folding; Repressor Proteins; Urea
PubMed: 31083972
DOI: 10.1021/acs.biochem.9b00089 -
Biophysical Chemistry Jan 2016Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common... (Review)
Review
Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results. It would always be preferable to measure the whole stability curve. The calculation of this curve is greatly facilitated whenever it is possible to observe cold denaturation. Using Yfh1, one of the few proteins whose cold denaturation occurs at neutral pH and low ionic strength, we could measure the variation of its full stability curve under several environmental conditions. Here we show the advantages of gauging stability as a function of external variables using stability curves.
Topics: Cold Temperature; Hydrogen-Ion Concentration; Iron-Binding Proteins; Models, Molecular; Osmolar Concentration; Protein Denaturation; Protein Stability; Temperature; Frataxin
PubMed: 26026885
DOI: 10.1016/j.bpc.2015.05.007 -
The Journal of Physical Chemistry. B Jul 2023Water is considered integral for the stabilization and function of proteins, which has recently attracted significant attention. However, the microscopic aspects of...
Water is considered integral for the stabilization and function of proteins, which has recently attracted significant attention. However, the microscopic aspects of water ranging up to the second hydration shell, including strongly and weakly bound water at the sub-nanometer scale, are not yet well understood. Here, we combined terahertz spectroscopy, thermal measurements, and infrared spectroscopy to clarify how the strongly and weakly bound hydration water changes upon protein denaturation. With denaturation, that is, the exposure of hydrophobic groups in water and entanglement of hydrophilic groups, the number of strongly bound hydration water decreased, while the number of weakly bound hydration water increased. Even though the constraint of water due to hydrophobic hydration is weak, it extends to the second hydration shell as it is caused by the strengthening of hydrogen bonds between water molecules, which is likely the key microscopic mechanism for the destabilization of the native state due to hydration.
Topics: Water; Proteins; Hydrophobic and Hydrophilic Interactions; Hydrogen Bonding; Protein Denaturation
PubMed: 37417885
DOI: 10.1021/acs.jpcb.3c02970 -
Proceedings of the National Academy of... Jun 1991The standard enthalpy or entropy change upon transfer of a small nonpolar molecule from a nonaqueous phase into water at a given temperature is generally different for...
The standard enthalpy or entropy change upon transfer of a small nonpolar molecule from a nonaqueous phase into water at a given temperature is generally different for different solute species. However, if the heat capacity change is independent of temperature, there exists a temperature at which the enthalpy or the entropy change becomes the same for all solute species within a given class. Similarly, the enthalpy or the entropy change of protein denaturation, when extrapolated to high temperature assuming a temperature-independent heat capacity change, shows a temperature at which its value becomes the same for many different globular proteins on a per weight basis. It is shown that the existence of these temperatures can be explained from a common formalism based on a linear relationship between the thermodynamic quantity and a temperature-independent molecular property that characterizes the solute or the protein. For the small nonpolar molecule transfer processes, this property is the surface area or the number of groups that are brought in contact with water. For protein denaturation, it is suggested that this property measures the polar/nonpolar mix of the internal interaction within the protein interior. Under a certain set of assumptions, this model leads to the conclusion that the nonpolar and the polar groups of the protein contribute roughly equally to the stability of the folded state of the molecule and that the solvent-accessible surface area of the denatured form of a protein is no more than about two-thirds that of the fully extended form.
Topics: Mathematics; Protein Denaturation; Temperature; Thermodynamics
PubMed: 2052594
DOI: 10.1073/pnas.88.12.5154