Revealing the nature of the native state ensemble through cold denaturation

Recent advances in NMR methodology have enabled the structural analysis of proteins at temperatures far below the freezing point of water, thus opening a window to the cold denaturation process. Although the phenomenon of cold denaturation has been known since the mid-1970s, the freezing point of water has prevented detailed and structurally resolved studies without application of additional significant perturbations of the protein ensemble. As a result, the cold-denatured state and the process of cold denaturation have gone largely unstudied. Here, the structural and thermodynamic basis of cold denaturation is explored with emphasis placed on the insights that are uniquely ascertained from low-temperature studies. It is shown that the noncooperative cold-induced unfolding of protein results in the population of partially folded states that cannot be accessed by other techniques. The structurally resolved view of the cold denaturation process therefore can provide direct access to the cooperative substructures within the protein molecule and provide an unprecedented structurally resolved picture of the states that comprise the native state ensemble.     

Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of ΔC p of protein unfolding

Cold and heat denaturation of the double mutant Arg 3→Glu/Leu 66→Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D ¹H NMR spectroscopy in the temperature range from −12°C in supercooled water up to +70°C. The fraction of unfolded protein, f _u, was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be −11°C and 39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, ΔC _p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.     

Calorimetric study of the heat and cold denaturation of beta-lactoglobulin.

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold denaturation of proteins

This article summarizes all experimental facts concerning the cold denaturation of single-domain, multi-domain, and multimeric globular proteins in aqueous solutions with and without urea and guanidine hydrochloride. The facts obtained by various experimental techniques are analyzed thermodynamically and it is shown that the cold denaturation is a general phenomenon caused by the very specific and strongly temperature-dependent interaction of protein nonpolar groups with water. Hydration of these groups, in contrast to expectations, is favorable thermodynamically, i.e., the Gibbs energy of hydration is negative and increases in magnitude at a temperature decrease. As a result, the polypeptide chain, tightly packed in a compact native structure, unfolds at a sufficiently low temperature, exposing internal nonpolar groups to water. The reevaluation of the hydration effect on the base of direct calorimetric studies of protein denaturation and of transfer of non-polar compounds into water leads to revision of the conventional conception on the mechanism of hydrophobic interaction. The last appears to be a complex effect in which the positive contributor is van der Waals interactions between the nonpolar groups and not the hydration of these groups as it was usually supposed.     

The Hydrophobic Effect: A New Insight from Cold Denaturation and a Two-State Water Structure

Herein we provide a new insight into the hydrophobic effect in protein folding. Our proposition explains the molecular basis of cold denaturation, and of intermediate states in heat and their absence in cold denaturation. The exposure of non-polar surface reduces the entropy and enthalpy of the system, at low and at high temperatures. At low temperatures the favorable reduction in enthalpy overcomes the unfavorable reduction in entropy, leading to cold denaturation. At high temperatures, folding/unfolding is a two-step process: in the first, the entropy gain leads to hydrophobic collapse, in the second, the reduction in enthalpy due to protein-protein interactions leads to the native state. The different entropy and enthalpy contributions to the Gibbs energy change at each step at high, and at low, temperatures can be conveniently explained by a two-state model of the water structure. The model provides a clear view of the dominant factors in protein folding and stability. Consequently, it appears to provide a microscopic view of the hydrophobic effect and is consistently linked to macroscopic thermodynamic parameters.     

The hydrophobic effect: a new insight from cold denaturation and a two-state water structure

Herein we provide a new insight into the hydrophobic effect in protein folding. Our proposition explains the molecular basis of cold denaturation, and of intermediate states in heat and their absence in cold denaturation. The exposure of non-polar surface reduces the entropy and enthalpy of the system, at low and at high temperatures. At low temperatures the favorable reduction in enthalpy overcomes the unfavorable reduction in entropy, leading to cold denaturation. At high temperatures, folding/unfolding is a two-step process: in the first, the entropy gain leads to hydrophobic collapse, in the second, the reduction in enthalpy due to protein-protein interactions leads to the native state. The different entropy and enthalpy contributions to the Gibbs energy change at each step at high, and at low, temperatures can be conveniently explained by a two-state model of the water structure. The model provides a clear view of the dominant factors in protein folding and stability. Consequently, it appears to provide a microscopic view of the hydrophobic effect and is consistently linked to macroscopic thermodynamic parameters.     

Cold Denaturation of Protein

Abstract

This article summarizes all experimental facts concerning the cold denaturation of single-domain, multi-domain, and multimeric globular proteins in aqueous solutions with and without urea and guanidine hydrochloride. The facts obtained by various experimental techniques are analyzed thermodynamically and it is shown that the cold denaturation is a general phenomenon caused by the very specific and strongly termperature-dependent interaction of protein nonpolar groups with water. Hydration of these groups, in contrast to expectations, is favorable thermodynamically, i.e., the Gibbs energy of hydration is negative and increases in magnitude at a temperature! decrease. As a result, the polypeptide chain, tightly packed in a compact native structure, unfolds at a sufficiently low temperature, exposing internal nonpolar groups to water. The reevaluation of the hydration effect on the base of direct calorimetric studies of protein denaturation and of transfer of non-polar compounds into water leads to revision of the conventional conception on the mechanism of hydrophobic interaction. The last appears to be a complex effect in which the positive con- tributor is van der Waals interactions between the nonpolar groups and not the hydration of these groups as it was usually supposed.     

The Mechanism of N-Terminal Acetylation of Protein

Abstract

This article summarizes all experimental facts concerning the cold denaturation of single-domain, multi-domain, and multimeric globular proteins in aqueous solutions with and without urea and guanidine hydrochloride. The facts obtained by various experimental techniques are analyzed thermodynamically and it is shown that the cold denaturation is a general phenomenon caused by the very specific and strongly termperature-dependent interaction of protein nonpolar groups with water. Hydration of these groups, in contrast to expectations, is favorable thermodynamically, i.e., the Gibbs energy of hydration is negative and increases in magnitude at a temperature decrease. As a result, the polypeptide chain, tightly packed in a compact native structure, unfolds at a sufficiently low temperature, exposing internal nonpolar groups to water. The reev-aluation of the hydration effect on the base of direct calorimetric studies of protein denaturation and of transfer of non-polar compounds into water leads to revision of the conventional conception on the mechanism of hydrophobic interaction. The last appears to be a complex effect in which the positive contributor is van der Waals interactions between the nonpolar groups and not the hydration of these groups as it was usually supposed.     

Essential dynamics of the cold denaturation: pressure and temperature effects in yeast frataxin

The cold denaturation of globular proteins is a process that can be caused by increasing pressure or decreasing the temperature. Currently, the action mechanism of this process has not been clearly understood, raising an interesting debate on the matter. We have studied the process of cold denaturation using molecular dynamics simulations of the frataxin system Yfh1, which has a dynamic experimental characterization of unfolding at low and high temperatures. The frataxin model here studied allows a comparative analysis using experimental data. Furthermore, we monitored the cold denaturation process of frataxin and also investigated the effect under the high‐pressure regime. For a better understanding of the dynamics and structural properties of the cold denaturation, we also analyzed the MD trajectories using essentials dynamic. The results indicate that changes in the structure of water by the effect of pressure and low temperatures destabilize the hydrophobic interaction modifying the solvation and the system volume leading to protein denaturation. Proteins 2016; 85:125–136. © 2016 Wiley Periodicals, Inc.     

Effect of hydrophobic environments on the hypothesized liquid-liquid critical point of water

The complex behavior of liquid water, along with its anomalies and their crucial role in the existence of life, continue to attract the attention of researchers. The anomalous behavior of water is more pronounced at subfreezing temperatures and numerous theoretical and experimental studies are directed towards developing a coherent thermodynamic and dynamic framework for understanding supercooled water. The existence of a liquid–liquid critical point in the deep supercooled region has been related to the anomalous behavior of water. However, the experimental study of supercooled water at very low temperatures is hampered by the homogeneous nucleation of the crystal. Recently, water confined in nanoscopic structures or in solutions has attracted interest because nucleation can be delayed. These systems have a tremendous relevance also for current biological advances; e.g., supercooled water is often confined in cell membranes and acts as a solvent for biological molecules. In particular, considerable attention has been recently devoted to understanding hydrophobic interactions or the behavior of water in the presence of apolar interfaces due to their fundamental role in self-assembly of micelles, membrane formation and protein folding. This article reviews and compares two very recent computational works aimed at elucidating the changes in the thermodynamic behavior in the supercooled region and the liquid–liquid critical point phenomenon for water in contact with hydrophobic environments. The results are also compared to previous reports for water in hydrophobic environments.     

The Effects of Temperature and Heavy Water on Cell Division in Heat-Synchronized Cells of Tetrahymena*

SYNOPSIS. A study was undertaken of the rates of cell division of heat-synchronized cells of Tetrahymena pyriformis GL at various temperatures in water and 20, 30 and 40% heavy water. The results suggest that division rate is limited by a protein which undergoes both high and low temperature denaturation and that this protein is partially stabilized against heat-denaturation while becoming more susceptible to cold denaturation in the presence of heavy water. Thus, the optimum temperature for division shifts upward as the heavy water concentration is increased, with a maximum shift of 1 C occurring in 40% heavy water. In addition, division activity occurs in heavy water at 34 C, a temperature at which cells kept in water are blocked. Furthermore, the sharp increase in slope seen in the low temperature portion of Arrhenius plots of the data, occurs at higher temperatures when heavy water is present. Finally, at virtually all temperatures, except the highest, heavy water has a depressive effect on division rate indicating a general inhibitory influence of deuterium-substituted water on rate processes within the cell.     

Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation

The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching -35 degrees C. The cold-induced unfolding of ubiquitin is found to be highly noncooperative, in distinct contrast to the thermal melting of this and other proteins. These results demonstrate the potential of cold denaturation as a means to dissect the cooperative substructures of proteins and to provide a rigorous framework for testing statistical thermodynamic treatments of protein stability, dynamics and function.     

The unusually stable coiled-coil domain of COMP exhibits cold and heat denaturation in 4-6 M guanidinium chloride

A high thermal stability is observed for the five-stranded alpha-helical coiled-coil domain of cartilage oligomeric matrix protein COMP. It does not unfold in non-denaturing buffer between 0 and 100 degrees C and thermal denaturation is only achieved at high concentrations of guanidinium chloride (4-6 M). In these solutions the protein structure is lost at decreasing (cold denaturation) and increasing temperatures (heat denaturation). In the cold denaturation region, the melting profile showed deviations from the theory of Privalov et al. [P.L. Privalov, V. Griko Yu, S. Venyaminov, V.P. Kutyshenko, Cold denaturation of myoglobin, J. Mol. Biol. 190 (1986) 487-498] probably due to deviations from a two-state mechanism. High thermal stability as well as cold and heat denaturation was also observed for a mutant of the coiled-coil domain of COMP in which glutamine 54 was replaced by isoleucine but it still forms pentamer. The melting temperatures in plain buffer for the heat denaturation of COMP coiled-coil domain and its mutant obtained by extrapolation to zero molar guanidinium chloride concentration are approximately 160 and 220 degrees C, respectively, which groups them among the most stable proteins.     

Protein stability and hydrophobic interactions

The paper summarizes results of calorimetric studies of protein denaturation and of dissolution of non-polar substances in water. The analysis of the available experimental data shows that the positive contribution of the hydrophobic interactions in stabilization of the protein compact state is due to van der Waals interactions between the protein non-polar groups, while the contribution of water solvation by these groups, in spite of the widely spread opinion, appears to be always negative. This destabilizing action of water solvation on the protein increases as the temperature decreases, and at a significantly low temperature causes unfolding of the compact structure of protein, i. e. cold denaturation.     

Water at Interface with Proteins

Water is essential for the activity of proteins. However, the effect of the properties of water on the behavior of proteins is only partially understood. Recently, several experiments have investigated the relation between the dynamics of the hydration water and the dynamics of protein. These works have generated a large amount of data whose interpretation is debated. New experiments measure the dynamics of water at low temperature on the surface of proteins, finding a qualitative change (crossover) that might be related to the slowing down and stop of the protein’s activity (protein glass transition), possibly relevant for the safe preservation of organic material at low temperature. To better understand the experimental data several scenarios have been discussed. Here, we review these experiments and discuss their interpretations in relation with the anomalous properties of water. We summarize the results for the thermodynamics and dynamics of supercooled water at an interface. We consider also the effect of water on protein stability, making a step in the direction of understanding, by means of Monte Carlo simulations and theoretical calculations, how the interplay of water cooperativity and hydrogen bonds interfacial strengthening affects the protein cold denaturation.     

Cold hardiness and deep supercooling in xylem of shagbark hickory

Differential thermal analysis, differential scanning calorimetry, pulsed nuclear magnetic resonance spectroscopy, and low temperature microscopy are utilized to investigate low temperature freezing points or exotherms which occur near −40 C in the xylem of cold-acclimated shagbark hickory (Carya ovata L.). Experiments using these methods demonstrate that the low temperature exotherm results from the freezing of cellular water in a manner predicted for supercooled dilute aqueous solutions. Heat release on freezing, nuclear magnetic resonance relaxation times, and freezing and thawing curves for hickory twigs all point to a supercooled fraction in the xylem at subfreezing temperatures. Calorimetric and low temperature microscopic analyses indicate that freezing occurs intracellularly in the xylem ray parenchyma. The supercooled fraction is found to be extremely stable, even at temperatures only slightly above the homogeneous nucleation temperature for water (−38 C). Xylem water is also observed to be resistant to dehydration when exposed to 80% relative humidity at 20 C. D₂O exchange experiments find that only a weak kinetic barrier to water transport exists in the xylem rays of shagbark hickory.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

Two types of arrestins expressed in medaka rod photoreceptors

Globular protein thermostability is characterized the cold denaturation, maximal stability (Tms) and heat denaturation temperatures. For mesophilic globular proteins, Tms typically ranges from -25 degrees C to +35 degrees C. We show that the indirect estimate of Tms from calorimetry and the direct estimate from chemical denaturation performed in a range of temperatures are in close agreement. The heat capacity change of unfolding per mol residue (delta Cp) alone is shown to accurately predict Tms. Delta Cp and hence Tms can be predicted solely from the protein sequence. The average difference in free energy of unfolding at the observed and predicted values of Tms is 1.0 kcal mol(-1), which is small compared to typical values of the total free energy of unfolding.     

Molecular basis of the heat denaturation of photosystem II

The thermal denaturation of the photosystem II (PSII) membrane protein complex is investigated by assigning the endothermic transitions observed by differential scanning calorimetry (DSC) to the denaturation of particular proteins of the PSII complex. In a prior DSC study of PSII membranes [Thompson, L. K., Sturtevant, J. M., & Brudvig, G. W. (1986) Biochemistry 25, 6161], five DSC peaks were observed in the 30-70 degrees C temperature range (A1, A2, B, C, and D). The A2 peak was assigned to denaturation of a component essential for water oxidation and the B peak to denaturation of a component critical to the remainder of the electron-transport chain. We have now extended these studies with thermal gel analysis and electron paramagnetic resonance (EPR) measurements. Thermal gel analysis, a technique which relies on a change in the solubility properties of a membrane protein upon denaturation, has been used to determine the temperatures of denaturation of all of the major membrane proteins of the PSII complex. EPR experiments have been used to monitor chlorophyll photooxidation and the stability of TyrD+. Peaks B, C, and D in the DSC denaturation profile are each assigned to the denaturation of several proteins, which provides information on the organization of the PSII complex into structural and functional units. Peak B corresponds to the denaturation of peripheral core proteins and closely associated antenna proteins, peak C to the PSII core, and peak D to the loosely associated antenna proteins. No membrane protein is observed to denature during the A2 peak. The A2 peak is altered by the presence of catalase, superoxide dismutase, low chloride, and high pH. These results suggest that the abnormally sharp A2 peak occurs when the highly oxidizing, sequestered Mn complex (the active site in water oxidation) becomes accessible to the aqueous phase, at elevated temperatures. We propose a mechanism for the reaction of the Mn complex with hydroxide ions, which involves peroxide or superoxide and results in the reduction and release of Mn. The proposed model provides insight into the well-known instability of the Mn complex and the role of chloride in stabilizing the complex. This may enable the future development of purification procedures and may explain the sensitivity of the water-oxidizing apparatus of PSII to heat denaturation.