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.     

High-resolution nuclear magnetic resonance studies of proteins

The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, alpha-lactalbumin and troponin C were the model proteins investigated.     

Unusual cold denaturation of a small protein domain

A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.     

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.     

Experimental comparison of energy landscape features of ubiquitin family proteins

Small ubiquitin-related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence-structure-stability-function relationship. Here, we used chemical denaturation in comparing the folding-unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W-pseudo wild-type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant-dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two-state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow-down their folding rates. The denaturant-dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent-accessible surface area, as quantified by the Tanford β _u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding-unfolding rates within a structurally homologous protein family.     

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.     

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.     

What causes hyperfluorescence: folding intermediates or conformationally flexible native states?

Hyperfluorescent intensity maxima during protein unfolding titrations are often taken as a sign for a thermodynamic folding intermediate. Here we explore another possibility: that hyperfluorescence could be the signature of a "pretransition" conformationally loosened native state. To model such native states, we study mutants of a fluorescent ubiquitin variant, placing cavities at various distances from the tryptophan fluorophore. We examine the correlation between protein flexibility and enhanced fluorescence intensity by using circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy measurements, and molecular dynamics. Based on experiment and simulation, we propose a simple model for hyperfluorescence in terms of static and dynamic conformational properties of the native state during unfolding. Apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation are discussed as examples. Our results do not preclude the existence of thermodynamic intermediates but do raise caution that by itself, hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.     

Elimination of the C-cap in ubiquitin - structure, dynamics and thermodynamic consequences

Single amino acid substitutions rarely produce substantial changes in protein structure. Here we show that substitution of the C-cap residue in the alpha-helix of ubiquitin with proline (34P variant) leads to dramatic structural changes. The resulting conformational perturbation extends over the last two turns of the alpha-helix and leads to enhanced flexibility for residues 27-37. Thermodynamic analysis of this ubiquitin variant using differential scanning calorimetry reveals that the thermal unfolding transition remains highly cooperative, exhibiting two-state behavior. Similarities with the wild type in the thermodynamic parameters (heat capacity change upon unfolding and m-value) of unfolding monitored by DSC and chemical denaturation suggests that the 34P variant has comparable buried surface area. The hydrophobic core of 34P variant is not packed as well as that of the wild type protein as manifested by a lower enthalpy of unfolding. The increased mobility of the polypeptide chain of this ubiquitin variant allows the transient opening of the hydrophobic core as evidenced by ANS binding. Taken together, these results suggest exceptional robustness of cooperativity in protein structures.     

Macromolecular crowding stabilizes the molten globule form of apomyoglobin with respect to both cold and heat unfolding

At pH 2 apomyoglobin is extensively unfolded. Addition of increasing concentration of salts has been shown to convert the protein into molten globule form(s), which can undergo both heat-induced and cold-induced unfolding. Increasing concentrations of an inert polymer, dextran, lead to increased formation of molten globule and stabilizes the protein with respect to both heat-induced and cold-induced denaturation. The transitions were studied by circular dichroism. Two-state analysis of the data shows that the effects of salt and polymer are additive, and that stabilization by the polymer is independent of temperature, as predicted by excluded volume theory.     

Thermodynamics of denaturation of hisactophilin, a beta-trefoil protein

Hisactophilin is a histidine-rich pH-dependent actin-binding protein from Dictyostelium discoideum. The structure of hisactophilin is typical of the beta-trefoil fold, a common structure adopted by diverse proteins with unrelated primary sequences and functions. The thermodynamics of denaturation of hisactophilin have been measured using fluorescence- and CD-monitored equilibrium urea denaturation curves, pH-denaturation, and thermal denaturation curves, as well as differential scanning calorimetry. Urea denaturation is reversible from pH 5.7 to pH 9.7; however, thermal denaturation is highly reversible only below pH approximately 6.2. Reversible denaturation by urea and heat is well fit using a two-state transition between the native and the denatured states. Urea denaturation curves are best fit using a quadratic dependence of the Gibbs free energy of unfolding upon urea concentration. Hisactophilin has moderate, roughly constant stability from pH 7.7 to pH 9.7; however, below pH 7.7, stability decreases markedly, most likely due to protonation of histidine residues. Enthalpic effects of histidine ionization upon unfolding also appear to be involved in the occurrence of cold unfolding of hisactophilin under relatively mild solution conditions. The stability data for hisactophilin are compared with data on hisactophilin function, and with data for two other beta-trefoil proteins, human interleukin-1beta, and basic fibroblast growth factor.     

The effect of aqueous methanol cryosolvents on the heat- and cold-induced denaturation of lactate dehydrogenase

The effect on the low-temperature-induced denaturation temperature of various concentrations of methanol has been studied for lactate dehydrogenase. The results have been compared to similar data for the thermal denaturation temperature. Extrapolations of the low-temperature data show that, in a physiological buffer in the absence of methanol, the cold denaturation temperature would be -30 degrees C. Data obtained with high concentrations of methanol indicate that residues are exposed to a similar degree upon either heat- or cold-induced denaturation. Aggregation does not occur in the cold-denatured protein. Cold-induced denaturation is fully reversible at a protein concentration of 250 micrograms/ml. The spectra of the two denatured forms are similar.     

Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability.

We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface.     

Conformational stability of HPr: the histidine-containing phosphocarrier protein from Bacillus subtilis.

The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (delta Hg, Tg, delta Sg, and delta Cp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520-2525) was used to estimate delta Cp as was a global analysis of the thermal- and urea-induced unfolding data. Each method used to analyze the data gives a similar value for delta Cp (1,170 +/- 50 cal mol-1K-1). Despite the high melting temperature for HPr (Tg = 73.5 degrees C), the maximum stability of the protein, which occurs at 26 degrees C, is quite modest (delta Gs = 4.2 kcal mol-1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of delta Cp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685-691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.     

FTIR study on heat-induced and pressure-assisted cold-induced changes in structure of bovine alpha-lactalbumin: stabilizing role of calcium ion

The second derivative FTIR study of heat-induced and pressure-assisted cold-induced changes in the secondary structure of bovine alpha-lactalbumin was carried out for native holoprotein and calcium ion depleted apoprotein. The secondary structure and compactness of alpha-lactalbumin were examined in a temperature range from 20 to 80 degrees C during the heat treatment and 20 to -15 degrees C during the pressure-assisted cold treatment. This was the first FTIR study on the pressure-assisted cold denaturation of a protein. Because protein solutions had close to neutral pD and low ionic strength, the apoprotein remained in the molten globule state and the holoform maintained its native tertiary structure. In order to distinguish between unfolding-related and partially deuterated exchange-related spectral changes, we examined both the fully deuterated holoform and the partially deuterated holoform. The quantitative analysis of the spectral changes in the amide I/I' vibrational band revealed that the 3(10) helices were more prone to thermal unfolding than the alpha helices. We observed that the protein's compactness and secondary structure were both considerably stabilized against an increase and decrease in temperature by the presence of a calcium ion. Under the conditions of this study, only the apoprotein was susceptible to the cold denaturation. In contrast to this, an unexpected linear increase of the alpha-helical content was observed upon the cooling of the holoprotein under high pressure. The results were discussed in reference to the existing crystallographic data for crystals of human alpha-lactalbumin grown at two different temperatures.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

Highly anomalous energetics of protein cold denaturation linked to folding-unfolding kinetics

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a "mirror image" of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.     

Protein stability curves

The stability curve of a protein is defined as the plot of the free energy of unfolding as a function of temperature. For most proteins the change in heat capacity on denaturation, or unfolding, is large but approximately constant. When unfolding is s two-state process, most of the salient features of the stability curves of proteins can be derived from this fact. A number of relations are obtained, including the special features of low-temperature denaturation, the properties of the maximum in stability, and the interrelationships of the characteristic temperatures of the protein. The paper closes with a formula that permits one to calculate small changes in stabilization free energy from changes in the melting temperature of the protein.     

Pressure denaturation of the yeast prion protein Ure2

Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure. Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl. Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state. Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar.     

Thermodynamic characterization of the human acidic fibroblast growth factor: evidence for cold denaturation.

The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrations of urea using fluorescence and far-UV CD circular dichroism (CD) spectroscopy. The equilibrium unfolding transitions at pH 7.0 are adequately described by a two-state (native <--> unfolded state) mechanism. The stability of the protein is pH-dependent, and the protein unfolds completely below pH 3.0 (at 25 degrees C). hFGF-1 is shown to undergo a two-state transition only in a narrow pH range (pH 7.0-8.0). Under acidic (pH <6.0) and basic (pH >8.0) conditions, hFGF-1 is found to unfold noncooperatively, involving the accumulation of intermediates. The average temperature of maximum stability is determined to be 295.2 K. The heat capacity change (DeltaC(p)()) for the unfolding of hFGF-1 is estimated to be 2.1 +/- 0.5 kcal.mol(-1).K(-1). Temperature denaturation experiments in the absence and presence of urea show that hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional (1)H-(15)N HSQC spectra of hFGF-1 acquired at subzero temperatures clearly show that hFGF-1 unfolds under low-temperature conditions. The significance of the noncooperative unfolding under acidic conditions and the cold denaturation process observed in hFGF-1 are discussed in detail.