Osmolyte effects on protein stability and solubility: a balancing act between backbone and side-chains.

In adaptation biology the discovery of intracellular osmolyte molecules that in some cases reach molar levels, raises questions of how they influence protein thermodynamics. We've addressed such questions using the premise that from atomic coordinates, the transfer free energy of a native protein (ΔG(tr,N)) can be predicted by summing measured water-to-osmolyte transfer free energies of the protein's solvent exposed side chain and backbone component parts. ΔG(tr,D) is predicted using a self avoiding random coil model for the protein, and ΔG(tr,D)-ΔG(tr,N), predicts the m-value, a quantity that measures the osmolyte effect on the N⇌D transition. Using literature and newly measured m-values we show 1:1 correspondence between predicted and measured m-values covering a range of 12 kcal/mol/M in protein stability for 46 proteins and 9 different osmolytes. Osmolytes present a range of side chain and backbone effects on N and D solubility and protein stability key to their biological roles.

Backbone additivity in the transfer model of protein solvation.

The transfer model implying additivity of the peptide backbone free energy of transfer is computationally tested. Molecular dynamics simulations are used to determine the extent of change in transfer free energy (DeltaG(tr)) with increase in chain length of oligoglycine with capped end groups. Solvation free energies of oligoglycine models of varying lengths in pure water and in the osmolyte solutions, 2M urea and 2M trimethylamine N-oxide (TMAO), were calculated from simulations of all atom models, and DeltaG(tr) values for peptide backbone transfer from water to the osmolyte solutions were determined. The results show that the transfer free energies change linearly with increasing chain length, demonstrating the principle of additivity, and provide values in reasonable agreement with experiment. The peptide backbone transfer free energy contributions arise from van der Waals interactions in the case of transfer to urea, but from electrostatics on transfer to TMAO solution. The simulations used here allow for the calculation of the solvation and transfer free energy of longer oligoglycine models to be evaluated than is currently possible through experiment. The peptide backbone unit computed transfer free energy of -54 cal/mol/M compares quite favorably with -43 cal/mol/M determined experimentally.

Hydrogen bonding progressively strengthens upon transfer of the protein urea-denatured state to water and protecting osmolytes.

Using osmolyte cosolvents, we show that hydrogen-bonding contributions can be separated from hydrophobic interactions in the denatured state ensemble (DSE). Specifically, the effects of urea and the protecting osmolytes sarcosine and TMAO are reported on the thermally unfolded DSE of Nank4-7*, a truncated notch ankyrin protein. The high thermal energy of this state in the presence and absence of 6 M urea or 1 M sarcosine solution is sufficient to allow large changes in the hydrodynamic radius (R(h)) and secondary structure accretion without populating the native state. The CD change at 228 nm is proportional to the inverse of the volume of the DSE, giving a compact species equivalent to a premolten globule in 1 M sarcosine. The same general effects portraying hierarchical folding observed in the DSE at 55 degrees C are also often seen at room temperature. Analysis of Nank4-7* DSE structural energetics at room temperature as a function of solvent provides rationale for understanding the structural and dimensional effects in terms of how modulation of the solvent alters solvent quality for the peptide backbone. Results show that while the strength of hydrophobic interactions changes little on transferring the DSE from 6 M urea to water and then to 1 M TMAO, backbone-backbone (hydrogen-bonding) interactions are greatly enhanced due to progressively poorer solvent quality for the peptide backbone. Thus, increased intrachain hydrogen bonding guides secondary structure accretion and DSE contraction as solvent quality is decreased. This process is accompanied by increasing hydrophobic contacts as chain contraction gathers hydrophobes into proximity and the declining urea-backbone free energy gradient reaches urea concentrations that are energetically insufficient to keep hydrophobes apart in the DSE.

Osmolyte-induced conformational changes in the Hsp90 molecular chaperone.

Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native-state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X-ray scattering we observe dramatic osmolyte-dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well-established osmolyte principles that govern stability also apply to large-scale conformational changes, a proposition that is corroborated by structure-based fitting of the scattering data, surface area comparisons and m-value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain-domain interaction. Hsp90 adopts an additional ligand-specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.

Rational modulation of conformational fluctuations in adenylate kinase reveals a local unfolding mechanism for allostery and functional adaptation in proteins.

Elucidating the complex interplay between protein structure and dynamics is a prerequisite to an understanding of both function and adaptation in proteins. Unfortunately, it has been difficult to experimentally decouple these effects because it is challenging to rationally design mutations that will either affect the structure but not the dynamics, or that will affect the dynamics but not the structure. Here we adopt a mutation approach that is based on a thermal adaptation strategy observed in nature, and we use it to study the binding interaction of Escherichia coli adenylate kinase (AK). We rationally design several single-site, surface-exposed glycine mutations to selectively perturb the excited state conformational repertoire, leaving the ground-state X-ray crystallographic structure unaffected. The results not only demonstrate that the conformational ensemble of AK is significantly populated by a locally unfolded state that is depopulated upon binding, but also that the excited-state conformational ensemble can be manipulated through mutation, independent of perturbations of the ground-state structures. The implications of these results are twofold. First, they indicate that it is possible to rationally design dynamic allosteric mutations, which do not propagate through a pathway of structural distortions connecting the mutated and the functional sites. Secondly and equally as important, the results reveal a general strategy for thermal adaptation that allows enzymes to modulate binding affinity by controlling the amount of local unfolding in the native-state ensemble. These findings open new avenues for rational protein design and fundamentally illuminate the role of local unfolding in function and adaptation.

Structure and energetics of the hydrogen-bonded backbone in protein folding.

We seek to understand the link between protein thermodynamics and protein structure in molecular detail. A classical approach to this problem involves assessing changes in protein stability resulting from added cosolvents. Under any given conditions, protein molecules in aqueous buffer are in equilibrium between unfolded and folded states, U(nfolded) <==> N(ative). Addition of organic osmolytes, small uncharged compounds found throughout nature, shift this equilibrium. Urea, a denaturing osmolyte, shifts the equilibrium toward U; trimethylamine N-oxide (TMAO), a protecting osmolyte, shifts the equilibrium toward N. Using the Tanford Transfer Model, the thermodynamic response to many such osmolytes has been dissected into groupwise free energy contributions. It is found that the energetics involving backbone hydrogen bonding controls these shifts in protein stability almost entirely, with osmolyte cosolvents simply dialing between solvent-backbone versus backbone-backbone hydrogen bonds, as a function of solvent quality. This reciprocal relationship establishes the essential link between protein thermodynamics and the protein's hydrogen-bonded backbone structure.

Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides.

Protein stability and solubility depend strongly on the presence of osmolytes, because of the protein preference to be solvated by either water or osmolyte. It has traditionally been assumed that only this relative preference can be measured, and that the individual solvation contributions of water and osmolyte are inaccessible. However, it is possible to determine hydration and osmolyte solvation (osmolation) separately using Kirkwood-Buff theory, and this fact has recently been utilized by several researchers. Here, we provide a thermodynamic assessment of how each surface group on proteins contributes to the overall hydration and osmolation. Our analysis is based on transfer free energy measurements with model-compounds that were previously demonstrated to allow for a very successful prediction of osmolyte-dependent protein stability. When combined with Kirkwood-Buff theory, the Transfer Model provides a space-resolved solvation pattern of the peptide unit, amino acids, and the folding/unfolding equilibrium of proteins in the presence of osmolytes. We find that the major solvation effects on protein side-chains originate from the osmolytes, and that the hydration mostly depends on the size of the side-chain. The peptide backbone unit displays a much more variable hydration in the different osmolyte solutions. Interestingly, the presence of sucrose leads to simultaneous accumulation of both the sugar and water in the vicinity of peptide groups, resulting from a saccharide accumulation that is less than the accumulation of water, a net preferential exclusion. Only the denaturing osmolyte, urea, obeys the classical solvent exchange mechanism in which the preferential interaction with the peptide unit excludes water.

Anatomy of energetic changes accompanying urea-induced protein denaturation.

Because of its protein-denaturing ability, urea has played a pivotal role in the experimental and conceptual understanding of protein folding and unfolding. The measure of urea's ability to force a protein to unfold is given by the m value, an experimental quantity giving the free energy change for unfolding per molar urea. With the aid of Tanford's transfer model [Tanford C (1964) J Am Chem Soc 86:2050-2059], we use newly obtained group transfer free energies (GTFEs) of protein side-chain and backbone units from water to 1 M urea to account for the m value of urea, and the method reveals the anatomy of protein denaturation in terms of residue-level free energy contributions of groups newly exposed on denaturation. The GTFEs were obtained by accounting for solubility and activity coefficient ratios accompanying the transfer of glycine from water to 1 M urea. Contrary to the opinions of some researchers, the GTFEs show that urea does not denature proteins through favorable interactions with nonpolar side chains; what drives urea-induced protein unfolding is the large favorable interaction of urea with the peptide backbone. Although the m value is said to be proportional to surface area newly exposed on denaturation, only approximately 25% of the area favorably contributes to unfolding (because of newly exposed backbone units), with approximately 75% modestly opposing urea-induced denaturation (originating from side-chain exposure). Use of the transfer model and newly determined GTFEs achieves the long-sought goal of predicting urea-dependent cooperative protein unfolding energetics at the level of individual amino acid residues.

Application of the transfer model to understand how naturally occurring osmolytes affect protein stability.

A primary thermodynamic goal in protein biochemistry is to attain a predictive understanding of the energetic changes responsible for solvent-induced folding and unfolding. This chapter demonstrates the use of Tanford's transfer model to predict solvent-dependent cooperative protein folding/unfolding free energy changes (m values). This approach provides a thermodynamic description of these free energy changes in terms of individual contributions from the peptide backbone and residue side chains. The quantitative success of the transfer model has been hindered for many years because of unresolved issues involving proper measurement of the group transfer-free energies of amino acid side chains and the peptide backbone unit. This chapter demonstrates what is necessary to design experiments properly so that reliable values of group transfer-free energies are obtainable. It then demonstrates how to derive a prediction of the m value for the description of protein folding/unfolding cooperativity and that the calculated values using the transfer model agree quite well with experimentally measured values.

Molecular basis of the apparent near ideality of urea solutions.

Activity coefficients of urea solutions are calculated to explore the mechanism of its solution properties, which form the basis for its well-known use as a strong protein denaturant. We perform free energy simulations of urea solutions in different urea concentrations using two urea models (OPLS and KBFF models) to calculate and decompose the activity coefficients. For the case of urea, we clarify the concept of the ideal solution in different concentration scales and standard states and its effect on our subsequent analysis. The analytical form of activity coefficients depends on the concentration units and standard states. For both models studied, urea displays a weak concentration dependence for excess chemical potential. However, for the OPLS force-field model, this results from contributions that are independent of concentration to the van der Waals and electrostatic components whereas for the KBFF model those components are nontrivial but oppose each other. The strong ideality of urea solutions in some concentration scales (incidentally implying a lack of water perturbation) is discussed in terms of recent data and ideas on the mechanism of urea denaturation of proteins.

Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase.

During cell volume regulation, intracellular concentration changes occur in both inorganic and organic osmolytes in order to balance the extracellular osmotic stress and maintain cell volume homeostasis. Generally, salt and urea increase the Km's of enzymes and trimethylamine N-oxide (TMAO) counteracts these effects by decreasing Km's. The hypothesis to account for these effects is that urea and salt shift the native state ensemble of the enzyme toward conformers that are substrate-binding incompetent (BI), while TMAO shifts the ensemble toward binding competent (BC) species. Km's are often complex assemblies of rate constants involving several elementary steps in catalysis, so to better understand osmolyte effects we have focused on a single elementary event, substrate binding. We test the conformational shift hypothesis by evaluating the effects of salt, urea, and TMAO on the mechanism of binding glycerol 3-phosphate, a substrate analogue, to yeast triosephosphate isomerase. Temperature-jump kinetic measurements promote a mechanism consistent with osmolyte-induced shifts in the [BI]/[BC] ratio of enzyme conformers. Importantly, salt significantly affects the binding constant through its effect on the activity coefficients of substrate, enzyme, and enzyme-substrate complex, and it is likely that TMAO and urea affect activity coefficients as well. Results indicate that the conformational shift hypothesis alone does not account for the effects of osmolytes on Km's.

Protein phase diagrams II: nonideal behavior of biochemical reactions in the presence of osmolytes.

In the age of biochemical systems biology, proteomics, and high throughput methods, the thermodynamic quantification of cytoplasmatic reaction networks comes into reach of the current generation of scientists. What is needed to efficiently extract the relevant information from the raw data is a robust tool for evaluating the number and stoichiometry of all observed reactions while providing a good estimate of the thermodynamic parameters that determine the molecular behavior. The recently developed phase-diagram method, strictly speaking a graphical representation of linkage or Maxwell Relations, offers such capabilities. Here, we extend the phase diagram method to nonideal conditions. For the sake of simplicity, we choose as an example a reaction system involving the protein RNase A, its inhibitor CMP, the osmolyte urea, and water. We investigate this system as a function of the concentrations of inhibitor and osmolyte at different temperatures ranging from 280 K to 340 K. The most interesting finding is that the protein-inhibitor binding equilibrium depends strongly on the urea concentration--by orders-of-magnitude more than expected from urea-protein interaction alone. Moreover, the m-value of ligand binding is strongly concentration-dependent, which is highly unusual. It is concluded that the interaction between small molecules like urea and CMP can significantly contribute to cytoplasmic nonideality. Such a finding is highly significant because of its impact on renal tissue where high concentrations of cosolutes occur regularly.

Mixed osmolytes: the degree to which one osmolyte affects the protein stabilizing ability of another.

Mixtures of organic osmolytes occur in cells of many organisms, raising the question of whether their actions on protein stability are independent or synergistic. To investigate this question it is desirable to develop a system that permits evaluation of the effect of one osmolyte on the efficacy of another to either force-fold or denature a protein. A means of evaluating the efficacy of an osmolyte is provided by its m-value, an experimental quantity that measures the ability of the osmolyte to force a protein to unfold or fold. An experimental system is presented that enables evaluations of the m-values of osmolytes in the presence and absence of a second osmolyte. The experimental system involves use of a marginally stable protein in 10 mM buffer (pH 7, 200 mM salt, and 34 degrees C) that is at the midpoint of its native to denatured transition. These conditions enable determination of m-values for protecting and denaturing osmolytes in the presence and absence of a second osmolyte, permitting assessment of the extent to which the two osmolytes affect each other's efficacy. The two osmolytes investigated in this work are the denaturing osmolyte, urea, and the protecting osmolyte, sarcosine. Results show unequivocally that neither osmolyte alters the efficacy of the other in forcing the protein to fold or unfold-the osmolytes act independently on the protein despite their combined concentrations being in the multi-molar range. These osmolytes avoid altering one another's efficacy at these high concentrations because the number of osmolyte interaction sites on the protein is large and the binding constants are quite small. Consequently, the site occupancies are low enough in number that the two osmolytes neither compete nor cooperate in interacting with the protein.

A molecular mechanism for osmolyte-induced protein stability.

Osmolytes are small organic compounds that affect protein stability and are ubiquitous in living systems. In the equilibrium protein folding reaction, unfolded (U) native (N), protecting osmolytes push the equilibrium toward N, whereas denaturing osmolytes push the equilibrium toward U. As yet, there is no universal molecular theory that can explain the mechanism by which osmolytes interact with the protein to affect protein stability. Here, we lay the groundwork for such a theory, starting with a key observation: the transfer free energy of protein backbone from water to a water/osmolyte solution, Deltagtr, is negatively correlated with an osmolyte's fractional polar surface area. Deltagtr measures the degree to which an osmolyte stabilizes a protein. Consequently, a straightforward interpretation of this correlation implies that the interaction between the protein backbone and osmolyte polar groups is more favorable than the corresponding interaction with nonpolar groups. Such an interpretation immediately suggests the existence of a universal mechanism involving osmolyte, backbone, and water. We test this idea by using it to construct a quantitative solvation model in which backbone/solvent interaction energy is a function of interactant polarity, and the number of energetically equivalent ways of realizing a given interaction is a function of interactant surface area. Using this model, calculated Deltagtr values show a strong correlation with measured values (R = 0.99). In addition, the model correctly predicts that protecting/denaturing osmolytes will be preferentially excluded/accumulated around the protein backbone. Taken together, these model-based results rationalize the dominant interactions observed in experimental studies of osmolyte-induced protein stabilization and denaturation.

Metrics that differentiate the origins of osmolyte effects on protein stability: a test of the surface tension proposal.

Osmolytes that are naturally selected to protect organisms against environmental stresses are known to confer stability to proteins via preferential exclusion from protein surfaces. Solvophobicity, surface tension, excluded volume, water structure changes and electrostatic repulsion are all examples of forces proposed to account for preferential exclusion and the ramifications exclusion has on protein properties. What has been lacking is a systematic way of determining which force(s) is(are) responsible for osmolyte effects. Here, we propose the use of two experimental metrics for assessing the abilities of various proposed forces to account for osmolyte-mediated effects on protein properties. Metric 1 requires prediction of the experimentally determined ability of the osmolyte to bring about folding/unfolding resulting from the application of the force in question (i.e. prediction of the m-value of the protein in osmolyte). Metric 2 requires prediction of the experimentally determined ability of the osmolyte to contract or expand the Stokes radius of the denatured state resulting from the application of the force. These metrics are applied to test separate claims that solvophobicity/solvophilicity and surface tension are driving forces for osmolyte-induced effects on protein stability. The results show clearly that solvophobic/solvophilic forces readily account for protein stability and denatured state dimensional effects, while surface tension alone fails to do so. The agreement between experimental and predicted m-values involves both positive and negative m-values for three different proteins, and as many as six different osmolytes, illustrating that the tests are robust and discriminating. The ability of the two metrics to distinguish which forces account for the effects of osmolytes on protein properties and which do not, provides a powerful means of investigating the origins of osmolyte-protein effects.

Predicting the energetics of osmolyte-induced protein folding/unfolding.

A primary thermodynamic goal in protein biochemistry is to attain predictive understanding of the detailed energetic changes that are responsible for folding/unfolding. Through use of recently determined free energies of side-chain and backbone transfer from water to osmolytes and Tanford's transfer model, we demonstrate that the long-sought goal of predicting solvent-dependent cooperative protein folding/unfolding free-energy changes (m values) can be achieved. Moreover, the approach permits dissection of the folding/unfolding free-energy changes into individual contributions from the peptide backbone and residue side chains.

Additive transfer free energies of the peptide backbone unit that are independent of the model compound and the choice of concentration scale.

With knowledge of individual transfer free energies of chemical groups that become newly exposed on protein denaturation and assuming the group transfer free energy contributions are additive, it should be possible to predict the stability of a protein in the presence of denaturant. Unfortunately, several unresolved issues have seriously hampered quantitative development of this transfer model for protein folding/unfolding. These issues include the lack of adequate demonstration that group transfer free energies (DeltaG(tr)) are additive and independent of the choice of model compound, the problem arising from dependence of DeltaG(tr) on concentration scales, the lack of knowledge of activity coefficients, and the validity of the mathematical constructs used in obtaining DeltaG(tr) values. Regarding transfer from water to 1 M concentrations of the naturally occurring osmolytes, trimethylamine-N-oxide (TMAO), sarcosine, betaine, proline, glycerol, sorbitol, sucrose, trehalose, and urea, using cyclic glycylglycine, zwitterionic glycine peptides, and N-acetylglycine amide peptides as models for the peptide backbone of proteins, we set out to address these issues and obtain DeltaG(tr) values for the peptide backbone unit. We demonstrate experimental approaches that obviate the choice of concentration scale and demonstrate additivity in DeltaG(tr) of the peptide backbone unit for all solvent systems studied. Evidence is presented to show that the DeltaG(tr) values are independent of the chemical model studied, and experimental conditions are given to illustrate when the mathematical constructs are valid and when activity coefficients can be ignored. Resolution of the long-standing issues that have stymied development of the transfer model now make it possible to design transfer experiments that yield reliable and quantitative values for the interactions between osmolyte-containing solvents and native and unfolded protein.

Hydrogen exchange kinetics of RNase A and the urea:TMAO paradigm.

A key paradigm in the biology of adaptation holds that urea affects protein function by increasing the fluctuations of the native state, while trimethylamine N-oxide (TMAO) affects function in the opposite direction by decreasing the normal fluctuations of the native ensemble. Using urea and TMAO separately and together, hydrogen exchange (HX) studies on RNase A at pH* 6.35 were used to investigate the basic tenets of the urea:TMAO paradigm. TMAO (1 M) alone decreases HX rate constants of a select number of sites exchanging from the native ensemble, and low urea alone increases the rate constants of some of the same sites. Addition of TMAO to urea solutions containing RNase A also suppresses HX rate constants. The data show that urea and TMAO independently or in combination affect the dynamics of the native ensemble in opposing ways. The results provide evidence in support of the counteraction aspect of the urea:TMAO paradigm linking structural dynamics with protein function in urea-rich organs and organisms. RNase A is so resistant to urea denaturation at pH* 6.35 that even in the presence of 4.8 M urea, the native ensemble accounts for >99.5% of the protein. An essential test, devised to determine the HX mechanism of exchangeable protons, shows that over the 0-4.8 M urea concentration range nearly 80% of all observed sites convert from EX2 to EX1. The slow exchange sites are all EX1; they do not exhibit global exchange even at urea concentrations (5.8 M) well into the denaturation transition zone, and their energetically distinct activated complexes leading to exchange gives evidence of residual structure. Under these experimental conditions, the use of DeltaG(HX) as a basis for HX analysis of RNase A urea denaturation is invalid.