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.     

Designing a High Throughput Refolding Array Using a Combination of the GroEL Chaperonin and Osmolytes

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL–GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin–substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a ‘folding array’ to identify superior folding conditions. The stable chaperonin–substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (~mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.     

Observing the osmophobic effect in action at the single molecule level

Protecting osmolytes are widespread small organic molecules able to stabilize the folded state of most proteins against various denaturing stresses in vivo. The osmophobic model explains thermodynamically their action through a preferential exclusion of the osmolyte molecules from the protein surface, thus favoring the formation of intrapeptide hydrogen bonds. Few works addressed the influence of protecting osmolytes on the protein unfolding transition state and kinetics. Among those, previous single molecule force spectroscopy experiments evidenced a complexation of the protecting osmolyte molecules at the unfolding transition state of the protein, in apparent contradiction with the osmophobic nature of the protein backbone. We present single-molecule evidence that glycerol, which is a ubiquitous protecting osmolyte, stabilizes a globular protein against mechanical unfolding without binding into its unfolding transition state structure. We show experimentally that glycerol does not change the position of the unfolding transition state as projected onto the mechanical reaction coordinate. Moreover, we compute theoretically the projection of the unfolding transition state onto two other common reaction coordinates, that is, the number of native peptide bonds and the weighted number of native contacts. To that end, we augment an analytic Ising-like protein model with support for group-transfer free energies. Using this model, we find again that the position of the unfolding transition state does not change in the presence of glycerol, giving further support to the conclusions based on the single-molecule experiments.     

Effects of a protecting osmolyte on the ion atmosphere surrounding DNA duplexes

Osmolytes are small, chemically diverse, organic solutes that function as an essential component of cellular stress response. Protecting osmolytes enhance protein stability via preferential exclusion, and nonprotecting osmolytes, such as urea, destabilize protein structures. Although much is known about osmolyte effects on proteins, less is understood about osmolyte effects on nucleic acids and their counterion atmospheres. Nonprotecting osmolytes destabilize nucleic acid structures, but effects of protecting osmolytes depend on numerous factors including the type of nucleic acid and the complexity of the functional fold. To begin quantifying protecting osmolyte effects on nucleic acid interactions, we used small-angle X-ray scattering (SAXS) techniques to monitor DNA duplexes in the presence of sucrose. This protecting osmolyte is a commonly used contrast matching agent in SAXS studies of protein-nucleic acid complexes; thus, it is important to characterize interaction changes induced by sucrose. Measurements of interactions between duplexes showed no dependence on the presence of up to 30% sucrose, except under high Mg(2+) conditions where stacking interactions were disfavored. The number of excess ions associated with DNA duplexes, reported by anomalous small-angle X-ray scattering (ASAXS) experiments, was sucrose independent. Although protecting osmolytes can destabilize secondary structures, our results suggest that ion atmospheres of individual duplexes remain unperturbed by sucrose.     

Naturally occurring osmolytes modulate the nanomechanical properties of polycystic kidney disease domains

Polycystin-1 (PC1) is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. However, it is known that the native conformation of proteins can be stabilized by protecting osmolytes that are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here, we used single-molecule force spectroscopy to study directly the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and trimethylamine N-oxide can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated polycystin-2 channel or other intracellular events mediated by PC1.     

The osmophobic effect: natural selection of a thermodynamic force in protein folding

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments and these osmolytes protect intracellular macromolecules against the denaturing environmental stress. In natural selection of organic osmolytes as protein stabilizers, it appears that the osmolyte property selected for is the unfavorable interaction between the osmolyte and the peptide backbone, a solvophobic thermodynamic force that we call the osmophobic effect. Because the peptide backbone is highly exposed to osmolyte in the denatured state, the osmophobic effect preferentially raises the free energy of the denatured state, shifting the equilibrium in favor of the native state. By focusing the solvophobic force on the denatured state, the native state is left free to function relatively unfettered by the presence of osmolyte. The osmophobic effect is a newly uncovered thermodynamic force in nature that complements the well-recognized hydrophobic interactions, hydrogen bonding, electrostatic and dispersion forces that drive protein folding. In organisms whose survival depends on the intracellular presence of osmolytes that can counteract denaturing stresses, the osmophobic effect is as fundamental to protein folding as these well-recognized forces.     

Temperature-mediated switching of protectant-denaturant behavior of trimethylamine-N-oxide and consequences on protein stability from a replica exchange molecular dynamics simulation study

The detailed mechanism of protein folding–unfolding processes with the aid of osmolytes has been a leading topic of discussion over many decades. We have used replica-exchange molecular dynamics simulation to propose the molecular mechanism of interaction of a 20-residue mini-protein with urea and trimethylamine N-oxide (TMAO) that act as denaturing and protecting osmolyte, respectively, in binary osmolyte solutions. Urea is found to exert its action by interacting directly with the protein residues. Temperature tolerance of TMAO’s action is particularly emphasised in this study. At lower range of temperature, TMAO acts as a successful protein protectant. Interestingly, the study discloses the tendency of TMAO molecules to prefer self-association at the protein surface at elevated temperature. A greater number of TMAO molecules in the protein hydration shell at higher temperature is also observed. Dihedral angle principal component analysis and free energy landscape plots sampled all possible conformations adopted by the protein that reveal highly folded behaviour of the protein in pure water and binary TMAO solutions and highly unfolded behaviour in presence of urea.     

An analysis of the molecular origin of osmolyte-dependent protein stability

Protein solvation is the key determinant for isothermal, concentration-dependent effects on protein equilibria, such as folding. The required solvation information can be extracted from experimental thermodynamic data using Kirkwood-Buff theory. Here we derive and discuss general properties of proteins and osmolytes that are pertinent to their biochemical behavior. We find that hydration depends very little on osmolyte concentration and type. Strong dependencies on both osmolyte concentration and type are found for osmolyte self-solvation and protein-osmolyte solvation changes upon unfolding. However, solvation in osmolyte solutions does not involve complex concentration dependencies as found in organic molecules that are not used as osmolytes in nature. It is argued that the simple solvation behavior of naturally occurring osmolytes is a prerequisite for their usefulness in osmotic regulation in vivo.     

Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principle

Amyloid proteins are converted from their native‐fold to long β‐sheet‐rich fibrils in a typical sigmoidal time‐dependent protein aggregation curve. This reaction process from monomer or dimer to oligomer to nuclei and then to fibrils is the subject of intense study. The main results of this work are based on the use of a well‐studied model amyloid protein, insulin, which has been used in vitro by others. Nine osmolyte molecules, added during the protein aggregation process for the production of amyloid fibrils, slow‐down or speed up the process depending on the molecular structure of each osmolyte. Of these, all stabilizing osmolytes (sugars) slow down the aggregation process in the following order: tri > di > monosaccharides, whereas destabilizing osmolytes (urea, guanidium hydrochloride) speed up the aggregation process in a predictable way that fits the trend of all osmolytes. With respect to kinetics, we illustrate, by adapting our earlier reaction model to the insulin system, that the intermediates (trimers, tetramers, pentamers, etc.) are at very low concentrations and that nucleation is orders of magnitude slower than fibril growth. The results are then collated into a cogent explanation using the preferential exclusion and accumulation of osmolytes away from and at the protein surface during nucleation, respectively. Both the heat of solution and the neutral molecular surface area of the osmolytes correlate linearly with two fitting parameters of the kinetic rate model, that is, the lag time and the nucleation rate prior to fibril formation. These kinetic and thermodynamic results support the preferential exclusion model and the existence of oligomers including nuclei and larger structures that could induce toxicity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

High concentrations of viscogens decrease the protein folding rate constant by prematurely collapsing the coil

In several studies, viscogenic osmolytes have been suggested to decrease the folding rate constant of polypeptides by slowing their motion through the solvent. Here, we show that osmolytes may slow protein folding by prematurely collapsing the coil. At low or moderate concentrations of osmolytes (<30%), folding of the two-state protein CI2 becomes faster with increasing osmolyte concentrations, suggesting that the kinetics are governed by protein stability. However, at higher concentrations of osmolyte, the coil collapses in the dead-time of the refolding experiment, causing a dramatic drop in the folding rate. The collapsed state is non-native and appears to be different for different osmolytes.     

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.     

Living with urea stress

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments. These osmolytes protect intracellular macromolecules against denaturing environmental stress. In contrast to the usually benign effects of most organic osmolytes, the waste product urea is a well-known perturbant of macromolecules. Although urea is a perturbing solute which inhibits enzyme activity and stability, it is employed by some species as a major osmolyte. The answer to this paradox was believed to be the discovery of protective osmolytes (methylamines). We review the current state of knowledge on the various ways of counteracting the harmful effects of urea in nature and the mechanisms for this. This review ends with the mechanistic idea that cellular salt (KCl/NaCl) plays a crucial role in counteracting the effects of urea, either by inducing required chaperones or methylamines, or by thermodynamic interactions with urea-destabilised proteins. We also propose future opportunities and challenges in the field.     

Osmolyte-induced protein folding free energy changes

Upon addition of protecting osmolyte to an aqueous solution of an intrinsically unstructured protein, spectral observables are often seen to change in a sigmoid fashion as a function of increasing osmolyte concentration. Commonly, such data are analyzed using the linear extrapolation model (LEM), a method that defines a scale from 0%-100% folded species at each osmolyte concentration by means of extending pre- and post-folding baselines into the transition region. Defining the 0%-100% folding scale correctly for each osmolyte is an important part of the analysis, leading to evaluation of the fraction of folded protein existing in the absence of osmolytes. In this study, we used reduced and carboxyamidated RNase T1 (RCAM-T1) as an intrinsically unstructured protein, and determined the thermodynamic stability of RCAM-T1 induced by naturally occurring osmolytes. Because the folded fraction of the protein population determined by experiments of thermal and urea-induced denaturation is nonzero in the absence of osmolytes at 15 degrees C, the commonly used LEM can lead to false values of DeltaG[stackD-->N0] for protein folding due to the arbitrary assumption that the protein is 100% unfolded in the presence of buffer alone. To correct this problem, titration of the protein solution with urea and extrapolating back to zero urea concentration gives the spectral value for 100% denatured protein. With fluorescence as the observable we redefine F/F0 to F/F0extrap = 1.0 and require that the denatured-state baseline have this value as its intercept. By so doing, the 0%-100% scale-corrected DeltaG[D-->N0] values of RCAM-T1 folding in the presence of various osmolytes are then found to be identical, with small error, demonstrating that DeltaG[D-->N0] is independent of the osmolytes used. Such a finding is an important step in validating this quantity derived from the LEM as having the properties expected of an authentic thermodynamic parameter. The rank order of osmolyte efficacies in stabilizing RCAM-T1 is sarcosine > sucrose > sorbitol > proline > betaine > glycerol.     

Protein and DNA destabilization by osmolytes: the other side of the coin

Osmolytes are naturally occurring small molecules accumulated intracellularly to protect organisms from various denaturing stresses. Similar to the two faces of a coin, several of these osmolytes are stabilizing and destabilizing proteins depending on the concentrations and/or solvent conditions. For example, the well known stabilizing osmolyte, trehalose destabilizes some proteins at high concentration and/or high pH. In spite of the fact that destabilizing aspects of osmolytes can modulate many cellular processes including regulation of protein homeostasis (proteostasis), protein-protein interaction, and protein-DNA interaction, researchers have mostly focused on the stabilizing aspects of osmolytes. Thus, it is important to look into both aspects of osmolytes to determine their precise role under physiological conditions. In this article, we have discussed both stabilizing and destabilizing/denaturant aspects of osmolytes to uncover both sides of the coin.     

Second virial coefficients as a measure of protein--osmolyte interactions

The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte. To better understand protein--smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein. From these experiments, we determined the second virial coefficients for a series of osmolytes. We have interpreted the second virial coefficient as a measure of both excluded volume and protein--osmolyte binding. We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins.     

An osmolyte mitigates the destabilizing effect of protein crowding

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by dehydration-stressed cells.     

Strategies for folding of affinity tagged proteins using GroEL and osmolytes

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.     

BPPred: a Web-based computational tool for predicting biophysical parameters of proteins

We exploit the availability of recent experimental data on a variety of proteins to develop a Web-based prediction algorithm (BPPred) to calculate several biophysical parameters commonly used to describe the folding process. These parameters include the equilibrium m-values, the length of proteins, and the changes upon unfolding in the solvent-accessible surface area, in the heat capacity, and in the radius of gyration. We also show that the knowledge of any one of these quantities allows an estimate of the others to be obtained, and describe the confidence limits with which these estimations can be made. Furthermore, we discuss how the kinetic m-values, or the Beta Tanford values, may provide an estimate of the solvent-accessible surface area and the radius of gyration of the transition state for protein folding. Taken together, these results suggest that BPPred should represent a valuable tool for interpreting experimental measurements, as well as the results of molecular dynamics simulations.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.