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.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Osmophobic effect of glycerol on irreversible thermal denaturation of rabbit creatine kinase

Protein stability plays an extremely important role not only in its biological function but also in medical science and protein engineering. Osmolytes provide a general method to protect proteins from the unfolding and aggregation induced by extreme environmental stress. In this study, the effect of glycerol on protection of the model enzyme creatine kinase (CK) against heat stress was investigated by a combination of spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal inactivation and aggregation in a concentration-dependent manner. The spectroscopic measurements suggested that the protective effect of glycerol was a result of enhancing the structural stability of native CK. A further thermodynamic analysis using the activated-complex theory suggested that the effect of glycerol on preventing CK against aggregation was consistent with those previously established mechanisms in reversible systems. The osmophobic effect of glycerol, which preferentially raised the free energy of the activated complex, shifted the equilibrium between the native state and the activated complex in favor of the native state. A comparison of the inactivation rate and the denaturation rate suggested that the protection of enzyme activity by glycerol should be attributed to the enhancement of the structural stability of the whole protein rather than the flexible active site.     

Worm-like Ising model for protein mechanical unfolding under the effect of osmolytes

We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.     

Role of osmolytes as chemical chaperones during the refolding of aminoacylase.

The refolding and reactivation of aminoacylase is particularly difficult because of serious off-pathway aggregation. The effects of 4 osmolytes--dimethylsulphoxide, glycerol, proline, and sucrose--on the refolding and reactivation of guanidine-denatured aminoacylase were studied by measuring aggregation, enzyme activity, intrinsic fluorescence spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra, and circular dichroism (CD) spectra. The results show that all the osmolytes not only inhibit aggregation but also recover the activity of aminoacylase during refolding in a concentration-dependent manner. In particularly, a 40% glycerol concentration and a 1.5 mol/L sucrose concentration almost completely suppressed the aminoacylase aggregation. The enzyme activity measurements revealed that the influence of glycerol is more significant than that of any other osmolyte. The intrinsic fluorescence results showed that glycerol, proline, and sucrose stabilized the aminoacylase conformation effectively, with glycerol being the most effective. All 4 kinds of osmolytes reduced the exposure of the hydrophobic surface, indicating that osmolytes facilitate the formation of protein hydrophobic collapse. The CD results indicate that glycerol and sucrose facilitate the return of aminoacylase to its native secondary structure. The results of this study suggest that the ability of the various osmolytes to facilitate the refolding and renaturation of aminoacylase is not the same. A survey of the results in the literature, as well as those presented here, suggests that although the protective effect of osmolytes on protein activity and structure is equal for different osmolytes, the ability of osmolytes to facilitate the refolding of various proteins differs from case to case. In all cases, glycerol was found to be the best stabilizer and a folding aid.     

Trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures

A number of naturally occurring small organic molecules, primarily involved in maintaining osmotic pressure in the cell, display chaperone-like activity, stabilizing the native conformation of proteins, and protecting them from various kinds of stress. Most of them are sugars, polyols, amino acids or methylamines. Similar to molecular chaperones, most of these compounds have no substrate specificity, but some specifically stabilize certain proteins. In the present work, the capacity of trehalose and glycerol, two well-known osmolytes, to stabilize and renature inorganic pyrophosphatase is demonstrated. Both trehalose and glycerol significantly protect pyrophosphatase against thermoinactivation achieved by incubating the enzyme at temperatures up to 95 degrees C, and allow the enzyme already inactivated in the presence of these osmolytes to renature upon incubation at low temperatures. To the best of our knowledge, there are no data on the effects of these compounds on renaturation of thermoinactivated proteins. The correlation between the recovery of enzyme activity and structural changes indicated by fluorescence spectroscopy contribute to better understanding of the protein stabilization mechanism.     

Osmolytes and crowders regulate aggregation of the cancer-related L106R mutant of the Axin protein

Protein aggregation is involved in a variety of diseases, including neurodegenerative diseases and cancer. The cellular environment is crowded by a plethora of cosolutes comprising small molecules and biomacromolecules at high concentrations, which may influence the aggregation of proteins in vivo. To account for the effect of cosolutes on cancer-related protein aggregation, we studied their effect on the aggregation of the cancer-related L106R mutant of the Axin protein. Axin is a key player in the Wnt signaling pathway, and the L106R mutation in its RGS domain results in a native molten globule that tends to form native-like aggregates. This results in uncontrolled activation of the Wnt signaling pathway, leading to cancer. We monitored the aggregation process of Axin RGS L106R in vitro in the presence of a wide ensemble of cosolutes including polyols, amino acids, betaine, and polyethylene glycol crowders. Except myo-inositol, all polyols decreased RGS L106R aggregation, with carbohydrates exerting the strongest inhibition. Conversely, betaine and polyethylene glycols enhanced aggregation. These results are consistent with the reported effects of osmolytes and crowders on the stability of molten globular proteins and with both amorphous and amyloid aggregation mechanisms. We suggest a model of Axin L106R aggregation in vivo, whereby molecularly small osmolytes keep the protein as a free soluble molecule but the increased crowding of the bound state by macromolecules induces its aggregation at the nanoscale. To our knowledge, this is the first systematic study on the effect of osmolytes and crowders on a process of native-like aggregation involved in pathology, as it sheds light on the contribution of cosolutes to the onset of cancer as a protein misfolding disease and on the relevance of aggregation in the molecular etiology of cancer.     

Manipulating the amyloid-beta aggregation pathway with chemical chaperones.

Amyloid-beta (Abeta) assembly into fibrillar structures is a defining characteristic of Alzheimer's disease that is initiated by a conformational transition from random coil to beta-sheet and a nucleation-dependent aggregation process. We have investigated the role of organic osmolytes as chemical chaperones in the amyloid pathway using glycerol to mimic the effects of naturally occurring molecules. Osmolytes such as the naturally occurring trimethylamine N-oxide and glycerol correct folding defects by preferentially hydrating partially denatured proteins and entropically stabilize native conformations and polymeric states. Trimethylamine N-oxide and glycerol were found to rapidly accelerate the Abeta random coil-to-beta-sheet conformational change necessary for fiber formation. This was accompanied by an immediate conversion of amorphous unstructured aggregates into uniform globular and possibly nucleating structures. Osmolyte-facilitated changes in Abeta hydration also affected the final stages of amyloid formation and mediated transition from the protofibrils to mature fibers that are observed in vivo. These findings suggest that hydration forces can be used to control fibril assembly and may have implications for the accumulation of Abeta within intracellular compartments such as the endoplasmic reticulum and in vitro modeling of the amyloid pathway.     

Natural osmolytes remodel the aggregation pathway of mutant huntingtin exon 1

In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types to regulate the intracellular solvent quality and to counteract the deleterious effect on the stability and function of cellular proteins. Given the evidence that destabilization of the native state of a protein either by mutation or by environmental changes triggers the aggregation in the neurodegenerative pathologies, the modulation of the intracellular solute composition with osmolytes is an attractive strategy to stabilize an aggregating protein. Here we report the effect of three natural osmolytes on the in vivo and in vitro aggregation landscape of huntingtin exon 1 implicated in the Huntington's disease. Trimethylamine N-oxide (TMAO) and proline redirect amyloid fibrillogenesis of the pathological huntingtin exon 1 to nonamyloidogenic amorphous assemblies via two dissimilar molecular mechanisms. TMAO causes a rapid formation of bulky amorphous aggregates with minimally exposed surface area, whereas proline solubilizes the monomer and suppresses the accumulation of early transient aggregates. Conversely, glycine-betaine enhances fibrillization in a fashion reminiscent of the genesis of functional amyloids. Strikingly, none of the natural osmolytes can completely abrogate the aggregate formation; however, they redirect the amyloidogenesis into alternative, nontoxic aggregate species. Our study reveals new insights into the complex interactions of osmoprotectants with polyQ aggregates.     

Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.5 that does not show any cooperative transition upon heating. Does the presence of some of these osmolytes at pH 2.5 help in the retention of structure that is typical of native state? To answer this question, the effect of ethylene glycol (EG), glycerol, xylitol, sorbitol, trehalose and glucose at pH 2.5 on the structure and stability of yeast hexokinase A was investigated using spectroscopy and calorimetry. In presence of the above osmolytes, except EG, yeast hexokinase at pH 2.5 retains native secondary structure and hydrophobic core and unfolds with excessive heat absorption upon thermal denaturation. However, the cooperative structure binds to ANS suggesting that it is an intermediate between MG and the native state. Further, we show that at high concentration of polyols at pH 2.5, except EG, which populates a non-native ensemble, ΔH_cal/ΔH_van approaches unity indicative of two-state unfolding. The results suggest that osmolytes stabilize cooperative protein structure relative to non-cooperative ensemble. These findings have implications toward the structure formation, folding and stability of proteins produced under stress in cellular systems.     

Testing polyols' compatibility with Gibbs energy of stabilization of proteins under conditions in which they behave as compatible osmolytes

It is generally believed that compatible osmolytes stabilize proteins by shifting the denaturation equilibrium, native state <--> denatured state toward the left. We show here that if osmolytes are compatible with the functional activity of the protein at a given pH and temperature, they should not significantly perturb this denaturation equilibrium under the same experimental conditions. This conclusion was reached from the measurements of the activity parameters (K(m) and k(cat)) and guanidinium chloride-induced denaturations of lysozyme and ribonuclease-A in the presence of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at pH 7.0 and 25 degrees C.     

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.     

The protective effects of osmolytes on arginine kinase unfolding and aggregation

Osmolytes are a series of different kinds of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. In this study, the protective effects of four compatible osmolytes, i.e., proline, sucrose, DMSO and glycerol, were studied during arginine kinase (EC 2.7.3.3) unfolding and aggregation. The results showed that all the osmolytes applied in this study obviously prevented AK unfolding and inactivation that was due to a GdnHCl denaturant by reducing the inactivation rate constants (k_i), increasing the transition free energy changes (ΔΔG_i) and increasing the value for the midpoint of denaturation (C_m). Furthermore, the osmolytes remarkably prevented AK aggregation in a concentration-dependent manner during AK refolding. Our results strongly indicated that osmolytes were not only metabolism substrates, but they were also important compounds with significant physiological protective functions for proteins, especially in some extremely harsh environments.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Osmolytes induce structure in an early intermediate on the folding pathway of barstar

Osmolytes stabilize proteins against denaturation, but little is known about how their stabilizing effect might affect a protein folding pathway. Here, we report the effects of the osmolytes, trimethylamine-N-oxide, and sarcosine on the stability of the native state of barstar as well as on the structural heterogeneity of an early intermediate ensemble, IE, on its folding pathway. Both osmolytes increase the stability of the native protein to a similar extent, with stability increasing linearly with osmolyte concentration. Both osmolytes also increase the stability of IE but to different extents. Such stabilization leads to an acceleration in the folding rate. Both osmolytes also alter the structure of IE but do so differentially; the fluorescence and circular dichroism properties of IE differ in the presence of the different osmolytes. Because these properties also differ from those of the unfolded form in refolding conditions, different burst phase changes in the optical signals are seen for folding in the presence of the different osmolytes. An analysis of the urea dependence of the burst phase changes in fluorescence and circular dichroism demonstrates that the formation of IE is itself a multistep process during folding and that the two osmolytes act by stabilizing, differentially, different structural components present in the IE ensemble. Thus, osmolytes can alter the basic nature of a protein folding pathway by discriminating, through differential stabilization, between different members of an early intermediate ensemble, and in doing so, they thereby appear to channel folding along one route when many routes are available.     

Effects of osmolytes on arginine kinase from Euphausia superba: A study on thermal denaturation and aggregation

Investigations of energy-related enzymatic properties may provide valuable information about the mechanisms that are involved in the adaptation to extreme climatic environments. The protective effects of osmolytes on the thermal denaturation and aggregation of arginine kinase from E. superba (ESAK) was investigated. When the concentration of glycine, proline and glycerol increased, the relative activation was significantly enhanced, while the aggregation of ESAK during thermal denaturation was decreased. Spectrofluorometry results showed that the presence of these three osmolytes significantly decreased the tertiary structural changes of ESAK and that thermal denaturation directly induced ESAK aggregation. The results demonstrated that glycine, proline and glycerol not only prevented ESAK from inactivation and unfolding but also inhibited aggregation by stabilizing the ESAK conformation. We measured the ORF gene sequence of ESAK by RACE, and built the 3D structure of ESAK and osmolytes by homology models. The results showed that the docking energy was relatively low and that the clustering groups were spread to the surface of ESAK, indicating that osmolytes directly protect the surface of the protein. Our study provides important insight into the protective effects of osmolytes on ESAK folding.     

The metastable states of proteins

The intriguing process of protein folding comprises discrete steps that stabilize the protein molecules in different conformations. The metastable state of protein is represented by specific conformational characteristics, which place the protein in a local free energy minimum state of the energy landscape. The native‐to‐metastable structural transitions are governed by transient or long‐lived thermodynamic and kinetic fluctuations of the intrinsic interactions of the protein molecules. Depiction of the structural and functional properties of metastable proteins is not only required to understand the complexity of folding patterns but also to comprehend the mechanisms of anomalous aggregation of different proteins. In this article, we review the properties of metastable proteins in context of their stability and capability of undergoing atypical aggregation in physiological conditions.     

Effects of naturally occurring osmolytes on protein stability and solubility: issues important in protein crystallization

Protein solubility and stability are issues of consideration in attempts to crystallize proteins. These two properties of proteins are also at issue in the cells of organisms that have adapted to water stress conditions that could ordinarily denature or inactivate some proteins. Most organisms that have adapted to environmental stresses have done so by production and accumulation of certain small organic molecules, known as osmolytes, that arose by natural selection and have the ability to stabilize intracellular proteins against the environmental stress. Here, concepts developed to understand the special properties of the naturally occurring osmolytes in effecting protein stability and solubility, and the principles that have come from studies of these compounds have been presented. Along with excluded volume and preferential interaction parameters, identification of the osmophobic effect and the attenuation of this effect by favorable interactions of solute with side-chains appear to contribute to the full set of effects protecting osmolytes have on protein stability and solubility. With these concepts in mind and the fact that urea interacts favorably with the peptide backbone we note that: (1) osmolyte-induced effects on protein stability ranging from denaturation to forcing proteins to fold can be achieved experimentally and the underlying principles understood at near molecular-level detail, and (2) osmolyte-mediated solubility effects ranging from protein precipitation to protein solubilization are predictable based on these principles. These effects are contrasted and compared with effects of 2-methyl-2,4-pentanediol and polyethylene glycol on proteins, and how the principles found for the naturally occurring osmolytes can be applied to these two commonly used protein crystallizing agents.     

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.     

Chaperone Effects on Prion and Nonprion Aggregates

Exposure to high temperature or other stresses induces a synthesis of heat shock proteins. Many of these proteins are molecular chaperones and some of them help cells to cope with heat-induced denaturation and aggregation of other proteins. In the last decade, chaperones have received increased attention in connection with their role in maintenance and propagation of the Saccharomyces cerevisiae prions, infectious or heritable agents transmitted at the protein level. Recent data suggest that functioning of the chaperones in reactivation of heat-damaged proteins and in propagation of prions is based on the same molecular mechanisms but may lead to different consequences depending on the type of aggregate. In both cases the concerted and balanced action of “chaperones'' team,” including Hsp104, Hsp70, Hsp40 and possibly other proteins, determines whether a misfolded protein is to be incorporated into an aggregate, rescued to the native state or targeted for degradation.Key Words: Amyloid, Hsp40, Hsp70, Hsp104, stress response, yeast