Osmolyte trimethylamine-N-oxide does not affect the strength of hydrophobic interactions: origin of osmolyte compatibility

Osmolytes are small organic solutes accumulated at high concentrations by cells/tissues in response to osmotic stress. Osmolytes increase thermodynamic stability of folded proteins and provide protection against denaturing stresses. The mechanism of osmolyte compatibility and osmolyte-induced stability has, therefore, attracted considerable attention in recent years. However, to our knowledge, no quantitative study of osmolyte effects on the strength of hydrophobic interactions has been reported. Here, we present a detailed molecular dynamics simulation study of the effect of the osmolyte trimethylamine-N-oxide (TMAO) on hydrophobic phenomena at molecular and nanoscopic length scales. Specifically, we investigate the effects of TMAO on the thermodynamics of hydrophobic hydration and interactions of small solutes as well as on the folding-unfolding conformational equilibrium of a hydrophobic polymer in water. The major conclusion of our study is that TMAO has almost no effect either on the thermodynamics of hydration of small nonpolar solutes or on the hydrophobic interactions at the pair and many-body level. We propose that this neutrality of TMAO toward hydrophobic interactions-one of the primary driving forces in protein folding-is at least partially responsible for making TMAO a "compatible" osmolyte. That is, TMAO can be tolerated at high concentrations in organisms without affecting nonspecific hydrophobic effects. Our study implies that protein stabilization by TMAO occurs through other mechanisms, such as unfavorable water-mediated interaction of TMAO with the protein backbone, as suggested by recent experimental studies. We complement the above calculations with analysis of TMAO hydration and changes in water structure in the presence of TMAO molecules. TMAO is an amphiphilic molecule containing both hydrophobic and hydrophilic parts. The precise balance of the effects of hydrophobic and hydrophilic segments of the molecule appears to explain the virtual noneffect of TMAO on the strength of hydrophobic interactions.     

The osmolyte trimethylamine‐N‐oxide stabilizes the Fyn SH3 domain without altering the structure of its folding transition state

Trimethylamine‐N‐oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins against denaturation. Although the impact of TMAO on the folding thermodynamics of many proteins has been well characterized, far fewer studies have investigated its effects on protein folding kinetics. In particular, no previous studies have used Φ‐value analysis to determine whether TMAO may alter the structure of the folding transition state. Here we have measured the effects on folding kinetics of 16 different amino acid substitutions distributed across the structure of the Fyn SH3 domain both in the presence and absence of TMAO. The folding and unfolding rates in TMAO, on average, improved to equivalent degrees, with a twofold increase in the protein folding rate accompanied by a twofold decrease in the unfolding rate. Importantly, TMAO caused little alteration to the Φ‐values of the mutants tested, implying that this compound minimally perturbs the folding transition state structure. Furthermore, the solvent accessibility of the transition state was not altered as reflected in an absence of a TMAO‐induced change in the denaturant β factors. Through TMAO‐induced folding studies, a β factor of 0.5 was calculated for this compound, suggesting that the protein backbone, which is the target of action of TMAO, is 50% exposed in the transition state as compared to the native state. This finding is consistent with the equivalent effects of TMAO on the folding and unfolding rates. Through thermodynamic analysis of mutants, we also discovered that the stabilizing effect of TMAO is lessened with increasing temperature.     

Counteraction of Urea by Trimethylamine N-Oxide Is Due to Direct Interaction

Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins, induces folding, and counteracts the denaturing effects of urea, pressure, and ice. To establish the mechanism behind these effects, isotopic substitution neutron-scattering measurements were performed on aqueous solutions of TMAO and 1:1 TMAO-urea at a solute mole fraction of 0.05. The partial pair distribution functions were extracted using the empirical potential structure refinement method. The results were compared with previous results obtained with isosteric tert-butanol, as well as the available data from spectroscopy and molecular-dynamics simulations. In solution, the oxygen atom of TMAO is strongly hydrogen-bonded to, on average, between two and three water molecules, and the hydrogen-bond network is tighter in water than in pure water. In TMAO-urea solutions, the oxygen atom in TMAO preferentially forms hydrogen bonds with urea. This explains why the counteraction is completed at a 2:1 urea/TMAO concentration ratio, independently of urea concentration. These results strongly support models for the effect of TMAO on the stability of proteins based on a modification of the simultaneous equilibria that control hydrogen bonding between the peptide backbone and water or intramolecular sites, without any need for direct interaction between TMAO and the protein.     

Effects of the osmolyte trimethylamine-N-oxide on conformation,self-association,and two-dimensional crystallization of myelin basic protein

The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring "chemical chaperone" that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys-->Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light-scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination.     

No effect of trimethylamine N-oxide on the internal dynamics of the protein native fold

Trimethylamine N-oxide (TMAO) is a natural osmolyte accumulated in cells of organisms as they adapt to environmental stresses. In vitro, TMAO increases protein stability and forces partially unfolded structures to refold. Its effects on the native fold are unknown. To investigate the interrelationship between protein stability, internal dynamics and function, the influence of TMAO on the flexibility of the native fold was examined with four different proteins by Trp phosphorescence spectroscopy. Its influence on conformational dynamics was assessed by both the intrinsic phosphorescence lifetime, which reports on the local structure about the triplet probe, and the acrylamide bimolecular quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results demonstrate that for apoazurin, alcohol dehydrogenase, alkaline phosphatase and glyceraldehydes-3-phosphate dehydrogenase 1.8 M TMAO does not perturb the flexibility of these macromolecules in a temperature range between - 10 degreesC and up to near the melting temperature. This unexpected finding contrasts with the dampening effect observed with polyols as well as with the expectations based on the preferential exclusion of the osmolyte from the protein surface.     

Effects of osmolytes on hexokinase kinetics combined with macromolecular crowding: test of the osmolyte compatibility hypothesis towards crowded systems

We investigated the effect of compatible and non-compatible osmolytes in combination with macromolecular crowding on the kinetics of yeast hexokinase. This was motivated by the fact that almost all studies concerning the osmolyte effects on enzyme activity have been performed in diluted buffer systems, which are far from the physiological conditions within cells, where the cytosol contains several hundred mg protein ml(-1). Four organic (glycerol, betaine, TMAO and urea) and one inorganic (NaCl) osmolyte were tested. It was concluded that the effect of compatible osmolytes (glycerol, betaine and TMAO) on V(max) and K(M) was practically equivalent in pure buffer and in 200-250 mg BSA ml(-1) supporting the view that these small organic osmolytes do minimal perturbance on enzyme function in physiological solutions. The effect of urea on enzyme kinetics was not independent of protein concentration, since the presence of 250 mg BSA ml(-1) partly compensated the perturbing effect of urea. Even though the organic osmolytes glycerol, betaine and TMAO are generally considered compatible with enzyme function, especially glycerol did have a significant effect on hexokinase kinetics, decreasing both k(cat), K(M) and k(cat)/K(M). The osmolytes decreased k(cat)/K(M) in the order: NaCl>Urea>TMAO/glycerol>betaine. For the organic osmolytes this order correlates with the degree of exclusion from protein-water interfaces. Thus, the stronger the exclusion the weaker the perturbing effects on k(cat)/K(M).     

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.     

Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide

Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N-oxide (TMAO). The structure of CspA has five strands of beta-sheet. Strands beta1-beta4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all-or-none global unfolding mechanism. By contrast, the protection of amide protons from strand beta5 is too weak to measure in water. Strand beta5 is hydrogen bonded to strands beta3 and beta4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands beta1-beta4 but fail to account for the weak protection in strand beta5. The most conspicuous feature of strand beta5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands beta1-beta4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand beta5 and the loops connecting the beta-strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen-bonding interactions in the native structure.     

Osmolyte trimethylamine N-oxide converts recombinant alpha-helical prion protein to its soluble beta-structured form at high temperature

The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.     

The effects of cosolutes on protein dynamics: the reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide

The protein stabilizing effects of the small molecule osmolyte, trimethylamine N-oxide, against chemical denaturant was investigated by NMR spin-relaxation measurements and model-free analysis. In the presence of 0.7 M guanidine hydrochloride increased picosecond-nanosecond dynamics are observed in the protein ribonuclease A. These increased fluctuations occur throughout the protein, but the most significant increases in flexibility occur at positions believed to be the first to unfold. Addition of 0.35 M trimethylamine N-oxide to this destabilized form of ribonuclease results in significant rigidification of the protein backbone as assessed by (1)H-(15)N order parameters. Statistically, these order parameters are the same as those measured in native ribonuclease indicating that TMAO reduces the amplitude of backbone fluctuations in a destabilized protein. These data suggest that TMAO restricts the bond vector motions on the protein energy landscape to resemble those motions that occur in the native protein and points to a relation between stability and dynamics in this enzyme.     

The Osmolyte TMAO Stabilizes Native RNA Tertiary Structures in the Absence of Mg: Evidence for a Large Barrier to Folding from Phosphate Dehydration

The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ∼ 0 for a hairpin secondary structure and between 0.70 and 1.85 kcal mol^− 1m^− 1 for four RNA tertiary structures (30-86 nt). Further analysis of two RNAs by small-angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg²⁺ and that the structures stabilized by TMAO and Mg²⁺ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg²⁺ ion chelation site formed in part by a buried phosphate, even though Mg²⁺ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy, + 211 cal mol^− 1m^− 1 for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66-173 water molecules in the RNA structures studied) and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg²⁺ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.     

The determinants of bond angle variability in protein/peptide backbones: A comprehensive statistical/quantum mechanics analysis

The elucidation of the mutual influence between peptide bond geometry and local conformation has important implications for protein structure refinement, validation, and prediction. To gain insights into the structural determinants and the energetic contributions associated with protein/peptide backbone plasticity, we here report an extensive analysis of the variability of the peptide bond angles by combining statistical analyses of protein structures and quantum mechanics calculations on small model peptide systems. Our analyses demonstrate that all the backbone bond angles strongly depend on the peptide conformation and unveil the existence of regular trends as function of ψ and/or φ. The excellent agreement of the quantum mechanics calculations with the statistical surveys of protein structures validates the computational scheme here employed and demonstrates that the valence geometry of protein/peptide backbone is primarily dictated by local interactions. Notably, for the first time we show that the position of the H^α hydrogen atom, which is an important parameter in NMR structural studies, is also dependent on the local conformation. Most of the trends observed may be satisfactorily explained by invoking steric repulsive interactions; in some specific cases the valence bond variability is also influenced by hydrogen‐bond like interactions. Moreover, we can provide a reliable estimate of the energies involved in the interplay between geometry and conformations. Proteins 2015; 83:1973–1986. © 2015 Wiley Periodicals, Inc.     

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.     

Osmolyte effects on helix formation in peptides and the stability of coiled-coils

The ability of several naturally occurring substances known as osmolytes to induce helix formation in an alanine-based peptide have been investigated. As predicted by the osmophobic effect hypothesis, the osmolytes studies here do induce helix formation. Trimethylamine-N-oxide (TMAO) is the best structure-inducing osmolytes investigated here, but it is not as effective in promoting helix formation as the common cosolvent trifluoroethanol (TFE). We also provide a semiquantitative study of the ability of TMAO to induce helix formation and urea, which acts as a helix (and protein) denaturant. We find that on a molar basis, these agents are exactly counteractive as structure inducing and unfolding agents. Finally, we extend the investigations to the effects of urea and TMAO on the stability of a dimeric coiled-coil peptide and find identical results. Together these results support the tenets of the osmophobic hypothesis and highlight the importance of the polypeptide backbone in protein folding and stability.     

An effective solvent theory connecting the underlying mechanisms of osmolytes and denaturants for protein stability

An all-atom Gō model of Trp-cage protein is simulated using discontinuous molecular dynamics in an explicit minimal solvent, using a single, contact-based interaction energy between protein and solvent particles. An effective denaturant or osmolyte solution can be constructed by making the interaction energy attractive or repulsive. A statistical mechanical equivalence is demonstrated between this effective solvent model and models in which proteins are immersed in solutions consisting of water and osmolytes or denaturants. Analysis of these studies yields the following conclusions: 1), Osmolytes impart extra stability to the protein by reducing the entropy of the unfolded state. 2), Unfolded states in the presence of osmolyte are more collapsed than in water. 3), The folding transition in osmolyte solutions tends to be less cooperative than in water, as determined by the ratio of van 't Hoff to calorimetric enthalpy changes. The decrease in cooperativity arises from an increase in native structure in the unfolded state, and thus a lower thermodynamic barrier at the transition midpoint. 4), Weak denaturants were observed to destabilize small proteins not by lowering the unfolded enthalpy, but primarily by swelling the unfolded state and raising its entropy. However, adding a strong denaturant destabilizes proteins enthalpically. 5), The folding transition in denaturant-containing solutions is more cooperative than in water. 6), Transfer to a concentrated osmolyte solution with purely hard-sphere steric repulsion significantly stabilizes the protein, due to excluded volume interactions not present in the canonical Tanford transfer model. 7), Although a solution with hard-sphere interactions adds a solvation barrier to native contacts, the folding is nevertheless less cooperative for reasons 1–3 above, because a hard-sphere solvent acts as a protecting osmolyte.     

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.     

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.     

Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability

The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k _cat /K _m) by 290% at 0°C. In the presence of 1 M TMAO, the thermostability (t _1/2) of MCP-01 increased by two- to fivefold at 60∼40°C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40°C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T _m) of MCP-01 by 10.5°C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.