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.     

Effects of pH and ionic strength on the assembly and bundling of FtsZ protofilaments: a possible role of electrostatic interactions in the bundling of protofilaments

Assembly, bundling and stability of FtsZ protofilaments are important for the formation and functioning of the cytokinetic Z-ring during bacterial division. We found that the bundling of FtsZ protofilaments decreased strongly with increasing pH from 6.0 to 7.9, while the assembly of FtsZ monomers did not decrease considerably. In addition, the disassembly of FtsZ protofilaments was strongly suppressed at pH 6.0 as compared to the elevated pHs. The far-UV circular dichroism spectra of the native FtsZ and the tryptophan emission spectra of mutated FtsZ (Y371W) did not change by increasing pH from 6 to 7.9 indicating that the structure of FtsZ was not altered significantly. Further, the inhibition of bundling of FtsZ protofilaments predominantly, and the inhibition of assembly to a lesser extent by salt indicated that electrostatic interactions are important for the assembly and bundling of FtsZ protofilaments. These observations are supported by the results of computational docking of Escherichia coli dimer structure. The results suggest that the basic intracellular pH (7.4-7.8) of E. coli may play a role in regulating the assembly dynamics of FtsZ in the Z-ring by reducing protofilament stability and bundling in bacterial cytoplasm.     

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.     

Curcumin inhibits FtsZ assembly: an attractive mechanism for its antibacterial activity

The assembly and stability of FtsZ protofilaments have been shown to play critical roles in bacterial cytokinesis. Recent evidence suggests that FtsZ may be considered as an important antibacterial drug target. Curcumin, a dietary polyphenolic compound, has been shown to have a potent antibacterial activity against a number of pathogenic bacteria including Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus. We found that curcumin induced filamentation in the Bacillus subtilis 168, suggesting that it inhibits bacterial cytokinesis. Further, curcumin strongly inhibited the formation of the cytokinetic Z-ring in B. subtilis 168 without detectably affecting the segregation and organization of the nucleoids. Since the assembly dynamics of FtsZ protofilaments plays a major role in the formation and functioning of the Z-ring, we analysed the effects of curcumin on the assembly of FtsZ protofilaments. Curcumin inhibited the assembly of FtsZ protofilaments and also increased the GTPase activity of FtsZ. Electron microscopic analysis showed that curcumin reduced the bundling of FtsZ protofilaments in vitro. Further, curcumin was found to bind to FtsZ in vitro with a dissociation constant of 7.3+/-1.8 microM and the agent also perturbed the secondary structure of FtsZ. The results indicate that the perturbation of the GTPase activity of FtsZ assembly is lethal to bacteria and suggest that curcumin inhibits bacterial cell proliferation by inhibiting the assembly dynamics of FtsZ in the Z-ring.     

Detection of an intermediate during unfolding of bacterial cell division protein FtsZ: loss of functional properties precedes the global unfolding of FtsZ

Using environment-sensitive fluorescence of 1-anilinonaphthalene-8-sulfonic acid, polarization of fluorescein 5'-isothiocyanate-labeled FtsZ, and far-UV circular dichroism spectroscopy, the chemical unfolding of FtsZ was found to proceed through two steps. The first step of the urea-induced unfolding produced an intermediate, which then unfolded at higher concentrations of urea. The intermediate state contains native-like secondary structure and much less tertiary structure compared with the native state. It is distinct from the native state as well as from the unfolded state. Similar to urea-induced unfolding of FtsZ, thermal unfolding of FtsZ also occurs in two steps. The midpoints for the first and second thermal unfolding transitions were found to be 38 +/- 4 and 77 +/- 5 degrees C, respectively. Further, the functional properties of FtsZ are extremely sensitive to urea, guanidium chloride, and sodium dodecyl sulfate. For example, 50% inhibition of the FtsZ assembly and GTP hydrolysis occurred at 0.1 and 0.2 m of urea, respectively. FtsZ lost its functional properties before any significant perturbation in the secondary or tertiary structure was detected by using several fluorescence techniques and far UV-CD indicating preferential local unfolding of the functional region(s). In addition, the unfolded FtsZ regains its ability to polymerize fully upon removal of urea. The data taken together suggest that FtsZ unfolds reversibly through a multistep process, and local responses that inhibit functional properties precede the global transition of FtsZ to the unfolded state.     

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.     

Glutamate counteracts the denaturing effect of urea through its effect on the denatured state

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea. d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.     

Acid-induced loss of functional properties of bacterial cell division protein FtsZ: evidence for an alternative conformation at acidic pH

Several types of bacteria live in highly acidic environments. Since the assembly of FtsZ is important for bacterial cytokinesis, the effects of pH on the assembly and structural properties of FtsZ were examined. FtsZ retained GTP binding ability but lost GTPase activity at pH 2.5. In the presence of GTP, FtsZ formed protofilaments at pH 7 while it formed aggregates instead of protofilaments at pH 2.5, indicating that GTP hydrolysis is important for the assembly of FtsZ into protofilaments. Further, the acid-inactivated state of FtsZ recovered its structural and functional properties upon refolding at pH 7, indicating that the cellular functions of FtsZ may be restored after removal of the external stress. In addition, the affinity of 1-anilinonaphthalene-8-sulfonic acid (ANS) binding to FtsZ was found to be higher at pH 2.5 than at pH 7. FtsZ-ANS complex had a higher quantum yield and lifetime at pH 2.5 than at pH 7. However, the secondary structures of FtsZ were similar at pH 7 and 2.5, indicating that FtsZ attained an alternatively folded state (A) at pH 2.5, which has some characteristics of a molten-globule-like state. The A state was more stable than the native state (N) against urea-induced unfolding. The transition from N to A state involves the formation of aggregates of FtsZ (I). The association of FtsZ monomers occurred in the narrow pH range (3.2-2.8) and it was found to be a fully reversible process. The results suggest that a productive intermediate (I) forms in the acid-induced unfolding pathway of FtsZ and that the unfolding pathway may be minimally described as N <==> I <==> A.     

SepF increases the assembly and bundling of FtsZ polymers and stabilizes FtsZ protofilaments by binding along its length

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90 degrees light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZDelta16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366-382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division.     

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).     

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.     

Effect of osmolytes on protein dynamics in the lactate dehydrogenase-catalyzed reaction

Laser-induced temperature jump relaxation spectroscopy was used to probe the effect of osmolytes on the microscopic rate constants of the lactate dehydrogenase-catalyzed reaction. NADH fluorescence and absorption relaxation kinetics were measured for the lactate dehydrogenase (LDH) reaction system in the presence of varying amounts of trimethylamine N-oxide (TMAO), a protein-stabilizing osmolyte, or urea, a protein-destabilizing osmolyte. Trimethylamine N-oxide (TMAO) at a concentration of 1 M strongly increases the rate of hydride transfer, nearly nullifies its activation energy, and also slightly increases the enthalpy of hydride transfer. In 1 M urea, the hydride transfer enthalpy is almost nullified, but the activation energy of the step is not affected significantly. TMAO increases the preference of the closed conformation of the active site loop in the LDH·NAD(+)·lactate complex; urea decreases it. The loop opening rate in the LDH·NADH·pyruvate complex changes its temperature dependence to inverse Arrhenius with TMAO. In this complex, urea accelerates the loop motion, without changing the loop opening enthalpy. A strong, non-Arrhenius decrease in the pyruvate binding rate in the presence of TMAO offers a decrease in the fraction of the open loop, pyruvate binding competent form at higher temperatures. The pyruvate off rate is not affected by urea but decreases with TMAO. Thus, the osmolytes strongly affect the rates and thermodynamics of specific events along the LDH-catalyzed reaction: binding of substrates, loop closure, and the chemical event. Qualitatively, these results can be understood as an osmolyte-induced change in the energy landscape of the protein complexes, shifting the conformational nature of functional substates within the protein ensemble.     

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.     

Ruthenium red-induced bundling of bacterial cell division protein, FtsZ

The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.     

TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal

Concentrations of trimethylamine oxide (TMAO) and other 'compatible' osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration.     

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.     

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.     

Measuring the stability of partly folded proteins using TMAO

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

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.     

Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization

Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO). Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered. The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar. Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species. Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble. Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding. Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability. These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate. This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state.