Trehalose favors a cutinase compact intermediate off-folding pathway

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.     

Thermodynamics and mechanism of cutinase stabilization by trehalose

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy-enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding.     

Improving cutinase stability in aqueous solution and in reverse micelles by media engineering

The stability of a recombinant cutinase from the fungus Fusarium solani was evaluated in aqueous media and in reverse micelles. Thermal unfolding in aqueous solution is a two-state process at the pH values tested and trehalose increased the temperature at the mid-point of the unfolding transitions. Irreversible inactivation is a first-order process at pH 9.2, but two inactivation phases were resolved at pH 4.5. Trehalose did not change the irreversible inactivation pathway but increased the kinetics of the irreversible inactivation step. Unfolding of cutinase induced by guanidine hydrochloride was more complex, showing a stable intermediate, molten globule in character, within the transition region. Trehalose did not change the three-state nature of the unfolding process. Encapsulation of cutinase in AOT reverse micelles induced unfolding at room temperature due to an enzyme location at the micellar interface. The presence of 1-hexanol as co-surfactant delayed or even prevented the unfolding of cutinase by promoting the establishment of a new equilibrium in the system. Cutinase is encapsulated in a 10-fold larger AOT/hexanol reverse micelle built up by the fusion of empty reverse micelles. When tested in a membrane reactor in the presence of 1-hexanol, an operational half-life of 674 days was achieved.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model.

The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl. This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP. These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible. From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits.     

Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.     

The place of inactivated actin and its kinetic predecessor in actin folding-unfolding

The kinetics of actin unfolding induced by guanidine hydrochloride of different concentrations was studied. The parametric representation of the kinetic dependencies of tryptophan fluorescence intensity changes recorded at two wavelengths allowed us to detect and characterize a new essentially unfolded kinetic intermediate. Its characteristics suggested that this intermediate state is a premolten globule. It was shown that the equilibrium transition between inactivated and completely unfolded states is also a two-step process and proceeds via an essentially unfolded kinetic intermediate. The new kinetic pathway of actin unfolding--refolding was proposed. According to it, the founded essentially unfolded kinetic state is the on-pathway intermediate, while inactivated actin is the off-pathway misfolded state stabilized by aggregation of partially folded macromolecules of protein.     

Probing Conformational Stability and Dynamics of Erythroid and Nonerythroid Spectrin: Effects of Urea and Guanidine Hydrochloride

We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔG_u ^H ₂ ⁰) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

The unfolding of alpha-momorcharin proceeds through the compact folded intermediate

The unfolding of alpha-momorcharin was systematically investigated using steady-state and time-resolved tryptophan fluorescence, circular dichroism and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. These spectroscopic studies demonstrated that alpha-momorcharin unfolded through a compact folded intermediate state. The content of alpha-helix was increased, Trp192 approached closer to the side of active site and its rotational motion was restricted by being equilibrated with 2-3 M of guanidine hydrochloride. Furthermore, the binding of ANS with alpha-momorcharin was more suppressed to show that the hydrophobic parts would not be accessed to the protein surface but rather be sealed off in this specific conformation state. These results suggest that the structure of alpha-momorcharin holds the more compact conformation as an incipient state for unfolding, which is the sharp contrast to beta-momorcharin that gives the characteristics of the generally known molten globule state.     

Guanidine hydrochloride induced equilibrium unfolding studies of colicin B and its channel-forming fragment

The conformational stabilities of full-length colicin B and its isolated C-terminal domain were studied by guanidine hydrochloride induced unfolding. The unfolding/refolding was monitored by far-UV CD and intrinsic tryptophan fluorescence spectroscopies. At pH 7.4, the disruption of the secondary structure of full-length colicin B is monophasic, while changes in tertiary structure occur in two separate transitions. The intermediate species, which is well-populated around 2.2 M guanidine hydrochloride, exhibits secondary and tertiary structures distinct from both native and unfolded states. Whereas the domain structure of native full-length colicin B is reflected in its DSC profile, the folding intermediate of the same protein exhibits a single unresolved peak. These observations have led us to propose an unfolding model for full-length colicin B where the first transition between 0 and 2.5 M GuHCl with an associated free energy of 3 kcal/mol correlates with the partial unfolding of the R/T domain. The stability of full-length colicin B is weakened due to the presence of the R/T domain in both the native [Ortega, A., Lambotte, S., and Bechinger, B. (2001) J. Biol. Chem. 276 (17), 13563-13572] and the intermediate states. The second transition between 2.5 and 5 M GuHCl involves unfolding of the C-terminal domain (Delta = 7 kcal/mol). The isolated colicin B C-terminal domain consists of two subdomains, and the two parts of this protein fragment unfold sequentially through the formation of at least one intermediate. The significance of these results for membrane insertion of colicin B is discussed.     

Folding of a de novo designed native-like four-helix bundle protein

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.     

Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability

The urea and guanidine hydrochloride (GdnHCl)-induced denaturation of tetrameric concanavalin A (ConA) at pH 7.2 has been studied by using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and size-exclusion chromatography. The equilibrium denaturation pathway of ConA, as monitored by steady state fluorescence, exhibits a three-state mechanism involving an intermediate state, which has been characterized as a structured monomer of the protein by ANS binding, far-UV CD and gel filtration size analysis. The three-state equilibrium is analyzed in terms of two distinct and separate dissociation (native tetramer<-->structured monomer) and unfolding (structured monomer<-->unfolded monomer) reaction steps, with the apparent transition midpoints (C(m)), respectively, at 1.4 and 4.5 M in urea, and at 0.8 and 2.4 M in GdnHCl. The results show that the free energy of stabilization of structured monomer relative to the unfolded state (-DeltaG(unf, aq)), is 4.4-5.5 kcal mol(-1), and that of native tetramer relative to structured monomer (-DeltaG(dis, aq)) is 7.2-7.4 kcal mol(-1), giving an overall free energy of stabilization (-DeltaG(dis&unf, aq)) of 11.6-12.9 kcal mol(-1) (monomer mass) for the native protein. However, the free energy preference at the level of quaternary tetrameric structure is found to be far greater than that at the tertiary monomeric level, which reveals that the structural stability of ConA is maintained mostly by subunit association.     

The partially unfolded state of beta-momorcharin characterized with steady-state and time-resolved fluorescence studies

The specific conformation of partially unfolded state of beta-momorcharin was characterized through the steady-state and time-resolved fluorescence spectroscopic studies on a single Trp-190 which located adjacently to the active site. The content of secondary structure was retained, the binding of ANS was remarkably enhanced, and the correlation time of entire protein rotation was prolonged at the partially unfolded state formed by being equilibrated with the mild concentration of guanidine hydrochloride. The time-resolved fluorescence depolarization and excitation energy transfer analysis suggest that Trp-190 approached 2 A closer to Tyr-70 and was hidden from the exposure to the protein surface, while the rotational correlation time and freedom of its segmental motion were shortened and enhanced, respectively. These results suggest that the transient folding/unfolding intermediate state of beta-momorcharin adopt the specific conformation at the vicinity of the active site, although it exhibits very similar properties with those of the generally known molten-globule state.     

Evidence that thermodynamic stability of papaya glutamine cyclase is only marginal

Papaya glutamine cyclase (PQC), a glycoprotein with a molecular mass of 32,980 Da, is a minor constituent of the papaya latex protein fraction. In neutral aqueous solutions, PQC adopts an all-beta conformation and exhibits high resistance to both proteolysis and denaturation. Complete unfolding of PQC requires a combination of an acidic medium and chemical denaturant such as urea or guanidine hydrochloride. The unfolding process takes place through formation of an intermediate A state that accumulates in the absence of chemical denaturants and displays all the features of a molten globule state. The different conformational states-N (native), A (acid-inactivated), and U (unfolded)-have been characterized by means of circular dichroism measurements, fluorescence spectroscopies, Stokes radii determinations, and 8-anilino-1-naphtalenesulfonic acid (ANS) binding characteristics. The unfolding pathways of the enzyme was further studied to estimate thermodynamic parameters characterizing both transitions N if A and A if U. In its A state, PQC is catalytically inefficient and highly susceptible to proteolysis. Also, its thermodynamic stability is decreased by some 3-5 kcal/mol. Conversion of the native to the A state involves digging up of five amino functions together with protonation of four to five acidic groups with pK(a)s, in the native state, around 2.7. It proceeds both cooperatively and reversibly although, in vitro, the refolding process is slow. Unfolding of the A state, on the other hand, occurs with a low degree of cooperativity. The intermediate A state thus seems to be only marginally more stable than the unfolded state. The role of suspected internal ion pairs in the stabilization of the native state of this enzyme is discussed.     

Equilibrium unfolding of class pi glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.     

Protein folding intermediates of invasin protein IbeA from Escherichia coli

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of beta-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with beta-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended beta-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.     

A reexamination of the conformational transitions of T4 thioredoxin

The unfolding and refolding of T4 thioredoxin was observed by equilibrium and kinetic size exclusion chromatographic measurements in guanidine hydrochloride at 4°C and pH 7.0. All the observed chromatographic profiles can be simulated by a cubic mechanism using a consistent set of equilibrium and kinetic parameters describing each of the coupled transitions. The four components in the folded protein and in the unfolded protein are interrelated by configurational transitions having parameters characteristic for proline peptide isomerizations. Only two of the four folded conformations are significantly populated at equilibrium. Each of the four unfolded components can refold by a unique Conformational transition. No transiently populated folding intermediates are detected having hydrodynamic volumes intermediate between those characteristic for the folded and unfolded protein. © 1993 John Wiley & Sons, Inc.     

Unfolding behavior of human alpha 1-acid glycoprotein is compatible with a loosely folded region in its polypeptide chain

The unfolding of human plasma alpha 1-acid glycoprotein (AGP) induced by heat or guanidine hydrochloride was studied under equilibrium conditions. In thermal unfolding, an intermediate state was detected by the appearance of unusual positive difference absorption bands in the 287-295-nm region, which occurred at lower temperatures than the common denaturation bands at 284 and 291 nm. The formation of this intermediate species apparently involves a local conformational change that perturbs the environment of tryptophyl residues, without affecting the secondary structure of the protein as judged from circular dichroism spectra. On the other hand, denaturation of the glycoprotein induced by guanidine hydrochloride seemed to follow a two-state model with no evidence of any intermediate species; however, the analysis of the transition curve indicated that the change in the accessibility to solvent of amino acid residues of AGP upon unfolding is significantly lower than those observed for other proteins. According to these results, it is proposed that part of the polypeptide chain in native AGP, namely, that from residue 122 to the C-terminus, may be "loosely" folded.     

Protein stabilization by osmolytes from hyperthermophiles: effect of mannosylglycerate on the thermal unfolding of recombinant nuclease a from Staphylococcus aureus studied by picosecond time-resolved fluorescence and calorimetry

2-O-alpha-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper)thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, pico-second time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A (SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 m potassium mannosylglycerate caused an increase of 7 degrees C in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65 degrees C and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed.