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Originally published In Press as doi:10.1074/jbc.M408806200 on September 4, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48680-48691, November 19, 2004
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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*

Tiago Q. Faria{ddagger}§, João C. Lima{ddagger}, Margarida Bastos||, António L. Maçanita{ddagger}**, and Helena Santos{ddagger}{ddagger}{ddagger}

From the {ddagger}Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, ||Centro de Investigação em Química da Universidade do Porto, Faculdade de Ciências, Universidade do Porto, R. Campo Alegre, 687, 4169-007 Porto, and the **Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal

Received for publication, August 2, 2004 , and in revised form, August 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 °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 °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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The accumulation of low molecular mass compounds is known to be a common strategy used by microorganisms to cope with environmental and stressful conditions, such as high osmolarity or elevated temperature. (Hyper)thermophiles accumulate compatible solutes rarely encountered in mesophiles (1–3). These solutes are generally negatively charged, whereas mesophiles accumulate primarily neutral solutes. This suggests that the unique charged solutes of hyperthermophiles were especially selected through evolution to preserve cell components at high temperature.

The use of osmolytes to improve protein stability is a well established practice, but the molecular mechanisms that govern this stabilization remain largely elusive. The demand for correctly folded and stable proteins from many biotechnological and clinical applications fosters the active research and vivid ongoing debate in this area (4–6).

A general mechanism proposed to explain the stabilizing effect of solutes upon proteins takes into account the exclusion of the solute from the protein hydration shell. Briefly, the theory states that a stabilizing solute raises the Gibbs energy of both native and unfolded states of the protein, but because the peptide backbone is more exposed to the osmolyte in the denatured state, is raises preferentially the Gibbs energy of the unfolded state, thus inducing a net stabilization effect, by shifting the equilibrium to the native state. This mechanism is particularly important with neutral osmolytes, or compounds known to act nonspecifically, i.e. independently of pH and the protein surface charge, and usually requires high concentrations to be effective. Fewer studies have been performed with charged solutes, but the ability of certain ions to bind and stabilize proteins has been reported, and it is generally believed that direct protein-solute interactions play a relevant role in the mechanism of protein stabilization by salts (7–10). Excellent reports on the principles that control protein stabilization by salts and electrically neutral molecules are available (4–6, 11–13).

In the present study we used time-resolved fluorescence spectroscopy (TRFS)1 and differential scanning calorimetry (DSC) to investigate the effect of mannosylglycerate (MG), a widespread compatible solute among microorganisms adapted to grow in hot environments (3), on the thermal denaturation of a model protein, nuclease A from Staphylococcus aureus (SNase). Steady-state fluorescence spectroscopy has been widely used to monitor protein conformational changes. However, derivation of thermodynamic quantities from these data involves a number of unproven assumptions on the unfolding mechanism and on the fluorescence properties of the native and denatured protein structures. Moreover, calculations depend critically on the extrapolation to the transition region of data obtained outside this region, the detection of intermediate unfolding states is rarely direct, and only the use of a combination of techniques can validate their presence (14). To overcome these drawbacks, we used TRFS, which has two important advantages. First, it is possible to discriminate the emission of the native and unfolded protein conformations through their decay times, a crucial bit of information for the characterization of the unfolding pathway. Second, the contribution of each species to the total fluorescence emission can be quantified and monitored during protein denaturation. TRFS provides data to directly determine the evolution of the unfolding equilibrium constant during the entire unfolding process, allowing for an accurate thermodynamic analysis of this process.

The fluorescence emission of the single tryptophan residue was used to probe the structural changes of SNase during thermal denaturation. The photophysical properties of the tryptophan residue in the protein were analyzed in comparison with those of N-acetyltryptophanamide (NAWA) in dioxanewater mixtures at different temperatures. NAWA is a consensual analogue for a tryptophan residue within a polypeptide chain (15), and dioxane-water mixtures mimic the range of microenvironments sensed by tryptophan in the folded and unfolded protein conformations.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—NAWA was purchased from Sigma. 1,4-Dioxane from Riedel-de-Haën was used after a distillation step to remove all emitting impurities, as confirmed by emission measurements. Mannosylglycerate (MG) was purified from cell extracts of Rhodothermus obamensis as described previously (7). To remove an absorbing contaminant present in the MG solution, a series of washing steps with dry activated charcoal were performed until no further absorbance decrease was observed. Typically, the solution with 0.5 M concentration had an absorbance of 0.05 at 280 nm. All other reagents were of analytical grade, and the buffer solutions were of spectroscopic grade.

Protein Expression and Purification—The pONF1 plasmid was a kind gift from Dr. Inouye (16). This plasmid has the nuclease gene cloned immediately after the ompA gene that encodes a signal excretion peptide and was transformed into Escherichia coli HB101 cells by heat shock. E. coli cells were grown in Luria-Bertani (LB) medium at 37 °C, and the recombinant protein production was induced by 1 mM isopropyl 1-thio-{beta}-D-galactopyranoside. The protein was purified by an osmotic shock procedure adapted from Takahara et al. (16) and Neu and Heppel (17) and briefly outlined here. Cells were washed three times with Tris-HCl buffer (10 mM, pH 7.6) and then suspended in sucrose buffer (20% sucrose, 10 mM Tris-HCl, 15 mM EDTA, pH 7.6). The suspension was incubated for 10 min on ice with stirring and then centrifuged. The resultant pellet was vigorously suspended in cold water and stirred for 10 min on ice. The periplasmic proteins (supernatant) were recovered by centrifugation, and the pellet was suspended in 1 M Tris-HCl, pH 7.6, and stirred on ice (30 min). The supernatant was separated from the cytoplasmic fraction by centrifugation. SDS-PAGE analysis revealed that the nuclease was fully released upon washing the membranes with 1 M Tris-HCl buffer. This fraction was dialyzed against 20 mM Tris-HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0, and loaded onto a Mono S column (HR5/5, Amersham Biosciences). SNase was eluted with ~0.3 M NaCl and was judged pure by SDS-PAGE stained with silver nitrate.

The N-terminal sequence was determined to confirm that no residue from the signal peptide was incorporated in the produced nuclease. To improve the reversibility of the thermal unfolding, nuclease was chemically denatured with 2 M guanidinium chloride and refolded by dilution with buffer and extensive dialysis. Pure nuclease was lyophilized and kept at –20 °C until use. The production yield was about 3 mg/liter of culture. Protein concentration was determined from UV absorbance at 280 nm, using an extinction coefficient of 0.93 (mg/ml)–1 cm–1.

Differential Scanning Calorimetry—Differential scanning calorimetry (DSC) was performed on a MicroCal VP-DSC MicroCalorimeter controlled by the VP-viewer program and equipped with 0.51 ml of cells. Calibration of temperature and heat flow was carried out according to MicroCal instructions. Stock solutions of nuclease were prepared by dissolving the lyophilized protein in phosphate buffer (10 mM, pH 7.5), and extensively "washing" with the same buffer in a Centricon tube (molecular mass of 10 kDa). A volume of 2 ml of phosphate buffer (10 mM, pH 7.5) without or with solute was prepared and dispensed into two Eppendorf tubes, 1 ml each. An aliquot of the concentrated protein solution or of phosphate buffer was added to each Eppendorf, and these solutions were used to fill up the sample and reference cells, respectively. Both solutions were degassed for 8 min under vacuum, prior to the calorimetric experiments. DSC scans were run at a constant heating rate of 1 °C/min from 20 to 80 °C when no solute was added and up to 90 °C when MG (or glycerol or trehalose) was present. An overpressure of about 30 p.s.i. was applied to the calorimeter cells. Reversibility of SNase thermal unfolding was assessed by performing two sequential DSC scans with each protein solution. In all assays, the area under the heat absorption peak of the second scan had, on average, at least 80% of that obtained in the first scan.

The thermodynamic parameters were determined in each case on the basis of five independent runs. Raw calorimetric data were converted to heat capacity by subtracting the buffer baseline, determined under identical conditions, and dividing by the scan rate and the sample protein concentration. The data were analyzed with EXAM software (18). This program uses a sigmoidal baseline to yield a van't Hoff enthalpy ({Delta}HvH), a transition temperature (Tm, the temperature at half the peak area), and the calorimetric enthalpy ({Delta}Hcal) that can be obtained from the ratio between the transition peak area and the number of moles of protein in the calorimetric cell. The change in heat capacity ({Delta}Cp) is calculated from the baseline shift by the software in the data-fitting procedure. The expression used in EXAM to fit the calorimetric data was,

(Eq. 1)
where dQ/dT is the derivative of the heat exchanged with respect to temperature, {alpha} is the extent of reaction (F({alpha}) = {alpha}(1–{alpha})), [Ba – Ba'(T – Tm)] is the pre-transition baseline, [Bb – Bb'(T – Tm)] is the posttransition baseline, {Delta}H is the van't Hoff enthalpy, and N is the number of protein moles present in the calorimeter cell. Protein concentrations around 16 µM were typically used.

Steady-state Fluorescence Spectroscopy—Measurements were carried out using a SPEX spectrofluorometer, Fluorolog 212I at 90° geometry with excitation and emission slits of 1 mm. Spectra were corrected for the wavelength dependence of the emission monochromator and the photomultiplier. Excitation was set to 280 nm for NAWA measurements and 292 nm for the SNase measurements to assure that only the tryptophan residue was excited. To remove oxygen NAWA solutions were left for 1 h under a nitrogen flow. Temperature was controlled with a circulating water bath. Fluorescence quantum yields of NAWA and nuclease were calculated from the integral of the emission bands, using p-terphenyl in cyclohexane at 20 °C as standard, {phi} = 0.77 (19). Nuclease fluorescence emission spectra were performed in phosphate buffer (10 mM, pH 7.5) in the absence and presence of 0.5 M MG at different temperatures. Nuclease concentration was adjusted to ~16 µM, which corresponds to an absorbance of ~0.1 at 292 nm.

Time-resolved Fluorescence Spectroscopy—Fluorescence decays were measured using a time-correlated single-photon-counting apparatus (20). Vertically polarized excitation light at 292 nm was obtained with the third harmonic of the output (876 nm) of a mode-locked Ti-sapphire laser (Spectra-Physics Tsunami) pumped by a Millenia X (Spectra-Physics) laser. Fluorescence emission was collected at 90° geometry, passed through a Glen-Thompson polarizer at 54.7° (magic angle) and a monochromator (360 nm) (Jobin-Yvon H20 Vis), and finally detected with a microchannel-plate photomultiplier (Hamamatsu R3809u-50). Automated alternate measurements (1 kilocount per cycle) of the excitation pulse, and sample emissions were made until 5–30 kilocounts at the maximum were reached. The fluorescence decays were deconvoluted from the excitation pulse using G. Striker's Sand program, which allows for individual and global analyses of the decays with individual shift optimization (21).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Differential Scanning Calorimetry—Thermodynamic parameters associated to SNase unfolding with and without MG were determined. Very high reproducibility of baselines was obtained both in the absence and presence of MG (Fig. 1). This is an important premise to obtain reliable parameters from DSC data. In the absence of solute, the van't Hoff enthalpy obtained from the fitting was 84 ± 4 kcal mol–1, and the calorimetric enthalpy was 83 ± 5 kcal mol–1, with a Tm of 53.9 ± 0.3 °C and a value of 1.7 ± 0.5 kcal K–1 mol–1 for the heat capacity change (Table I). The transition is thus perfectly described by a two-state model ({Delta}HvH/{Delta}Hcal = 1.0). The enthalpy values are in good agreement with those reported in the literature, which vary between 84.1 and 88.8 kcal mol–1 (22–24). The heat capacity change ({Delta}Cp) of SNase during thermal unfolding was determined from the calorimetric curve with the EXAM software using a sigmoidal baseline. The use of this procedure to extract {Delta}Cp is fully reasonable given the high sensitivity and reproducibility of the VP DSC calorimeter (25). The value of {Delta}Cp obtained for SNase, in the absence of solute, is within the range of values found in the literature, which vary between 1.7 kcal K–1 mol–1 (22) and 2.7 kcal K–1 mol–1 (26).



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  		FIG. 1.
Raw data of calorimetric scans performed in 10 mM phosphate buffer, pH 7.5, in the absence (A) and presence of 0.5 M mannosylglycerate (B). Repeats of baseline scans are also plotted.

 


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  		TABLE I
Thermodynamic parameters of SNase unfolding in 10 mM phosphate buffer, pH 7.5, with and without mannosylglycerate determined from DSC and TRFS {Delta}H values in kcal mol-1, {Delta}S in cal K-1 mol-1, and {Delta}Cp values in kcal K-1 mol-1.

 
In the presence of 0.5 M MG, Tm increased by 7.1 °C (to a value of 61.0 °C) indicating strong stabilization of SNase by MG. The calorimetric enthalpy at Tm was 88 ± 6 kcal mol–1 and the van't Hoff enthalpy was 95 ± 3 kcal mol–1. This slight deviation from a two-state model ({Delta}HvH/{Delta}Hcal = 1.07 ± 0.08) was not significant. An increase in {Delta}Cp of about 2-fold was observed (Table I).

DSC was also used to study the effect of MG concentration (up to 1 M) on the Tm of SNase (Table II). At 0.5 M concentration, glycerol or trehalose increased the Tm of SNase by 0.8 °C and 4.2 °C, respectively, a much lesser effect than that provided by MG (Table II).


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  		TABLE II
Effect of mannosylglycerate concentration on the melting temperature of SNase in 10 mM phosphate buffer, pH 7.5, determined by DSC

For comparison the Tm of the protein with 0.5 M glycerol or 0.5 M trehalose was also measured. The standard deviations refer to five independent measurements.

 
Absorption and Emission Spectra of SNase and NAWA—The absorption spectrum of SNase in phosphate buffer (10 mM, pH 7.5) at 20 °C reflects the presence of a single tryptophan and seven tyrosine residues (Fig. 2). As expected, the extinction coefficient of SNase at {lambda}max = 280 nm was identical to the weighted sum of the extinction coefficients of tyrosine and tryptophan. At wavelengths above 290 nm the absorption is mainly due to the single tryptophan residue. Above the melting temperature, a clear decrease of SNase absorption, caused primarily by the exposure of buried tyrosine residues to the aqueous external solvent, was observed (Fig. 2) (27, 28).



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  		FIG. 2.
Absorption and emission spectra of SNase in 10 mM phosphate buffer, pH 7.5, at 20 °C (solid lines) and at 80 °C (dashed lines). Fluorescence spectra of NAWA in dioxane at 20 °C (full circles) and in water at 80 °C (open circles). Fluorescence spectra at 80 °C are multiplied by a factor of 10.

 
Upon increasing the temperature, the fluorescence spectra of SNase in the same buffer, measured with 292-nm excitation, showed an increase of the maximum emission wavelength ({lambda}em) accompanied by a pronounced decrease of the quantum yield ({phi}f) (Figs. 2 and 3). This effect was particularly evident between 45 and 65 °C, the temperature range for SNase unfolding.



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  		FIG. 3.
Temperature dependence of fluorescence quantum yield (A) or wavelength of maximum emission (B) of NAWA in different dioxane-water mixtures (open triangles) and of SNase in 10 mM phosphate buffer with mannosylglycerate (open circles) or without (full circles). The following dioxane-water proportions were used: 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 (v/v).

 
Fluorescence quantum yields and maximum emission wavelengths of SNase, measured as a function of temperature, are presented in Fig. 3. This figure also shows the temperature dependence of the quantum yields and emission wavelengths of the model compound NAWA in different dioxane-water mixtures with polarities ranging from dioxane to water. The values of the maximum emission wavelength of NAWA increased with solvent polarity and, as expected, were independent of the temperature, whereas the quantum yield gradually decreased with either the increase of the solvent polarity (Table III) or temperature (Fig. 3).


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  		TABLE III
Fluorescence maximum wavelength ({lambda}em), quantum yield ({phi}f) and lifetime ({tau}f) of NAWA in dioxane-water mixtures at 23 °C

Values of the radiative (kf) and the sum of radiationless (knr) rate constant as well as the dielectric constant ({epsilon}) and refraction index (nD) of the mixtures are also shown.

 
Below 45 °C, the quantum yield and emission wavelength of SNase were similar to those of NAWA in a low polarity environment (~90:10 v/v dioxane-water mixture). However, in the 45–65 °C temperature range, the quantum yield of SNase decreased abruptly while the maximum emission wavelength increased; finally, above 65 °C, the quantum yield and emission wavelength of SNase became similar to the values of NAWA in an environment with polarity close to that of water. These observations clearly indicate that, within the 45–65 °C temperature range, SNase unfolds, exposing its tryptophan residue to water, whereas below 45 °C, the tryptophan residue inside the native SNase probes a relatively low polarity environment.

Fluorescence Decays of SNase and NAWA—Fluorescence decays of NAWA in dioxane-water mixtures were measured at temperatures ranging from 20 °C to 80 °C, with excitation at {lambda}exc = 292 nm, emission at {lambda}em = 360 nm and a time resolution of 24.3 ps/channel. The decays were strictly single exponentials, independent of the number of counts at the maximum (up to 30 kilocounts). Single exponential decay laws of NAWA in water were also observed in other laboratories (29, 30). The presence of 10 mM phosphate buffer or 0.5 M MG did not affect the NAWA decay in water, but the effect could not be assessed in dioxane or dioxane-water mixtures due to the low solubility of these compounds.

The fluorescence lifetime of NAWA at room temperature decreased from 5.2 ns in dioxane to 3.1 ns in water, following a trend with solvent polarity similar to that of the quantum yield (Table III). For each solvent or mixture, increasing temperature induced the expected decrease of the fluorescence lifetime (Fig. 4A). The radiative (kf) and the sum of the radiationless (knr) decay rates of NAWA in dioxane-water mixtures were calculated from quantum yields ({phi}f) and fluorescence lifetimes ({tau}f) by kf = {phi}f/{tau}f and knr = (1 – {phi}f)/{tau}f (Table III). The radiative rate constant (kf) shows the expected proportionality to the squared refractive index of the mixture, decreasing from dioxane to water, while knr increases with solvent polarity. The combined effect of the two variations induces the observed decrease of both fluorescence quantum yield and lifetime from dioxane to water. A similar increase of knr has been observed for other compounds in dioxane-water mixtures and has been attributed to the increase of the internal conversion rate constant upon increasing water content (31–35).



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  		FIG. 4.
A, fluorescence lifetime as a function of temperature of NAWA in different dioxane-water mixtures (open triangles) and of SNase in 10 mM phosphate buffer, pH 7.5 (full symbols). Full circles, native SNase ({tau}1); triangles, denatured SNase ({tau}2); and squares, denatured SNase ({tau}3). The following dioxane-water proportions were used: from top to bottom, 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 (v/v). B, pre-exponential coefficients of fluorescence decays for SNase in 10 mM phosphate buffer, pH 7.5. Circles, native SNase (a1); triangles and squares, denatured forms of SNase (a2 and a3). At low temperatures (below 40 °C) {tau}2 is poorly defined and not present in all samples.

 
The values of the radiative and radiationless rate constants of SNase at 23 °C were similar to those of NAWA in dioxane (Table III), thus indicating a low polarity environment of the tryptophan residue in the native protein. At 70 °C, the quantum yield and the major lifetime ({tau}2) of SNase were similar to those of NAWA in water at the same temperature, reflecting the high degree of exposure of the tryptophan residue to water in the unfolded protein. The quantum yield and emission wavelength profiles led to a similar conclusion.

The nature of the fluorescence decay of SNase in water at 23 °C, measured with a time resolution of 24.3 ps/channel, was not so simple. Typically, the decays could be fitted only with double-exponential functions, although the long-lived component (~5.5 ns) accounted for more than 95–99% of the total fluorescence emission. The minor component had a poorly defined lifetime ranging from 1 to 3 ns, whose weight varied with the protein preparation, but in one of the preparations it was undoubtedly absent (Fig. 5A). The observation of this single-exponential function means that the decay of the tryptophan fluorescence in the native form of SNase is genuinely single-exponential, and, therefore, the residual short time appearing in most SNase samples could arise from a small amount of denatured protein.



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  		FIG. 5.
A, single exponential fit of fluorescence intensity decay of SNase in 10 mM phosphate buffer at 23 °C. B, triple-exponential fit of SNase fluorescence decay at 54 °C. Decay times ({tau}), normalized pre-exponential coefficients (a) and fitting parameters ({chi}2) are also shown. AC, auto-correlation function. WR, weighted residue function.

 
Below 45 °C, the lifetime of the major component in the fluorescence decay of SNase ({tau}1) had values similar to those of NAWA in a 90:10 v/v dioxane-water mixture, hence it was assigned to the decay of tryptophan buried in the native form of the protein (Fig. 4A). Above 45 °C, three exponential functions were required to fit the decays of SNase (Fig. 5B). The third decay time ({tau}3) was significantly shorter than the lifetime of NAWA in water (Fig. 4A); moreover, the respective amplitude (a3) was always lower than that associated to the intermediate decay time (a2) (Fig. 4B). The longer decay time, {tau}1, was still close to the lifetime of NAWA in a low polarity environment (Fig. 4A). However, its amplitude (a1) decreased sharply with the temperature to values close to zero, whereas the amplitudes of the two remaining components (a2 and a3) increased, adding up to a value close to 1 at temperatures higher than ~60 °C (Fig. 4B).

In summary, the fluorescence decay of SNase in the range of temperature examined is characterized by three times, one ({tau}1) corresponding to the emission of tryptophan in the folded protein, and the other two times ({tau}2 and {tau}3) originating from exposed tryptophan residues in unfolded forms of the protein. The temperature dependence of the amplitudes reflects the transition from the folded to the unfolded state.

Effect of MG on the Fluorescence of SNase—The effect of MG on the photophysical properties of SNase was also studied by steady-state and time-resolved fluorescence spectroscopy. The fluorescence quantum yield showed identical values with and without MG at temperatures far from the unfolding transition; also, the same 20-nm red shift was observed for the variation of the maximum emission wavelength upon unfolding. However, the temperature range where these quantities change abruptly was clearly shifted toward higher temperatures upon MG addition (Fig. 3). From the quantum yield results, the SNase melting temperature increased by ~7 °C. In the temperature range where unfolding occurs, the emission spectrum of SNase is the sum of the spectra from the native and unfolded protein conformations weighted by the respective molar fractions. Because the quantum yield of the unfolded protein is much lower than that of the folded protein, a shift of the maximum emission wavelength is not detectable unless the population of the unfolded form is predominant. Despite this lack of sensitivity, it is interesting to point out that the curves representing the shift of the wavelength maximum in the presence and absence of MG were also separated by ~7 °C (Fig. 3B).

The temperature dependence of the lifetimes and amplitudes of the SNase fluorescence decays, in the presence of MG, are shown in Fig. 6. The evolution of the lifetimes was similar to that observed in the absence of solute, except for the higher temperature of the unfolding transition.



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  		FIG. 6.
A, temperature dependence of fluorescence decay times of NAWA in different dioxane-water mixtures (open triangles) and of SNase in 10 mM phosphate buffer, pH 7.5, containing 0.5 M mannosylglycerate (full symbols). Full circles, native SNase ({tau}1); triangles, denatured SNase ({tau}2); and squares, denatured SNase ({tau}3). The following dioxane-water proportions were used: from top to bottom, 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 (v/v). B, temperature dependence of pre-exponential coefficients of fluorescence decays of SNase in 10 mM phosphate buffer, pH 7.5, with 0.5 M mannosylglycerate. Circles, native form of SNase (a1); triangles and squares, denatured forms of SNase (a2 and a3). At low temperatures {tau}2 is poorly defined and not present in all samples.

 
Determination of Unfolding Equilibrium Constants by TRFS—The unfolding equilibrium constant for a two-state mechanism is defined as the ratio of concentrations of the unfolded, [U], and native, [N], proteins at equilibrium. The fluorescence decay functions are IN (t) = a1e–t/{tau}t/{tau}1 and IU(t) = a2e–t/{tau}2 + a3e–t/{tau}3 for the native and unfolded protein conformations, respectively. Under the assumption that the protein folding and unfolding rates are low with respect to the reciprocal fluorescence lifetimes of the buried and exposed tryptophan, at any temperature, the pre-exponential coefficient of each exponential term is proportional to the concentration, at t = 0, of the excited species to which that exponential term is assigned ([N*](0) or [U*](0)), multiplied by the radiative rate constant of each species (kfN or kfU). It is also necessary to account for the fraction of light that is detected at the experimental emission wavelength {lambda}em under steady-state conditions, fi({lambda}em), with respect to the total fluorescence emission, {int}I({lambda}){partial}{lambda}, of each species. These fractions are defined, for the native, fN({lambda}em), and unfolded protein, fU({lambda}em), as in Equations 2 and 3,

(Eq. 2)

(Eq. 3)
where IN({lambda}em) and IU({lambda}em) are the fluorescence intensities of the native and unfolded protein at {lambda}em (360 nm). The mathematical expression for the fluorescence decay of native, IN(t), and denatured, IU(t), protein conformations are given by Equations 4 and 5,

(Eq. 4)

(Eq. 5)
where c2 and c3 define the relative weight of the two exponential terms of the tryptophan fluorescence decay in the unfolded protein. Therefore, the amplitudes, ai, obtained from the fluorescence intensity decay fits are, apart from an instrumental constant, given by ai({lambda}em) = fi({lambda}em)·kfi·[i*](0). For the determination of [N*](0) and [D*](0), the amplitudes must be divided by fi({lambda}em)·kfi.

Taking NAWA in dioxane-water mixtures as an appropriate model system, the tryptophan environment in the native and unfolded protein can be represented by a 90:10 v/v dioxane-water mixture and water, respectively. Thus, the values of kfN and kfU were calculated as 4.9 x 107 s–1 and 3.8 x 107 s–1. The fraction of light emitted by NAWA at 360 nm in a 90:10 v/v dioxane-water mixture is ~70% lower than in water due to the large bathochromic shift and broadening of the emission spectrum from dioxane to water (Fig. 2). Accordingly, the amplitude of the low polarity component a1 was divided by 0.7 x 4.9 x 107 x s–1, and the amplitudes, a2 and a3, were divided by 3.8 x 107 x s–1. The corrected values were then normalized according to the condition a1 + a2 + a3 = 1 to obtain the molar fractions at t = 0 of the folded and unfolded forms in the excited state, respectively.

The concentration at t = 0 of each excited species is in turn proportional to the ground-state concentration of that species multiplied by its molar extinction coefficient at the excitation wavelength. Because there was no appreciable difference in the molar extinction coefficients of NAWA in dioxane and water at 292 nm, the corrected and normalized amplitude a1 is equal to the ground-state molar fraction of the folded SNase and the sum a2 + a3 is equal to the ground-state molar fraction of the unfolded SNase (Fig. 7).



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  		FIG. 7.
Molar fractions of folded (circles) and unfolded (squares) forms of SNase in 10 mM phosphate buffer, pH 7.5, with 0.5 M mannosylglycerate (open symbols) or without (full symbols).

 
The molar fractions of the folded and unfolded SNase were equal at 53.0 °C without solute, and 60.8 °C with 0.5 M MG. These values are in excellent agreement with the melting temperatures determined by DSC (Table I).

Thermodynamic Analysis of TRFS Data—The equilibrium constants of unfolding, calculated from the molar fractions of the folded and unfolded SNase at each temperature, can be used to calculate the temperature dependence of the Gibbs free energy, {Delta}G = –RT ln KU. The Gibbs energy function can also be described by the modified Gibbs-Helmholtz equation (Equation 6).

(Eq. 6)
Equilibrium data can then be fitted to a non-linear function, resulting from the combination of these two equations (Equation 7), to derive the values of Tm, {Delta}Hm, and {Delta}Cp associated to SNase unfolding in Equation 7.

(Eq. 7)

The fit of this function to the experimental points in the transition region leads to {Delta}Cp values with considerable uncertainty: {Delta}Cp = 2 ± 1 kcal K–1 mol–1 and 4 ± 2 kcal K–1 mol–1 for SNase unfolding in the absence and presence of MG, respectively. Nevertheless, these values agree well with those obtained from the DSC experiments, 1.7 ± 0.5 and 4 ± 1 kcal K–1 mol–1. The temperature dependences of KU, with and without solute, are shown in the van't Hoff plots of Fig. 8. The van't Hoff enthalpy, the melting temperature, and the heat capacity change values associated to SNase unfolding with and without MG are given in Table I together with the DSC results. In the absence of solute, the enthalpy values obtained by TRFS are only slightly lower than those determined by DSC. In the presence of MG, the TRFS enthalpy value is significantly lower than that obtained by DSC. A full explanation for this observation cannot be advanced at this point, but the discrepancy is perhaps not so important if one takes into consideration that TRFS only monitors the tryptophan-surrounding area, whereas DSC probes the unfolding of the whole protein and also senses possible contributions from the solvent.



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  		FIG. 8.
Van't Hoff plots of SNase unfolding in 10 mM phosphate buffer, pH 7.5, containing 0.5 M mannosylglycerate (open circles) and in the absence of mannosylglycerate (full circles). Solid lines are the best fits taking into account {Delta}Cp (see text, Equation 7); dashed lines represent the fits disregarding {Delta}Cp.

 
Protein-Solute Interaction: Contribution of Preferential Exclusion—We used the thermodynamic theory of preferential exclusion and analyzed our data of SNase stabilization by MG, following the formalism developed by Plaza-del-Pino and Sanchez-Ruiz (36). This analysis determines the preferential interaction parameter change ({Delta}{Phi}23) that can be used to calculate the denaturational change in the protein preferential hydration ({Delta}{Gamma}21). The thermodynamic equations to determine these quantities, at Tm, are,

(Eq. 8)

(Eq. 9)
where m3 is the molal concentration of solute, 1 is the molar volume of water, and {Pi} is the osmotic pressure of the water-solute mixture. The subscripts 1, 2, and 3 refer to water, protein, and solute (MG), respectively. Density measurements were performed to determine the solute molal concentration, and the effect of MG concentration on the Tm of SNase was studied up to 1 M MG (Table II). The polynomial equation that describes this variation is Tm =–(6.8 ± 0.4) m32 + (19.2 ± 0.6) m3 + (327.3 ± 0.1). Using Equation 8, the calculated value of {Delta}{Phi}23 in the presence of 0.5 M MG was 2.9 kcal kgwater/molprot molMG. Nothing is known about the osmotic pressure of the water-MG solutions, and, therefore, an estimation of this parameter must be made to determine the change in the protein preferential hydration. In an ideal solution, the variation of the osmotic pressure with the solute concentration can be calculated from {partial}{Pi}/{partial}m3 = RT/(1 (55.56 + m3)), which, for a MG concentration of 0.5 M, results in a value of 657.4 kcal kgwater/m3 molMG. Thus, the denaturational change in the protein preferential hydration is {Delta}{Gamma}21 = 248 molwater/molprot = 0.3 gwater/gprot, a value not far out the range found for most proteins (0.4–0.6 gwater/gprot) (36). According to the definition of Timasheff (4), superosmolytes induce an increase of the osmotic pressure at a higher rate than that observed in an ideal solution. Using the value of 843.3 kcal kgwater/m3 molMG proposed for the osmotic pressure increment of superosmolytes (37), the value obtained for the protein preferential hydration parameter is {Delta}{Gamma}21 = 191 molwater/molprot = 0.2 gwater/gprot. Thus, superosmolytes provide the same degree of protein stabilization with lower levels of solute exclusion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Characterization of SNase Unfolding by Time-resolved Fluorescence Spectroscopy—To evaluate the populations of the different protein forms, we need to know the fluorescence behavior of the tryptophan residue in the native and denatured protein conformations (see "Results"). This information was derived from the study of NAWA in dioxane-water mixtures by time-resolved and steady-state fluorescence spectroscopy. These solvent mixtures were used to represent the range of environment polarity sensed by tryptophan when the protein undergoes unfolding. Dioxane-water mixtures have been successfully used to mimic the microenvironment sensed by fluorescent probes in heterogeneous media, such as micelles (34) and micro-emulsions (35).

The good correlation observed between the emission properties of NAWA in dioxane-water mixtures and those of SNase during unfolding (Figs. 3 and 4) suggests that this is an adequate model system to represent the environment surrounding tryptophan in the protein structure, and hence, monitor structural changes. However, the fluorescence of NAWA decays as a single exponential, whereas SNase shows two or three lifetimes depending on the temperature range examined. Two lifetimes are required to fit the SNase data at temperatures well below the Tm. Many fluorescence studies with SNase can be found in the literature (24, 26, 38–41): at room temperature and neutral pH, the emission decay is characterized by a long-lived component (5.4–6.0 ns) and one or two additional components with short lifetimes (ranging from 1.0 to 3.3 ns). Generally, these short components are poorly defined and have variable amplitudes (5–30%). Multiple-exponential decays have also been reported for many other single-tryptophan proteins, but the interpretation of this observation in molecular terms is elusive. We performed a detailed analysis of the temperature dependence of the SNase fluorescence kinetics, resorting to data on the effect of solvent polarity on the lifetime of NAWA to ascribe the long ({tau}1) and short ({tau}2) lifetimes to the native and denatured forms, respectively. This assignment was corroborated by the temperature dependence of the amplitudes of the two components and the comparison of the lifetimes of the protein with those of NAWA in dioxane-water mixtures (Fig. 4).

Above Tm, SNase decays require an additional time ({tau}3), which can arise from: (i) existence of different unfolded conformations of the protein in which tryptophan fluorescence is quenched by neighboring residues like histidine, lysine, or tyrosine (42) or (ii) intramolecular electron transfer from the indole moiety in tryptophan to the peptide chain due to slow interconversion of the different tryptophan rotamers (43) that seems to occur with tyrosine residues in unfolded ubiquitin.2 At this stage, we are unable to provide a definite physical meaning for {tau}3, but this fact does not hamper an accurate analysis of the unfolding process.

Effect of MG on the Population of SNase Forms along Unfolding—We took advantage of the discriminating properties of TRFS to characterize the effect of MG on the unfolding pathway of SNase. The presence of MG did not alter the pattern of protein lifetimes nor the proportions of the species observed, after accounting for the shift in Tm. Therefore, despite the considerable degree of stabilization rendered by MG, we conclude that the unfolding pathway of SNase was essentially unaffected by this solute. In other words, the molecular mechanism for stabilization of SNase by mannosylglycerate is likely to involve subtle changes with no detectable impact in the population of protein states along denaturation.

Thermodynamic Basis for SNase Stabilization by Mannosylglycerate—Thermodynamic parameters of SNase unfolding were calculated from TRFS data and by DSC, a direct method that monitors the whole protein structure. The combination of these two techniques to study protein thermal unfolding allowed for a more thorough characterization of this process and a greater confidence on the data and conclusions produced.

The most notable changes induced by the presence of MG are the increases in Tm and the unfolding heat capacity of SNase. The temperature dependence of {Delta}G (according to Equation 6), considering that {Delta}Cp is temperature-independent, is shown in Fig. 9. The presence of MG led to a shift of the stability curve ({Delta}G versus T), as expected for a stabilizing osmolyte. The stability curve should be regarded with caution, because the assumption that {Delta}Cp remains constant may not hold over a large temperature range; yet, it is unlikely that the magnitude of the hypothetical change in {Delta}Cp is large enough to affect considerably the general trend. The entropic or enthalpic contributions to the Gibbs energy of stabilization provided by mannosylglycerate were evaluated from the temperature dependence of {Delta}H ={Delta}Hm +{Delta}Cp(T – Tm) and {Delta}S ={Delta}Sm +{Delta}Cp ln(T/Tm) (Fig. 10). Strong enthalpy-entropy compensation was observed, a common feature in phenomena involving the exposure of hydrophobic residues to the solvent.



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  		FIG. 9.
Unfolding Gibbs energy of SNase as a function of temperature calculated using the thermodynamic parameters derived from DSC measurements (Table I). Thick line, {Delta}G in the presence of 0.5 M mannosylglycerate; thin line, {Delta}G in the absence of mannosylglycerate.

 



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  		FIG. 10.
Temperature dependence of {Delta}H and T{Delta}S associated to SNase unfolding calculated using the thermodynamic parameters derived from DSC measurements (Table I). Thick solid line, {Delta}H in the presence of MG; thick dashed line, T{Delta}S in the presence of MG; thin solid line, {Delta}H in the absence of MG; and thin dashed line, T{Delta}S in the absence of MG.

 
Interestingly, the relative magnitudes of {Delta}H and {Delta}S, in the presence and absence of solute, depend on the temperature considered, because the respective lines cross at around 65 °C. Therefore, the major contribution for the Gibbs energy of stabilization depends on the specific temperature range: (i) between 40 and 65 °C, both the unfolding enthalpy and entropy decreased in the presence of MG, but the decrease in the unfolding entropy was more pronounced, and therefore, {Delta}GMG > {Delta}GNS; (ii) above 65 °C, the unfolding enthalpy and entropy increased in the presence of MG, but the increase in enthalpy outweighed that of entropy, hence the Gibbs energy of stabilization was positive. In summary, at temperatures between 40 and 65 °C the thermodynamic parameter responsible for stabilization is the entropy, but at higher temperatures the stabilization is enthalpic.

A 2-fold increase of the SNase unfolding heat capacity was observed in the presence of MG. The change in the unfolding heat capacity has been correlated with the variation of polar and apolar accessible surface area upon denaturation ({Delta}ASApol and {Delta}ASAap) and on their contributions for {Delta}Cp per unit surface area ( and ) by the equation . The polar and apolar contributions have different signs () and may change with the solute concentration. Therefore, the rationalization of the variation of {Delta}Cp values in the presence of solutes is not straightforward. The stabilization of ribonuclease A by sarcosine was accompanied with an increase of {Delta}Cp (36), whereas a decrease was observed for the unfolding of the same protein in the presence of sucrose (44) or sarcosine (45). Trehalose also caused a decrease of {Delta}Cp of five proteins (46). Other examples exist where {Delta}Cp values remain unchanged in the presence of stabilizing solutes (47). Hence, protein stabilization cannot be inferred from the variation that solutes induce on {Delta}Cp values. In a comparative study with proteins isolated from thermophiles and mesophiles, Zhou (48) observed that proteins from thermophiles have lower {Delta}Cp than their mesophilic counterparts, a fact also pointed out by other authors (49, 50). This observation could partly be attributed to stronger polar interactions in the native form (48) or to a higher propensity for residual structure and hydrophobic cluster formation in the denatured state of thermophilic proteins (50). The large increase in {Delta}Cp of SNase in the presence of MG could result from decreased polar interactions in the folded state as the positive charge of surface ionized groups is canceled by electrostatic interaction with MG. Also, the reduced electrostatic repulsion may lead to a more compact folded state and consequently to stabilization. In fact, a recent NMR study revealed an increase in the overall rigidity of rubredoxin induced by the presence of diglycerol phosphate, another negatively charged solute from hyperthermophiles (51).

Role of Preferential Hydration and Binding in the Stabilization of SNase by Mannosylglycerate—The phenomena of protein stabilization by solutes derive from differential interactions with the unfolded and folded forms of the protein, which could be attained either by preferential exclusion or binding.

The analysis of our thermodynamic data in the framework of the solute preferential exclusion theory revealed that exclusion definitely plays a role in the mechanism of stabilization by MG as indicated by the change of 0.3 gwater/gprot in the protein preferential hydration upon unfolding. In conclusion, the increase of protein stability in the presence of MG can be interpreted as resulting, to some extent, from destabilization of the denatured state caused by preferential exclusion of MG upon unfolding. Nevertheless, the contribution of MG binding to protein stabilization is likely to be relevant in the case of this anionic solute. The high degree of stabilization exerted even at moderate solute concentrations when compared with common, neutral osmolytes supports this hypothesis. For example, the Tm of SNase increased more than 7 °C in the presence of 0.5 M mannosylglycerate, whereas the same concentration of glycerol or trehalose induced much lower stabilization. A strong stabilization at low or moderate concentration is typical of situations that involve specific interactions, namely electrostatic (6, 45, 52, 53). Moreover, other authors have pointed out the need to invoke mechanisms other than increased surface tension to explain the stabilization rendered by carboxylic acid salts (54). The increase in the unfolding Gibbs energy in the presence of solutes results from an increase in the cavity formation energy (which is proportional to the surface tension) and also from the energy of the protein-solute interaction. If the increase in the surface tension were the sole factor responsible for stabilization, a linear correlation would be expected between the increase in the surface tension and the protein stabilization. Kaushik and Bhat (54) interpreted the non-linear correlation between the increase in water surface tension and the increment in Tm observed for carboxylic acid salts as evidence for a significant contribution of protein-solute interactions to protein stabilization.

At neutral pH, SNase has a high positive charge, therefore, a favorable interaction of fully ionized MG with the protein is expected. This interaction will reduce electrostatic repulsions, thereby stabilizing preferentially the native state, because the charges are closer, on average, than in the more extended denatured state. Electrostatic effects have also been invoked to explain the stabilization of RNase A rendered by MG only at pH values above the pKa of the solute (7). Also, recent studies on the stabilizing effect of mannosylglycerate and diglycerol phosphate in single residue mutants of rubredoxin showed that the effect on protein stability is strongly dependent on the specific protein/compatible solute system examined.3

Physiological Significance of Charged Solutes in Hyperthermophiles—Most of the compatible solutes accumulated by thermophiles and hyperthermophiles are negatively charged in opposition to mesophiles that accumulate neutral or zwitterionic compounds such as trehalose, glycerol, or proline. This observation raised the hypothesis that charge could play an important role in the protection of cells and their components against heat damage (3). The superior efficacy of negatively charged solutes (phosphodiester compounds or carboxylic acids) to enhance protein stability has been demonstrated in several studies from our and other groups (7, 51, 54–57). Therefore, it is interesting to note that hyperthermophiles have adapted their cellular components through evolution to be able to benefit from the most effective protectors against heat available. The use of charged solutes as cell stabilizers implies the intracellular accumulation of positive counterions such as potassium; high salt concentrations are known to be toxic for most mesophilic bacteria, hence, these organisms are unable to take advantage of the accumulation of charged solutes to a large extent. However, (hyper)thermophiles have acquired this attribute, which seems essential to endow them with the level of extrinsic stabilization needed for thriving in hot environments.


   FOOTNOTES
 
* This work was supported in part by the European Commission, Contracts QLK3-CT-2000-00640 and COOP-CT-2003-508644, and by Fundação para a Ciência e a Tecnologia (FCT), Portugal, and FEDER, Project POCTI 35131/BIO/2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Ph.D. grant from FCT, PRAXIS XXI/21524/99. Back

Present address: Rede de Química e Tecnologia/Centro de Química Fina E Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, Monte de Caparica, 2829-516 Caparica. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 351-21-446-9828; Fax: 351-21-442-8766; E-mail: santos{at}itqb.unl.pt.

1 The abbreviations used are: TRFS, time-resolved fluorescence spectroscopy; DSC, differential scanning calorimetry; MG, 2-O-{alpha}-mannosylglycerate (potassium salt); SNase, recombinant nuclease A from S. aureus; NAWA, N-acetyltryptophanamide. Back

2 Noronha, M., Lima, J. C., Bastos, M., Santos, H., and Maçanita, A. L. (2004) Biophys. J. 87, 2609–2620. Back

3 T. Pais, P. Lamosa, W. dos Santos, J. LeGall, D. Turner, and H. Santos, unpublished results. Back


   ACKNOWLEDGMENTS
 
We are grateful to Prof. M. Inouye, State University of New York, for the generous supply of pONF1. M. B. thanks Dr. Frederick P. Schwarz, from the National Institute of Standards and Technology/Center for Advanced Research in Biotechnology, USA, for providing the EXAM program.



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 DISCUSSION
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