Protein Stabilization by Osmolytes from HyperthermophilesEFFECT OF MANNOSYLGLYCERATE ON THE THERMAL UNFOLDING OF RECOMBINANTNUCLEASE A FROM STAPHYLOCOCCUS AUREUS STUDIED BY PICOSECONDTIME-RESOLVED FLUORESCENCE AND CALORIMETRY*

Received for publication, August 2, 2004, and in revised form, August 30, 2004Published, JBC Papers in Press, September 4, 2004, DOI 10.1074/jbc.M408806200

Tiago Q. Faria‡§, Joao C. Lima‡¶, Margarida Bastos�, Antonio L. Macanita‡**,and Helena Santos‡ ‡‡

From the ‡Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa, Rua da Quinta Grande 6,Apartado 127, 2780-156 Oeiras, �Centro de Investigacao em Quımica da Universidade do Porto, Faculdade de Ciencias,Universidade do Porto, R. Campo Alegre, 687, 4169-007 Porto, and the **Instituto Superior Tecnico, UniversidadeTecnica de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal

2-O-�-Mannosylglycerate, a negatively charged os-molyte widely distributed among (hyper)thermophilicmicroorganisms, is known to provide notable protectionto proteins against thermal denaturation. To study themechanism responsible for protein stabilization, pico-second time-resolved fluorescence spectroscopy wasused to characterize the thermal unfolding of a modelprotein, Staphylococcus aureus recombinant nuclease A(SNase), in the presence or absence of mannosylglycer-ate. The fluorescence decay times are signatures of theprotein state, and the pre-exponential coefficients areused to evaluate the molar fractions of the folded andunfolded states. Hence, direct determination of equilib-rium constants of unfolding from molar fractions wascarried out. Van’t Hoff plots of the equilibrium con-stants provided reliable thermodynamic data for SNaseunfolding. Differential scanning calorimetry was usedto validate this thermodynamic analysis. The presenceof 0.5 M potassium mannosylglycerate caused an in-crease of 7 °C in the SNase melting temperature and a2-fold increase in the unfolding heat capacity. Despitethe considerable degree of stabilization rendered by thissolute, the nature and population of protein states alongunfolding were not altered in the presence of mannosyl-glycerate, denoting that the unfolding pathway of SNasewas unaffected. The stabilization of SNase by mannosyl-glycerate arises from decreased unfolding entropy up to65 °C and from an enthalpy increase above this temper-ature. In molecular terms, stabilization is interpreted asresulting from destabilization of the denatured statecaused by preferential exclusion of the solute from theprotein hydration shell upon unfolding, and stabiliza-tion of the native state by specific interactions. Thephysiological significance of charged solutes in hyper-thermophiles is discussed.

The accumulation of low molecular mass compounds isknown to be a common strategy used by microorganisms tocope with environmental and stressful conditions, such as highosmolarity or elevated temperature. (Hyper)thermophiles ac-cumulate compatible solutes rarely encountered in mesophiles(1–3). These solutes are generally negatively charged, whereasmesophiles accumulate primarily neutral solutes. This sug-gests that the unique charged solutes of hyperthermophileswere especially selected through evolution to preserve cell com-ponents at high temperature.

The use of osmolytes to improve protein stability is a wellestablished practice, but the molecular mechanisms that gov-ern this stabilization remain largely elusive. The demand forcorrectly folded and stable proteins from many biotechnologicaland clinical applications fosters the active research and vividongoing debate in this area (4–6).

A general mechanism proposed to explain the stabilizingeffect of solutes upon proteins takes into account the exclusionof the solute from the protein hydration shell. Briefly, thetheory states that a stabilizing solute raises the Gibbs energy ofboth native and unfolded states of the protein, but because thepeptide backbone is more exposed to the osmolyte in the dena-tured state, is raises preferentially the Gibbs energy of theunfolded state, thus inducing a net stabilization effect, by shift-ing the equilibrium to the native state. This mechanism isparticularly important with neutral osmolytes, or compoundsknown to act nonspecifically, i.e. independently of pH and theprotein surface charge, and usually requires high concentra-tions to be effective. Fewer studies have been performed withcharged solutes, but the ability of certain ions to bind andstabilize proteins has been reported, and it is generally be-lieved that direct protein-solute interactions play a relevantrole in the mechanism of protein stabilization by salts (7–10).Excellent reports on the principles that control protein stabili-zation by salts and electrically neutral molecules are available(4–6, 11–13).

In the present study we used time-resolved fluorescencespectroscopy (TRFS)1 and differential scanning calorimetry(DSC) to investigate the effect of mannosylglycerate (MG), awidespread compatible solute among microorganisms adaptedto grow in hot environments (3), on the thermal denaturation ofa model protein, nuclease A from Staphylococcus aureus

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

§ Supported by a Ph.D. grant from FCT, PRAXIS XXI/21524/99.¶ Present address: Rede de Quımica e Tecnologia/Centro de Quımica

Fina E Biotecnologia, Faculdade de Ciencias e Tecnologia, Univer-sidade Nova de Lisboa, Quinta da Torre, Monte de Caparica, 2829-516Caparica.

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

1 The abbreviations used are: TRFS, time-resolved fluorescence spec-troscopy; DSC, differential scanning calorimetry; MG, 2-O-�-mannosyl-glycerate (potassium salt); SNase, recombinant nuclease A fromS. aureus; NAWA, N-acetyltryptophanamide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 47, Issue of November 19, pp. 48680–48691, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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(SNase). Steady-state fluorescence spectroscopy has beenwidely used to monitor protein conformational changes. How-ever, derivation of thermodynamic quantities from these datainvolves a number of unproven assumptions on the unfoldingmechanism and on the fluorescence properties of the nativeand denatured protein structures. Moreover, calculations de-pend critically on the extrapolation to the transition region ofdata obtained outside this region, the detection of intermediateunfolding states is rarely direct, and only the use of a combi-nation of techniques can validate their presence (14). To over-come these drawbacks, we used TRFS, which has two impor-tant advantages. First, it is possible to discriminate theemission of the native and unfolded protein conformationsthrough their decay times, a crucial bit of information for thecharacterization of the unfolding pathway. Second, the contri-bution of each species to the total fluorescence emission can bequantified and monitored during protein denaturation. TRFSprovides data to directly determine the evolution of the unfold-ing equilibrium constant during the entire unfolding process,allowing for an accurate thermodynamic analysis of thisprocess.

The fluorescence emission of the single tryptophan residuewas used to probe the structural changes of SNase duringthermal denaturation. The photophysical properties of thetryptophan residue in the protein were analyzed in comparisonwith those of N-acetyltryptophanamide (NAWA) in dioxane-water mixtures at different temperatures. NAWA is a consen-sual analogue for a tryptophan residue within a polypeptidechain (15), and dioxane-water mixtures mimic the range ofmicroenvironments sensed by tryptophan in the folded andunfolded protein conformations.

EXPERIMENTAL PROCEDURES

Materials—NAWA was purchased from Sigma. 1,4-Dioxane fromRiedel-de-Haen was used after a distillation step to remove all emittingimpurities, as confirmed by emission measurements. Mannosylglycer-ate (MG) was purified from cell extracts of Rhodothermus obamensis asdescribed previously (7). To remove an absorbing contaminant presentin the MG solution, a series of washing steps with dry activated char-coal were performed until no further absorbance decrease was observed.Typically, the solution with 0.5 M concentration had an absorbance of0.05 at 280 nm. All other reagents were of analytical grade, and thebuffer solutions were of spectroscopic grade.

Protein Expression and Purification—The pONF1 plasmid was akind gift from Dr. Inouye (16). This plasmid has the nuclease genecloned immediately after the ompA gene that encodes a signal excretionpeptide and was transformed into Escherichia coli HB101 cells by heatshock. E. coli cells were grown in Luria-Bertani (LB) medium at 37 °C,and the recombinant protein production was induced by 1 mM isopropyl1-thio-�-D-galactopyranoside. The protein was purified by an osmoticshock procedure adapted from Takahara et al. (16) and Neu and Heppel(17) and briefly outlined here. Cells were washed three times withTris-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 suspensionwas incubated for 10 min on ice with stirring and then centrifuged. Theresultant pellet was vigorously suspended in cold water and stirred for10 min on ice. The periplasmic proteins (supernatant) were recoveredby 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 thecytoplasmic fraction by centrifugation. SDS-PAGE analysis revealedthat the nuclease was fully released upon washing the membranes with1 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 NaCland was judged pure by SDS-PAGE stained with silver nitrate.

The N-terminal sequence was determined to confirm that no residuefrom the signal peptide was incorporated in the produced nuclease. Toimprove the reversibility of the thermal unfolding, nuclease was chem-ically denatured with 2 M guanidinium chloride and refolded by dilutionwith buffer and extensive dialysis. Pure nuclease was lyophilized andkept at �20 °C until use. The production yield was about 3 mg/liter ofculture. Protein concentration was determined from UV absorbance at280 nm, using an extinction coefficient of 0.93 (mg/ml)�1 cm�1.

Differential Scanning Calorimetry—Differential scanning calorime-try (DSC) was performed on a MicroCal VP-DSC MicroCalorimetercontrolled by the VP-viewer program and equipped with 0.51 ml of cells.Calibration of temperature and heat flow was carried out according toMicroCal instructions. Stock solutions of nuclease were prepared bydissolving 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 (10mM, pH 7.5) without or with solute was prepared and dispensed into twoEppendorf tubes, 1 ml each. An aliquot of the concentrated proteinsolution or of phosphate buffer was added to each Eppendorf, and thesesolutions were used to fill up the sample and reference cells, respec-tively. Both solutions were degassed for 8 min under vacuum, prior tothe calorimetric experiments. DSC scans were run at a constant heatingrate of 1 °C/min from 20 to 80 °C when no solute was added and up to90 °C when MG (or glycerol or trehalose) was present. An overpressureof about 30 p.s.i. was applied to the calorimeter cells. Reversibility ofSNase thermal unfolding was assessed by performing two sequentialDSC scans with each protein solution. In all assays, the area under theheat 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 thebasis of five independent runs. Raw calorimetric data were converted toheat capacity by subtracting the buffer baseline, determined underidentical conditions, and dividing by the scan rate and the sampleprotein concentration. The data were analyzed with EXAM software(18). This program uses a sigmoidal baseline to yield a van’t Hoffenthalpy (�HvH), a transition temperature (Tm, the temperature at halfthe peak area), and the calorimetric enthalpy (�Hcal) that can be ob-tained from the ratio between the transition peak area and the numberof moles of protein in the calorimetric cell. The change in heat capacity(�Cp) is calculated from the baseline shift by the software in the data-fitting procedure. The expression used in EXAM to fit the calorimetricdata was,

dQdT

� (1 � �)[Ba � B�a(T � Tm)] � N��H(T)2

RT2 �F(�)

� �[Bb � B�a(T � Tm)] (Eq. 1)

where dQ/dT is the derivative of the heat exchanged with respect totemperature, � is the extent of reaction (F(�) � �(1 � �)), [Ba � B�a(T �Tm)] is the pre-transition baseline, [Bb � B�b(T � Tm)] is the post-transition baseline, �H is the van’t Hoff enthalpy, and N is the numberof protein moles present in the calorimeter cell. Protein concentrationsaround 16 �M were typically used.

Steady-state Fluorescence Spectroscopy—Measurements were car-ried out using a SPEX spectrofluorometer, Fluorolog 212I at 90° geom-etry with excitation and emission slits of 1 mm. Spectra were correctedfor the wavelength dependence of the emission monochromator and thephotomultiplier. Excitation was set to 280 nm for NAWA measure-ments and 292 nm for the SNase measurements to assure that only thetryptophan residue was excited. To remove oxygen NAWA solutionswere left for 1 h under a nitrogen flow. Temperature was controlled witha circulating water bath. Fluorescence quantum yields of NAWA andnuclease were calculated from the integral of the emission bands, usingp-terphenyl in cyclohexane at 20 °C as standard, � � 0.77 (19). Nucle-ase fluorescence emission spectra were performed in phosphate buffer(10 mM, pH 7.5) in the absence and presence of 0.5 M MG at differenttemperatures. Nuclease concentration was adjusted to �16 �M, whichcorresponds to an absorbance of �0.1 at 292 nm.

Time-resolved Fluorescence Spectroscopy—Fluorescence decays weremeasured using a time-correlated single-photon-counting apparatus(20). Vertically polarized excitation light at 292 nm was obtained withthe third harmonic of the output (876 nm) of a mode-locked Ti-sapphirelaser (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) anda monochromator (360 nm) (Jobin-Yvon H20 Vis), and finally detectedwith a microchannel-plate photomultiplier (Hamamatsu R3809u-50).Automated alternate measurements (1 kilocount per cycle) of the exci-tation pulse, and sample emissions were made until 5–30 kilocounts atthe maximum were reached. The fluorescence decays were deconvo-luted from the excitation pulse using G. Striker’s Sand program, whichallows for individual and global analyses of the decays with individualshift optimization (21).

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RESULTS

Differential Scanning Calorimetry—Thermodynamic param-eters associated to SNase unfolding with and without MG weredetermined. Very high reproducibility of baselines was ob-tained both in the absence and presence of MG (Fig. 1). This isan important premise to obtain reliable parameters from DSCdata. In the absence of solute, the van’t Hoff enthalpy obtainedfrom the fitting was 84 � 4 kcal mol�1, and the calorimetricenthalpy was 83 � 5 kcal mol�1, with a Tm of 53.9 � 0.3 °C anda 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 (�HvH/�Hcal � 1.0). The enthalpy values are ingood agreement with those reported in the literature, whichvary between 84.1 and 88.8 kcal mol�1 (22–24). The heat ca-pacity change (�Cp) of SNase during thermal unfolding wasdetermined from the calorimetric curve with the EXAM soft-ware using a sigmoidal baseline. The use of this procedure toextract �Cp is fully reasonable given the high sensitivity andreproducibility of the VP DSC calorimeter (25). The value of�Cp obtained for SNase, in the absence of solute, is within therange of values found in the literature, which vary between 1.7kcal K�1 mol�1 (22) and 2.7 kcal K�1 mol�1 (26).

In the presence of 0.5 M MG, Tm increased by 7.1 °C (to avalue of 61.0 °C) indicating strong stabilization of SNase byMG. 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 (�HvH/�Hcal � 1.07 � 0.08)was not significant. An increase in �Cp of about 2-fold wasobserved (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 and4.2 °C, respectively, a much lesser effect than that provided byMG (Table II).

Absorption and Emission Spectra of SNase and NAWA—Theabsorption spectrum of SNase in phosphate buffer (10 mM, pH7.5) at 20 °C reflects the presence of a single tryptophan andseven tyrosine residues (Fig. 2). As expected, the extinctioncoefficient of SNase at �max � 280 nm was identical to theweighted sum of the extinction coefficients of tyrosine andtryptophan. At wavelengths above 290 nm the absorption ismainly due to the single tryptophan residue. Above the meltingtemperature, a clear decrease of SNase absorption, causedprimarily by the exposure of buried tyrosine residues to theaqueous external solvent, was observed (Fig. 2) (27, 28).

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

Fluorescence quantum yields and maximum emission wave-lengths of SNase, measured as a function of temperature, arepresented in Fig. 3. This figure also shows the temperaturedependence of the quantum yields and emission wavelengths ofthe model compound NAWA in different dioxane-water mix-tures with polarities ranging from dioxane to water. The valuesof the maximum emission wavelength of NAWA increased withsolvent polarity and, as expected, were independent of thetemperature, whereas the quantum yield gradually decreasedwith either the increase of the solvent polarity (Table III) ortemperature (Fig. 3).

Below 45 °C, the quantum yield and emission wavelength ofSNase were similar to those of NAWA in a low polarity envi-ronment (�90:10 v/v dioxane-water mixture). However, in the45–65 °C temperature range, the quantum yield of SNase de-creased abruptly while the maximum emission wavelengthincreased; finally, above 65 °C, the quantum yield and emissionwavelength of SNase became similar to the values of NAWA inan environment with polarity close to that of water. Theseobservations clearly indicate that, within the 45–65 °C temper-ature range, SNase unfolds, exposing its tryptophan residue towater, whereas below 45 °C, the tryptophan residue inside thenative SNase probes a relatively low polarity environment.

Fluorescence Decays of SNase and NAWA—Fluorescence de-cays of NAWA in dioxane-water mixtures were measured attemperatures ranging from 20 °C to 80 °C, with excitation at�exc � 292 nm, emission at �em � 360 nm and a time resolutionof 24.3 ps/channel. The decays were strictly single exponen-tials, independent of the number of counts at the maximum (upto 30 kilocounts). Single exponential decay laws of NAWA inwater were also observed in other laboratories (29, 30). Thepresence of 10 mM phosphate buffer or 0.5 M MG did not affectthe NAWA decay in water, but the effect could not be assessedin dioxane or dioxane-water mixtures due to the low solubilityof these compounds.

The fluorescence lifetime of NAWA at room temperaturedecreased from 5.2 ns in dioxane to 3.1 ns in water, following atrend with solvent polarity similar to that of the quantum yield(Table III). For each solvent or mixture, increasing tempera-ture induced the expected decrease of the fluorescence lifetime(Fig. 4A). The radiative (kf) and the sum of the radiationless

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

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(knr) decay rates of NAWA in dioxane-water mixtures werecalculated from quantum yields (�f) and fluorescence lifetimes(�f) by kf � �f/�f and knr � (1 � �f)/�f (Table III). The radiativerate constant (kf) shows the expected proportionality to thesquared refractive index of the mixture, decreasing from diox-ane to water, while knr increases with solvent polarity. Thecombined effect of the two variations induces the observeddecrease of both fluorescence quantum yield and lifetime fromdioxane to water. A similar increase of knr has been observedfor other compounds in dioxane-water mixtures and has beenattributed to the increase of the internal conversion rate con-stant upon increasing water content (31–35).

The values of the radiative and radiationless rate constantsof SNase at 23 °C were similar to those of NAWA in dioxane(Table III), thus indicating a low polarity environment of thetryptophan residue in the native protein. At 70 °C, the quan-tum yield and the major lifetime (�2) of SNase were similar tothose of NAWA in water at the same temperature, reflectingthe high degree of exposure of the tryptophan residue to waterin the unfolded protein. The quantum yield and emission wave-length profiles led to a similar conclusion.

The nature of the fluorescence decay of SNase in water at23 °C, measured with a time resolution of 24.3 ps/channel, wasnot so simple. Typically, the decays could be fitted only withdouble-exponential functions, although the long-lived compo-nent (�5.5 ns) accounted for more than 95–99% of the totalfluorescence emission. The minor component had a poorly de-

fined lifetime ranging from 1 to 3 ns, whose weight varied withthe protein preparation, but in one of the preparations it wasundoubtedly absent (Fig. 5A). The observation of this single-exponential function means that the decay of the tryptophanfluorescence in the native form of SNase is genuinely single-exponential, and, therefore, the residual short time appearingin most SNase samples could arise from a small amount ofdenatured protein.

Below 45 °C, the lifetime of the major component in thefluorescence decay of SNase (�1) had values similar to those ofNAWA in a 90:10 v/v dioxane-water mixture, hence it wasassigned to the decay of tryptophan buried in the native form ofthe protein (Fig. 4A). Above 45 °C, three exponential functionswere required to fit the decays of SNase (Fig. 5B). The thirddecay time (�3) was significantly shorter than the lifetime ofNAWA in water (Fig. 4A); moreover, the respective amplitude(a3) was always lower than that associated to the intermediatedecay time (a2) (Fig. 4B). The longer decay time, �1, was stillclose to the lifetime of NAWA in a low polarity environment(Fig. 4A). However, its amplitude (a1) decreased sharply withthe temperature to values close to zero, whereas the ampli-tudes 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 oftemperature examined is characterized by three times, one (�1)corresponding to the emission of tryptophan in the folded pro-

TABLE IThermodynamic parameters of SNase unfolding in 10 mM phosphate buffer, pH 7.5, with and without

mannosylglycerate determined from DSC and TRFS�H values in kcal mol�1, �S in cal K�1 mol�1, and �Cp values in kcal K�1 mol�1.

Tm �HcalTm �HvH

Tm �SvHTm �Cp

°C

DSC No solute 53.9 � 0.3 83 � 5 84 � 4 257 � 12 1.7 � 0.50.5 M MG 61.0 � 1.6 88 � 6 95 � 3 284 � 9 4 � 1

TRFS No solute 53.0 � 0.1 77 � 3 236 � 9 2 � 10.5 M MG 60.8 � 0.2 76 � 4 228 � 12 4 � 2

TABLE IIEffect 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 independentmeasurements.

Solute Mannosylglycerate Glycerol Trehalose

Concentration (M) 0 0.1 0.25 0.5 1.0 0.5 0.5Tm (°C) 53.9 � 0.3 54.9a 58.2a 61.0 � 1.6 67.4a 54.7a 58.1a

a Single experiments were carried out; the standard deviation of this type of measurements is lower than 2 °C.

FIG. 2. Absorption and emissionspectra of SNase in 10 mM phosphatebuffer, pH 7.5, at 20 °C (solid lines)and at 80 °C (dashed lines). Fluores-cence spectra of NAWA in dioxane at20 °C (full circles) and in water at 80 °C(open circles). Fluorescence spectra at80 °C are multiplied by a factor of 10.

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tein, and the other two times (�2 and �3) originating fromexposed tryptophan residues in unfolded forms of the protein.The temperature dependence of the amplitudes reflects thetransition from the folded to the unfolded state.

Effect of MG on the Fluorescence of SNase—The effect of MGon the photophysical properties of SNase was also studied by

steady-state and time-resolved fluorescence spectroscopy. Thefluorescence quantum yield showed identical values with andwithout MG at temperatures far from the unfolding transition;also, the same 20-nm red shift was observed for the variation ofthe maximum emission wavelength upon unfolding. However,the temperature range where these quantities change abruptly

FIG. 3. Temperature dependence offluorescence quantum yield (A) orwavelength of maximum emission (B)of NAWA in different dioxane-watermixtures (open triangles) and ofSNase in 10 mM phosphate bufferwith mannosylglycerate (open cir-cles) or without (full circles). The fol-lowing dioxane-water proportions wereused: 100:0, 80:20, 60:40, 40:60, 20:80,and 0:100 (v/v).

TABLE IIIFluorescence maximum wavelength (�em), quantum yield (�f) and lifetime (�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 (� ) and refraction index (nD) of themixtures are also shown.

� nD2 �em �f �f kf knr

nm ns 107 s�1

NAWA water (% v/v)0 2.2 2.021 326 0.27 5.2 5.0 14.220 10.9 2.011 333 0.26 5.1 4.8 14.540 27.8 1.995 338 0.24 4.8 4.7 16.060 45.0 1.966 341 0.20 4.1 4.6 19.580 63.0 1.889 345 0.17 3.6 4.3 23.4100 78.4 1.777 354 0.13 3.1 3.8 28.5

SNaseNo solute 329 0.27 5.6 4.8 13.0MG 329 0.27 5.7 4.7 12.8

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was clearly shifted toward higher temperatures upon MG ad-dition (Fig. 3). From the quantum yield results, the SNasemelting temperature increased by �7 °C. In the temperaturerange where unfolding occurs, the emission spectrum of SNaseis the sum of the spectra from the native and unfolded proteinconformations weighted by the respective molar fractions. Be-cause the quantum yield of the unfolded protein is much lowerthan that of the folded protein, a shift of the maximum emis-sion wavelength is not detectable unless the population of theunfolded form is predominant. Despite this lack of sensitivity,it is interesting to point out that the curves representing theshift of the wavelength maximum in the presence and absenceof MG were also separated by �7 °C (Fig. 3B).

The temperature dependence of the lifetimes and amplitudesof the SNase fluorescence decays, in the presence of MG, areshown in Fig. 6. The evolution of the lifetimes was similar tothat observed in the absence of solute, except for the highertemperature of the unfolding transition.

Determination of Unfolding Equilibrium Constants byTRFS—The unfolding equilibrium constant for a two-statemechanism is defined as the ratio of concentrations of theunfolded, [U], and native, [N], proteins at equilibrium. The fluores-

cence decay functions are IN (t) � a1e�t/�1 and IU(t) � a2e�t/�2 � a3e�t/�3

for the native and unfolded protein conformations, respectively.Under the assumption that the protein folding and unfolding ratesare low with respect to the reciprocal fluorescence lifetimes of theburied and exposed tryptophan, at any temperature, the pre-expo-nential coefficient of each exponential term is proportional to theconcentration, at t � 0, of the excited species to which that expo-nential term is assigned ([N*](0) or [U*](0)), multiplied by theradiative rate constant of each species (kfN or kfU). It is also neces-sary to account for the fraction of light that is detected at theexperimental emission wavelength �em under steady-state condi-tions, fi(�em), with respect to the total fluorescence emission,�I(�)��, of each species. These fractions are defined, for the native,fN(�em), and unfolded protein, fU(�em), as in Equations 2 and 3,

fN(�cm) �IN(�cm)

�IN(�)��(Eq. 2)

fU(�cm) �IU(�cm)

�IU(�)��(Eq. 3)

where IN(�em) and IU(�em) are the fluorescence intensities ofthe native and unfolded protein at �em (360 nm). The mathe-

FIG. 4. A, fluorescence lifetime as afunction of temperature of NAWA in dif-ferent dioxane-water mixtures (open tri-angles) and of SNase in 10 mM phosphatebuffer, pH 7.5 (full symbols). Full circles,native SNase (�1); triangles, denaturedSNase (�2); and squares, denatured SNase(�3). The following dioxane-water propor-tions 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 fluores-cence decays for SNase in 10 mM phos-phate buffer, pH 7.5. Circles, nativeSNase (a1); triangles and squares, dena-tured forms of SNase (a2 and a3). At lowtemperatures (below 40 °C) �2 is poorlydefined and not present in all samples.

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matical expression for the fluorescence decay of native, IN(t),and denatured, IU(t), protein conformations are given by Equa-tions 4 and 5,

IN(t) � fN(�cm)�kfN�[N*](t) � fN(�cm)�kfN�[N*](0)�e�t/�1 (Eq. 4)

IU(t) � fU(�cm)�kfU�[U*](t) � fU(�cm)�kfU�[U*](0)�(c2e�t/�2 � c3e�t/�3) (Eq. 5)

where c2 and c3 define the relative weight of the two exponen-tial terms of the tryptophan fluorescence decay in the unfoldedprotein. Therefore, the amplitudes, ai, obtained from the fluo-rescence intensity decay fits are, apart from an instrumentalconstant, given by ai(�em) � fi(�em)�kfi�[i*](0). For the determi-nation of [N*](0) and [D*](0), the amplitudes must be dividedby fi(�em)�kfi.

Taking NAWA in dioxane-water mixtures as an appropriatemodel 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 4.9 107 s�1 and 3.8 107 s�1. Thefraction of light emitted by NAWA at 360 nm in a 90:10 v/vdioxane-water mixture is �70% lower than in water due to thelarge bathochromic shift and broadening of the emission spec-trum from dioxane to water (Fig. 2). Accordingly, the amplitudeof the low polarity component a1 was divided by 0.7 4.9 107

s�1, and the amplitudes, a2 and a3, were divided by 3.8 107

s�1. The corrected values were then normalized according tothe condition a1 � a2 � a3 � 1 to obtain the molar fractions att � 0 of the folded and unfolded forms in the excited state,respectively.

The concentration at t � 0 of each excited species is in turnproportional to the ground-state concentration of that species

FIG. 5. A, single exponential fit of fluo-rescence intensity decay of SNase in 10mM phosphate buffer at 23 °C. B, triple-exponential fit of SNase fluorescence de-cay at 54 °C. Decay times (�), normalizedpre-exponential coefficients (a) and fittingparameters (2) are also shown. AC, auto-correlation function. WR, weighted resi-due function.

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multiplied by its molar extinction coefficient at the excitationwavelength. Because there was no appreciable difference in themolar extinction coefficients of NAWA in dioxane and water at292 nm, the corrected and normalized amplitude a1 is equal tothe ground-state molar fraction of the folded SNase and thesum a2 � a3 is equal to the ground-state molar fraction of theunfolded SNase (Fig. 7).

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 tem-peratures determined by DSC (Table I).

Thermodynamic Analysis of TRFS Data—The equilibriumconstants of unfolding, calculated from the molar fractions ofthe folded and unfolded SNase at each temperature, can beused to calculate the temperature dependence of the Gibbs freeenergy, �G � �RT ln KU. The Gibbs energy function can alsobe described by the modified Gibbs-Helmholtz equation(Equation 6).

�G � �Hm�1 �T

Tm� � �Cp�(T � Tm) � T ln

TTm� (Eq. 6)

Equilibrium data can then be fitted to a non-linear function,resulting from the combination of these two equations (Equa-tion 7), to derive the values of Tm, �Hm, and �Cp associatedto SNase unfolding in Equation 7.

R ln KU � ��Hm

Tm� �Cp(1 � ln Tm)� � (�CpTm � �Hm) �

1T

� �Cp ln T

(Eq. 7)

The fit of this function to the experimental points in thetransition region leads to �Cp values with considerable uncer-tainty: �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, respec-

FIG. 7. Molar fractions of folded (circles) and unfolded(squares) forms of SNase in 10 mM phosphate buffer, pH 7.5, with0.5 M mannosylglycerate (open symbols) or without (fullsymbols).

FIG. 6. A, temperature dependence offluorescence decay times of NAWA in dif-ferent dioxane-water mixtures (open tri-angles) and of SNase in 10 mM phosphatebuffer, pH 7.5, containing 0.5 M manno-sylglycerate (full symbols). Full circles,native SNase (�1); triangles, denaturedSNase (�2); and squares, denatured SNase(�3). The following dioxane-water propor-tions 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-expo-nential coefficients of fluorescence decaysof SNase in 10 mM phosphate buffer, pH7.5, with 0.5 M mannosylglycerate. Cir-cles, native form of SNase (a1); trianglesand squares, denatured forms of SNase(a2 and a3). At low temperatures �2 ispoorly defined and not present in allsamples.

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tively. Nevertheless, these values agree well with those ob-tained from the DSC experiments, 1.7 � 0.5 and 4 � 1 kcal K�1

mol�1. The temperature dependences of KU, with and withoutsolute, are shown in the van’t Hoff plots of Fig. 8. The van’t Hoffenthalpy, the melting temperature, and the heat capacitychange values associated to SNase unfolding with and withoutMG are given in Table I together with the DSC results. In theabsence of solute, the enthalpy values obtained by TRFS areonly slightly lower than those determined by DSC. In thepresence of MG, the TRFS enthalpy value is significantly lowerthan that obtained by DSC. A full explanation for this obser-vation cannot be advanced at this point, but the discrepancy isperhaps not so important if one takes into consideration thatTRFS only monitors the tryptophan-surrounding area,whereas DSC probes the unfolding of the whole protein andalso senses possible contributions from the solvent.

Protein-Solute Interaction: Contribution of Preferential Ex-clusion—We used the thermodynamic theory of preferentialexclusion and analyzed our data of SNase stabilization by MG,following the formalism developed by Plaza-del-Pino andSanchez-Ruiz (36). This analysis determines the preferentialinteraction parameter change (�23) that can be used to cal-culate the denaturational change in the protein preferentialhydration (��21). The thermodynamic equations to determinethese quantities, at Tm, are,

�23 �Hm

Tm��Tm

�m3�

pH

(Eq. 8)

��21 �23

V� 1(��/�m3)(Eq. 9)

where m3 is the molal concentration of solute, V� 1 is the molarvolume of water, and � is the osmotic pressure of the water-solute mixture. The subscripts 1, 2, and 3 refer to water, pro-tein, and solute (MG), respectively. Density measurementswere performed to determine the solute molal concentration,and the effect of MG concentration on the Tm of SNase wasstudied up to 1 M MG (Table II). The polynomial equation thatdescribes this variation is Tm � �(6.8 � 0.4) m3

2 � (19.2 � 0.6)m3 � (327.3 � 0.1). Using Equation 8, the calculated value of�23 in the presence of 0.5 M MG was 2.9 kcal kgwater/molprot

molMG. Nothing is known about the osmotic pressure of thewater-MG solutions, and, therefore, an estimation of this pa-rameter must be made to determine the change in the proteinpreferential hydration. In an ideal solution, the variation of theosmotic pressure with the solute concentration can be calcu-

lated from ��/�m3 � RT/(V� 1(55.56 � m3)), which, for a MG con-centration of 0.5 M, results in a value of 657.4 kcal kgwater/m

3

molMG. Thus, the denaturational change in the protein prefer-ential hydration is ��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 Ti-masheff (4), superosmolytes induce an increase of the osmoticpressure at a higher rate than that observed in an ideal solu-tion. Using the value of 843.3 kcal kgwater/m

3 molMG proposedfor the osmotic pressure increment of superosmolytes (37), thevalue obtained for the protein preferential hydration parame-ter is ��21 � 191 molwater/molprot � 0.2 gwater/gprot. Thus,superosmolytes provide the same degree of protein stabiliza-tion with lower levels of solute exclusion.

DISCUSSION

Characterization of SNase Unfolding by Time-resolved Fluo-rescence Spectroscopy—To evaluate the populations of the dif-ferent protein forms, we need to know the fluorescence behav-ior of the tryptophan residue in the native and denaturedprotein conformations (see “Results”). This information wasderived from the study of NAWA in dioxane-water mixtures bytime-resolved and steady-state fluorescence spectroscopy.These solvent mixtures were used to represent the range ofenvironment polarity sensed by tryptophan when the proteinundergoes unfolding. Dioxane-water mixtures have been suc-cessfully used to mimic the microenvironment sensed by fluo-rescent probes in heterogeneous media, such as micelles (34)and micro-emulsions (35).

The good correlation observed between the emission proper-ties of NAWA in dioxane-water mixtures and those of SNaseduring unfolding (Figs. 3 and 4) suggests that this is an ade-quate model system to represent the environment surroundingtryptophan in the protein structure, and hence, monitor struc-tural changes. However, the fluorescence of NAWA decays as asingle exponential, whereas SNase shows two or three lifetimesdepending on the temperature range examined. Two lifetimesare required to fit the SNase data at temperatures well belowthe Tm. Many fluorescence studies with SNase can be found inthe literature (24, 26, 38–41): at room temperature and neutralpH, the emission decay is characterized by a long-lived compo-nent (5.4–6.0 ns) and one or two additional components withshort lifetimes (ranging from 1.0 to 3.3 ns). Generally, theseshort components are poorly defined and have variable ampli-tudes (5–30%). Multiple-exponential decays have also been re-ported for many other single-tryptophan proteins, but the in-terpretation of this observation in molecular terms is elusive.We performed a detailed analysis of the temperature depend-ence of the SNase fluorescence kinetics, resorting to data on theeffect of solvent polarity on the lifetime of NAWA to ascribe thelong (�1) and short (�2) lifetimes to the native and denaturedforms, respectively. This assignment was corroborated by thetemperature dependence of the amplitudes of the two compo-nents and the comparison of the lifetimes of the protein withthose of NAWA in dioxane-water mixtures (Fig. 4).

Above Tm, SNase decays require an additional time (�3),which can arise from: (i) existence of different unfolded confor-mations of the protein in which tryptophan fluorescence isquenched by neighboring residues like histidine, lysine, or ty-rosine (42) or (ii) intramolecular electron transfer from theindole moiety in tryptophan to the peptide chain due to slowinterconversion of the different tryptophan rotamers (43) thatseems to occur with tyrosine residues in unfolded ubiquitin.2 Atthis stage, we are unable to provide a definite physical meaning

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

FIG. 8. Van’t Hoff plots of SNase unfolding in 10 mM phosphatebuffer, pH 7.5, containing 0.5 M mannosylglycerate (open circles)and in the absence of mannosylglycerate (full circles). Solid linesare the best fits taking into account �Cp (see text, Equation 7); dashedlines represent the fits disregarding �Cp.

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for �3, but this fact does not hamper an accurate analysis of theunfolding process.

Effect of MG on the Population of SNase Forms along Un-folding—We took advantage of the discriminating properties ofTRFS to characterize the effect of MG on the unfolding path-way of SNase. The presence of MG did not alter the pattern ofprotein lifetimes nor the proportions of the species observed,after accounting for the shift in Tm. Therefore, despite theconsiderable degree of stabilization rendered by MG, we con-clude that the unfolding pathway of SNase was essentiallyunaffected by this solute. In other words, the molecular mech-anism for stabilization of SNase by mannosylglycerate is likelyto involve subtle changes with no detectable impact in thepopulation of protein states along denaturation.

Thermodynamic Basis for SNase Stabilization by Mannosyl-glycerate—Thermodynamic parameters of SNase unfoldingwere calculated from TRFS data and by DSC, a direct methodthat monitors the whole protein structure. The combination ofthese two techniques to study protein thermal unfolding al-lowed for a more thorough characterization of this process anda greater confidence on the data and conclusions produced.

The most notable changes induced by the presence of MG arethe increases in Tm and the unfolding heat capacity of SNase.The temperature dependence of �G (according to Equation 6),considering that �Cp is temperature-independent, is shown inFig. 9. The presence of MG led to a shift of the stability curve(�G versus T), as expected for a stabilizing osmolyte. The sta-bility curve should be regarded with caution, because the as-sumption that �Cp remains constant may not hold over a largetemperature range; yet, it is unlikely that the magnitude of thehypothetical change in �Cp is large enough to affect consider-ably the general trend. The entropic or enthalpic contributionsto the Gibbs energy of stabilization provided by mannosylglyc-erate were evaluated from the temperature dependence of�H � �Hm � �Cp(T � Tm) and �S � �Sm � �Cp ln(T/Tm) (Fig.10). Strong enthalpy-entropy compensation was observed, acommon feature in phenomena involving the exposure of hy-drophobic residues to the solvent.

Interestingly, the relative magnitudes of �H and �S, in thepresence and absence of solute, depend on the temperatureconsidered, because the respective lines cross at around 65 °C.Therefore, the major contribution for the Gibbs energy ofstabilization depends on the specific temperature range: (i)

between 40 and 65 °C, both the unfolding enthalpy and en-tropy decreased in the presence of MG, but the decrease inthe unfolding entropy was more pronounced, and therefore,�GMG �GNS; (ii) above 65 °C, the unfolding enthalpy andentropy increased in the presence of MG, but the increase inenthalpy outweighed that of entropy, hence the Gibbs energy ofstabilization was positive. In summary, at temperatures be-tween 40 and 65 °C the thermodynamic parameter responsiblefor stabilization is the entropy, but at higher temperatures thestabilization is enthalpic.

A 2-fold increase of the SNase unfolding heat capacity wasobserved in the presence of MG. The change in the unfoldingheat capacity has been correlated with the variation of polarand apolar accessible surface area upon denaturation (�ASApol

and �ASAap) and on their contributions for �Cp per unitsurface area (�Cp

pol and �Cpap) by the equation �Cp �

�Cppol�ASApol � �Cp

ap�ASAap. The polar and apolar contribu-tions have different signs (�Cp

pol � 0 and �Cpap 0) and may

change with the solute concentration. Therefore, the rational-ization of the variation of �Cp values in the presence of solutesis not straightforward. The stabilization of ribonuclease A bysarcosine was accompanied with an increase of �Cp (36),whereas a decrease was observed for the unfolding of the sameprotein in the presence of sucrose (44) or sarcosine (45). Tre-halose also caused a decrease of �Cp of five proteins (46). Otherexamples exist where �Cp values remain unchanged in thepresence of stabilizing solutes (47). Hence, protein stabilizationcannot be inferred from the variation that solutes induce on�Cp values. In a comparative study with proteins isolated fromthermophiles and mesophiles, Zhou (48) observed that proteinsfrom thermophiles have lower �Cp than their mesophilic coun-terparts, a fact also pointed out by other authors (49, 50). Thisobservation could partly be attributed to stronger polar inter-actions in the native form (48) or to a higher propensity forresidual structure and hydrophobic cluster formation in thedenatured state of thermophilic proteins (50). The large in-crease in �Cp of SNase in the presence of MG could result fromdecreased polar interactions in the folded state as the positivecharge of surface ionized groups is canceled by electrostaticinteraction with MG. Also, the reduced electrostatic repulsionmay lead to a more compact folded state and consequently tostabilization. In fact, a recent NMR study revealed an increase

FIG. 9. Unfolding Gibbs energy of SNase as a function of tem-perature calculated using the thermodynamic parameters de-rived from DSC measurements (Table I). Thick line, �G in thepresence of 0.5 M mannosylglycerate; thin line, �G in the absence ofmannosylglycerate.

FIG. 10. Temperature dependence of �H and T�S associated toSNase unfolding calculated using the thermodynamic parame-ters derived from DSC measurements (Table I). Thick solid line,�H in the presence of MG; thick dashed line, T�S in the presence of MG;thin solid line, �H in the absence of MG; and thin dashed line, T�S inthe absence of MG.

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in the overall rigidity of rubredoxin induced by the presence ofdiglycerol phosphate, another negatively charged solute fromhyperthermophiles (51).

Role of Preferential Hydration and Binding in the Stabiliza-tion of SNase by Mannosylglycerate—The phenomena of pro-tein stabilization by solutes derive from differential interac-tions with the unfolded and folded forms of the protein, whichcould be attained either by preferential exclusion or binding.

The analysis of our thermodynamic data in the framework ofthe solute preferential exclusion theory revealed that exclusiondefinitely plays a role in the mechanism of stabilization by MGas indicated by the change of 0.3 gwater/gprot in the proteinpreferential hydration upon unfolding. In conclusion, the in-crease of protein stability in the presence of MG can be inter-preted as resulting, to some extent, from destabilization of thedenatured state caused by preferential exclusion of MG uponunfolding. Nevertheless, the contribution of MG binding toprotein stabilization is likely to be relevant in the case of thisanionic solute. The high degree of stabilization exerted even atmoderate solute concentrations when compared with common,neutral osmolytes supports this hypothesis. For example, theTm of SNase increased more than 7 °C in the presence of 0.5 M

mannosylglycerate, whereas the same concentration of glycerolor trehalose induced much lower stabilization. A strong stabi-lization at low or moderate concentration is typical of situa-tions that involve specific interactions, namely electrostatic (6,45, 52, 53). Moreover, other authors have pointed out the needto invoke mechanisms other than increased surface tension toexplain the stabilization rendered by carboxylic acid salts (54).The increase in the unfolding Gibbs energy in the presence ofsolutes results from an increase in the cavity formation energy(which is proportional to the surface tension) and also from theenergy of the protein-solute interaction. If the increase in thesurface tension were the sole factor responsible for stabiliza-tion, a linear correlation would be expected between the in-crease in the surface tension and the protein stabilization.Kaushik and Bhat (54) interpreted the non-linear correlationbetween the increase in water surface tension and the incre-ment in Tm observed for carboxylic acid salts as evidence for asignificant contribution of protein-solute interactions to proteinstabilization.

At neutral pH, SNase has a high positive charge, therefore,a favorable interaction of fully ionized MG with the protein isexpected. This interaction will reduce electrostatic repulsions,thereby stabilizing preferentially the native state, because thecharges are closer, on average, than in the more extendeddenatured state. Electrostatic effects have also been invoked toexplain the stabilization of RNase A rendered by MG only atpH values above the pKa of the solute (7). Also, recent studieson the stabilizing effect of mannosylglycerate and diglycerolphosphate in single residue mutants of rubredoxin showed thatthe effect on protein stability is strongly dependent on thespecific protein/compatible solute system examined.3

Physiological Significance of Charged Solutes in Hyperther-mophiles—Most of the compatible solutes accumulated by ther-mophiles and hyperthermophiles are negatively charged inopposition to mesophiles that accumulate neutral or zwitteri-onic compounds such as trehalose, glycerol, or proline. Thisobservation raised the hypothesis that charge could play animportant role in the protection of cells and their componentsagainst heat damage (3). The superior efficacy of negativelycharged solutes (phosphodiester compounds or carboxylic ac-ids) 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 haveadapted their cellular components through evolution to be ableto benefit from the most effective protectors against heat avail-able. The use of charged solutes as cell stabilizers implies theintracellular accumulation of positive counterions such as po-tassium; high salt concentrations are known to be toxic formost mesophilic bacteria, hence, these organisms are unable totake advantage of the accumulation of charged solutes to alarge extent. However, (hyper)thermophiles have acquired thisattribute, which seems essential to endow them with the levelof extrinsic stabilization needed for thriving in hotenvironments.

Acknowledgments—We are grateful to Prof. M. Inouye, State Uni-versity of New York, for the generous supply of pONF1. M. B. thanksDr. Frederick P. Schwarz, from the National Institute of Standards andTechnology/Center for Advanced Research in Biotechnology, USA, forproviding the EXAM program.

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