A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

A Comparative Infrared Spectroscopic Studyof Glycoside Hydrolases From ExtremophilicArchaea Revealed Different Molecular Mechanismsof Adaptation to High Temperatures

Alessio Ausili,1 Beatrice Cobucci-Ponzano,2 Barbara Di Lauro,2 Rossana D’Avino,2 Giuseppe Perugino,2

Enrico Bertoli,1 Andrea Scire,1 Mose Rossi,2,3 Fabio Tanfani,1* and Marco Moracci2

1Institute of Biochemistry, Universita Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy2Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131 Naples, Italy3Department of Biological Chemistry, University of Naples ‘‘Federico II’’, Via Mezzocannone 16, 80134, Naples, Italy

ABSTRACT The identification of the determi-nants of protein thermal stabilization is often pur-sued by comparing enzymes from hyperthermo-philes with their mesophilic counterparts whiledirect structural comparisons among proteins andenzymes from hyperthermophiles are ratheruncommon. Here, oligomeric b-glycosidases fromthe hyperthermophilic archaea Sulfolobus solfa-taricus (Ssb-gly), Thermosphaera aggregans (Tab-gly), and Pyrococcus furiosus (Pfb-gly), have beencompared. Studies of FTIR spectroscopy andkinetics of thermal inactivation showed that thethree enzymes had similar secondary structurecomposition, but Ssb-gly and Tab-gly (tempera-tures of melting 98.1 and 98.48C, respectively) wereless stable than Pfb-gly, which maintained its sec-ondary structure even at 99.58C. The thermaldenaturation of Pfb-gly, followed in the presenceof SDS, suggested that this enzyme is stabilized byhydrophobic interactions. A detailed inspection ofthe 3D-structures of these enzymes supported theexperimental results: Ssb-gly and Tab-gly are sta-bilized by a combination of ion-pairs networksand intrasubunit S-S bridges while the increasedstability of Pfb-gly resides in a more compact pro-tein core. The different strategies of protein stabi-lization give experimental support to recent theo-ries on thermophilic adaptation and suggest thatdifferent stabilization strategies could have beenadopted among archaea. Proteins 2007;67:991–1001. VVC 2007 Wiley-Liss, Inc.

Key words: b-glycosidase; Sulfolobus solfataricus;Thermosphaera aggregans; Pyrococcusfuriosus; protein structure; infrared

INTRODUCTION

The molecular mechanisms of protein stabilization arean interesting issue that have intrigued biochemists andbiophysics for the past 20 years. The reason for theseefforts is both for basic and applied research since thelack in the thermostability of proteins is one of the main

factors limiting their industrial application. The identifi-cation of the determinants of thermal stabilization hasbeen pursued by different methods; among the many, thecomparison of enzymes from hyperthermophiles (thrivingat temperatures above 808C) with their mesophilic coun-terparts at structural, biochemical, and, more recently,genomic level1–4 has led to the identification of a varietyof stabilizing factors, including increased numbers of ionpairs, reduction in size of loops and cavities, reduced ra-tio of surface area to volume, additional proline residuesin turns, increased hydrophobic interactions, and higherdegree of oligomerization.5,6 The comparison with themesophiles applied to enzymes belonging to the glycosidehydrolase family 1 (GH1) (http://afmb.cnrs-mrs.fr/CAZY/),allowed to conclude that an increased number of ion-pairscould stabilize the enzymes from hyperthermophiles7,8

while the b-glycosidase from the moderate thermophileThermus neoproteolyticus HG102 revealed a-helix stabili-zation, restricted loop flexibility and increase in the num-ber of ion-pairs and in Pro and Arg residues.9

Therefore, though the elements stabilizing the en-zymes from hyperthermophiles have been studied indetail at molecular level, simple trends and mechanismsof stabilization common to enzymes from hyperthermo-

Abbreviations: Amide I0, amide I band in a 2H2O medium; FTIR,Fourier transform infrared; Pfb-gly, recombinant Pyrococcus furio-sus b-glycosidase; SDS, sodium dodecyl sulphate; Ssb-gly, recombi-nant Sulfolobus solfataricus b-glycosidase; Tab-gly, recombinantThermosphaera aggregans b-glycosidase; Tm, temperature of melt-ing; Topt, temperature of optimal growth.

The Supplementary Material referred to in this article can befound at http://www.interscience.wiley.com/jpages/0887-3585/supp-mat/

Grant sponsor: Ministero dell’Istruzione, dell’Universita e dellaRicerca Scientifica (MIUR) [Progetti di ricerca di interesse nazionale(PRIN 2003)]; Grant sponsor: MIUR project ‘‘Folding di proteine:l’altra meta del codice genetico’’; Grant number: RBAU015B47.

*Correspondence to: Prof. Fabio Tanfani, Institute of Biochemis-try, Faculty of Sciences, Universita Politecnica delle Marche, ViaRanieri, 60131 Ancona, Italy. E-mail: [email protected]

Received 19 September 2006; Revised 1 December 2006; Accepted12 December 2006

Published online 13 March 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21368

VVC 2007 WILEY-LISS, INC.

PROTEINS: Structure, Function, and Bioinformatics 67:991–1001 (2007)

philes has not been unequivocally determined, suggest-ing that these organisms followed different routes to sta-bilize their proteins10 leaving the problem of the stabili-zation of hyperstable enzymes still open. Furthermore,the number of studies dealing with detailed enzymaticcomparisons among hyperthermophilic catalysts isremarkably low11 and they are focused on evolutionaryaspects.4 Therefore, the question of how different hyper-stable enzymes cope with temperatures near the boilingpoint of water still needs clarification.The aim of our study was to compare at structural and

functional level three archaeal enzymes belonging tofamily GH1 for which the three-dimensional (3D) struc-ture is available, namely the b-glycosidases from thehyperthermophiles Sulfolobus solfataricus (Ssb-gly),Thermosphaera aggregans (Tab-gly), and Pyrococcus fur-iosus (Pfb-gly). These proteins show high sequence simi-larity (sequence identity scores range between 52 and60%), have the same tetrameric functional unit, a con-served catalytic dyad12 and, since they belong to thesame domain of living organisms, they represent anideal model system to investigate the molecular basis ofprotein stability without evolutionary bias. The experi-ments of FTIR spectroscopy and the kinetics of thermalinactivation reported here revealed that Pfb-gly is morestable than Ssb-gly and Tab-gly under all the conditionstested and that it is sensitive to incubations in the pres-ence of SDS. These data, and the detailed inspection ofthe 3D-structures of these enzymes, allowed us to driveconclusions on their mechanisms of stabilization.

MATERIALS AND METHODS

Deuterium oxide (99.9% 2H2O) was purchased fromAldrich. All other reagents and solvents were commer-cial samples of the highest purity.

Preparation of Samples forInfrared Measurements

The enzymes were expressed in recombinant form andpurified as reported previously.12,13 In particular, thecharacteristics of Ssb-gly and Pfb-gly are similar tothose of the native enzymes,14,15 indicating that they canbe used as reliable model systems for the study of pro-tein stability. Typically, about 1.5 mg of Ssb-gly or Tab-gly, or Pfb-gly, dissolved in the buffer used for their puri-fication, were centrifuged in a ‘‘30K Centricon’’ microconcentrator (Amicon) at 3000 3 g and 48C, and concen-trated to a volume of approximately 40 lL. Then, 300 lLof 50 mM phosphate buffer (prepared in 2H2O), p2H 6.5,were added and the sample concentrated again; the p2Hvalue corresponds to the pH-meter reading þ0.4.16 Thisprocedure was repeated several times in order to com-pletely replace the original buffer with the 50 mM phos-phate buffer p2H 6.5. In the last washing the proteinsample was concentrated to a final volume of approxi-mately 35 lL and used for the infrared measurements.Pfb-gly showed the tendency to aggregate at high con-

centration. Indeed, during the preparation of the Pfb-gly

sample, i.e. during the concentration and dilution proce-dure, the protein underwent partial precipitation. We ana-lyzed both the precipitate and the protein that remained insolution. However, the addition of SDS prior to the concen-tration process subsequently avoided the precipitation ofthe protein. Hence, two samples were prepared in the pres-ence of different amounts of SDS. In particular, the deter-gent was added once to 1.6 mL of protein solution to a finalconcentration of 0.2% sample (A) or 0.5% sample (B). Thesamples were concentrated to about 40 lL and then dilutedby adding 300 lL of 50 mM phosphate buffer p2H 6.5. Theconcentration and dilution procedure was then repeatedseveral times as described above. Because of the formationof micelles, the majority of SDS remained in the micro con-centrator during the concentration process. However, partof the detergent probably passed through the pores of thefilter. Hence, the SDS concentration in the final concen-trated protein samples was checked using a calibrationcurve obtained by monitoring the intensity of the SDSsymmetric methylene stretching vibration band (2854cm�1)17 as a function of SDS concentration. The SDSresulted 1.2 and 5% in sample (A) and (B), respectively.

Infrared Spectra and Kinetics ofThermal Inactivation

The concentrated protein samples were placed in athermostated Graseby Specac 20500 cell (Graseby-Specac,Orpington, Kent, UK) fitted with CaF2 windows and a25 lm Teflon spacer. FTIR spectra were recorded by meansof a Perkin-Elmer 1760-x Fourier transform infrared spec-trometer using a deuterated triglycine sulphate detectorand a normal Beer-Norton apodization function. At least24 h earlier and during data acquisition the spectrometerwas continuously purged with dry air at a dew point of�708C. Spectra of buffers and samples were acquired at2 cm�1 resolution under the same scanning and tempera-ture conditions. In the thermal denaturation experiments,the temperature was raised in 58C steps from 20 to 958C.The actual temperature in the cell was controlled by athermocouple placed directly onto the CaF2 window. Spec-tra were collected and processed using the ‘‘Spectrum’’software from Perkin Elmer. The deconvoluted paramet-ers for the amide I band were set with a gamma value of2.5 and a smoothing length of 60. Second derivative spec-tra were calculated over a 9-data point range (9 cm�1).

The temperature of melting (Tm) in thermal denatura-tion curves was calculated as described.18

Kinetics of thermal inactivation were performed asreported previously by using 0.1 mg/mL of each enzymeat the indicated temperatures.19 Briefly, samples wereincubated in sodium phosphate buffer 50 mM pH 6.5 insealed tubes and taken at proper times, chilled in ice,and residual enzymatic activity was measured by follow-ing the b-galactosidase activity at 658C in 50 mM so-dium phosphate buffer at pH 6.5 with 4-nitrophenyl-b-D-galactopyranoside substrate at the final concentrationof 5 mM. Activity of the sample before heat incubationwas taken as 100%.

992 A. AUSILI ET AL.

PROTEINS: Structure, Function, and Bioinformatics DOI 10.1002/prot

In Silico Analysis

Tetrameric assembly of Ssb-gly and of Tab-gly wasgenerated using the DeepView/Swiss-PdbViewer Pro-gram20 by applying the BIOMT transformations given inthe pdb files to the crystallographic asymmetric unit(PDB code 1UQW and 1QVB, respectively).Analysis of intersubunit H-bonds and salt bridges was

performed on a Silicon Graphics O2 workstation byusing the InsightII molecular modeling system fromAccelrys (San Diego, USA) and with the program ProteinExplorer.21 Here, atoms of different charge (Arg NE, ArgNH, Lys NZ vs. Asp OD, Glu OE) are consideredinvolved in a salt bridge when they are within 4.0 A.Measurements of surfaces, contact areas and volumes,

and analysis of the cavities were performed by using theDeepView/Swiss-PdbViewer Program.20 The solvent ac-cessible surface and cavities area are measured as thecontact surface between a solvent probe (1.4 A diameter)and the molecule and computed using the Connolly sur-face algorithm. The buried hydrophobic surface area atthe interface was estimated by subtracting the valuecomputed for the tetramer from the sum of the valuescomputed for dimers. Hydrogen bonds were calculatedon a Silicon Graphics O2 workstation using the InsightIIpackage considering, in the absence of hydrogen atoms,the distance between two heavy atoms ranging between2.19 and 3.3 A.

RESULTS AND DISCUSSION

Secondary Structure of Protein Samples

Figure 1 shows the second derivative spectra of thesoluble and aggregated Pfb-gly. The lack of differences inthe amide I0 (1700–1620 cm�1) component bands indi-

cates that the soluble and insoluble sample have thesame secondary structure.

Figure 2 shows the second derivative (A) and deconvo-luted (B) spectra of Ssb-gly, Tab-gly, and Pfb-gly. All spec-tra display an a-helix band (close to 1655 cm�1) and b-sheet bands (close to 1638 cm�1 and close to 1630 cm�1).22

These bands have different intensities and positions, sug-gesting that the b-glycosidases may have different second-ary structure composition. The 1630 cm�1 band is wellvisible in Pfb-gly, while in Tab-gly and in Ssb-gly it ispresent as a shoulder, which is very small in the case ofthe second derivative spectrum of Ssb-gly. The band closeto 1682 cm�1 is probably due to b-sheet, but it may con-tain information also on turns since both turns andcoupled high frequency vibrations of b-segments can con-tribute in the 1670–1690 cm�1 spectral region.23 The bandclose to 1665 cm�1 may be assigned to turns, while theabsorption close to 1644 cm�1, which is well visible only inthe second derivative spectrum of Ssb-gly, is due to unor-dered structures.22 However, the presence of unorderedstructures in Tab-gly and of Pfb-gly is revealed by the

Fig. 1. Second derivative spectra of Pfb-gly. The concentration ofPfb-gly solution causes partial protein aggregation and precipitation.The precipitate and the protein that remained in solution were analyzedseparately. Continuous and dotted lines refer to the insoluble and solu-ble part of Pfb-gly, respectively. The spectra were obtained at 208C.The symbols (a) and (b) stand for a-helix and b-sheet, respectively.

Fig. 2. Second derivative (A) and deconvoluted (B) spectra of Ssb-gly, Tab-gly, and Pfb-gly. The spectra were obtained at 208C. The sym-bols (a), (b), (u), and (A II) stand for a-helix, b-sheet, unordered struc-tures, and residual amide II band, respectively.

993STRUCTURAL PROPERTIES OF THERMOPHILIC b-GLYCOSIDASES


deconvoluted spectra, which show a shoulder at about1644 cm�1. The absorptions below 1620 cm�1 are due toamino acid side chain absorption24 except for the bandclose to 1547 cm�1 that is due to residual amide II bandi.e. to the absorption of the amide II band after 1H/2Hexchange of the amide hydrogens of the polypeptide chain.In 1H2O medium the amide II band intensity is about 2/3of the intensity of amide I band (data not shown), while in2H2O medium its intensity decreases as a consequence ofthe exchange of amide hydrogens with deuterium.25 Thelarger the decrease in intensity of the amide II band, thelarger the 1H/2H exchange, that is the larger the accessi-bility of the solvent (2H2O) to the protein. Hence, the pres-ence of the peak close to 1547 cm�1 indicates that part ofamide hydrogens were not exchanged with deuterium dur-ing the preparation of the protein sample (see Materialsand Methods). The band close to 1515 cm�1 is due to tyro-sine residues, and the peaks close to 1565 and 1586 cm�1

are due to the ionized carboxyl groups of glutamic and as-partic acid, respectively.24

During the analysis of protein thermostability thedenaturation of Pfb-gly even at 99.58C was neverobserved (see below). Hence, the protein was analyzed inthe presence of SDS. Figure 3 compares the second de-rivative spectra of Pfb-gly in the absence and in thepresence of 1.2 or 5% SDS. The detergent affects the sec-ondary structure of the protein since it induces changesin the intensity and position of the secondary structuralelement bands. These changes are SDS concentration-de-pendent. Indeed, in the presence of 5% SDS the bandintensities are reduced remarkably and the 1630 cm�1

band is no longer visible. This is quite unusual since pro-teins from hyperthermophilic organisms often show arelevant stability and resistance towards common dena-turants, including detergents. Indeed, in previous stud-ies we showed that Ssb-gly maintains its secondary

structure even in the presence of 5% SDS.26 The residualamide II band intensity is also affected by the detergent.In the presence of 5% SDS it is not visible while in thepresence of 1.2% SDS it is very small when compared tothe control. Since the accessibility of the solvent (2H2O)to the protein increases when the protein undergoesdenaturation or when its tertiary/quaternary structure

Fig. 3. Second derivative spectra of Pfb-gly in the absence andpresence of 1.2 or 5% SDS. The spectrum Pfb-gly refers to the spec-trum of the protein without SDS (control). The spectra were obtained at208C. The symbols (a), (b), (u), and (A II) stand for a-helix, b-sheet,unordered structures, and residual amide II band, respectively.

Fig. 4. Deconvoluted amide I0 band contour with the best fittedGaussian/Lorentzian curves for Ssb-gly, Tab-gly, and Pfb-gly. Chisquare was 2.8 3 10�6, 1.2 3 10�5, and 9.5 3 10�6 for Ssb-gly, Tab-gly, and Pfb-gly, respectively.



becomes less compact, the decrease in intensity of resid-ual amide II band may be ascribed to a partial loss ofsecondary structures (Fig. 3) and/or to a relaxation ofthe Pfb-gly tertiary/quaternary structure induced bySDS.The composition of the secondary structure of the dif-

ferent protein samples was estimated by fitting the am-ide I0 band with Gaussian/Lorentzian curves (Fig. 4). Ta-ble I reports the results of the curve-fitting calculationswhich confirm the different content of the low frequencyb-sheets (band close to 1630 cm�1) in the protein sam-ples. On the other hand, Table I shows that the contentin a-helix is the same in all proteins, and that the b-sheets belonging to the main b-sheet band (band close to1638 cm�1) are very similar.

Thermal Denaturation

The changes in the infrared spectrum induced by thetemperature increase may reflect different phenomena.Typically, as shown in Figure 5, the temperature-depend-ent decrease in intensity of the a-helix and b-sheetbands are due to unfolding (denaturation) of these struc-tural elements whilst the appearance of two new bandsclose to 1618 and 1684 cm�1 are characteristic of proteinaggregation (intermolecular interaction) brought aboutby protein denaturation.13,27 On the other hand, thetemperature-dependent decrease in intensity of the re-sidual amide II band (close to 1550 cm�1) is due to fur-ther 1H/2H exchange caused by the temperature-depend-ent protein dynamics and/or protein denaturation.13 Fig-ure 5 shows that the increase in temperature leads todenaturation and aggregation of Ssb-gly and Tab-gly,but not of Pfb-gly. Indeed, both in Ssb-gly and in Tab-glyit is possible to observe a large decrease in intensity ofthe amide I0 band in the 90–99.58C temperature rangeand a concomitant increase in intensity of the bandsclose to 1618 and 1684 cm�1 reflecting denaturation andaggregation of the proteins. Moreover, in correspondenceof protein denaturation (90–99.58C), the decrease in in-tensity of the residual amide II band becomes greaterbecause of protein unfolding. At temperatures between20 and 908C, the decrease in intensity of the residual

amide II band is almost constant, most likely due tochanges in molecular dynamics induced by the increasein temperature.

In the case of Pfb-gly, the increase in temperaturedoes not lead to a large decrease in intensity of the am-ide I0 component bands, indicating that the protein is

TABLE I. Calculated Positions and Fractional Areas(%) of the Amide I’ Component Bands for Ssb-gly,

Tab-gly, and Pfb-gly

Ssb-gly Tab-gly Pfb-gly

Position % Position % Position %

1627.7 (b) 3.9 1631.3 (b) 7.3 1629.6 (b) 9.71636.1 (b) 15.0 1639.1 (b) 12.5 1638.1 (b) 16.71644.3 (u) 25.2 1646.8 (u) 26.1 1646.4 (u) 18.61654.8 (a) 34.2 1655.1 (a) 35.1 1654.7 (a) 34.51666.5 (t) 19.2 1667.2 (t) 15.7 1665.6 (t) 12.01681.9 (b/t) 2.2 1680.2 (b/t) 3.3 1674.9 (b/t) 5.2

1681.2 (b/t) 3.2

The symbols (a), (b), (u), (t), and (b/t) stand for a-helix, b-sheet,unordered structures, turns, and b-sheet and/or turns, respectively.

Fig. 5. Temperature-dependent changes in the deconvoluted infra-red spectra of Ssb-gly (A), Tab-gly (B), and Pfb-gly (C). In each panel,16 spectra in the 20–958C interval are shown, starting from 20 to 958Cwith 58C increments. Spectra recorded at 98, 99, and 99.58C are alsoshown. The symbols (a), (b), (a), and (A II) stand for a-helix, b-sheet,aggregation, and residual amide II band, respectively.



stable even at 99.58C. Conversely, the increase in tem-perature causes a constant decrease in intensity ofthe residual amide II band due to increased moleculardynamics. It is worth nothing that at 99.58C the residualamide II band is still visible (compare the Ssb-gly, Tab-gly, and Pfb-gly spectra). This finding may be due tothe fact that the protein retains its secondary structureand that it is particularly compact, and/or that somesegments may be particularly buried. These data arein agreement with the experiments of differential scan-ning calorimetry showing that the temperature of melt-ing (Tm) of this enzyme was 1088C. 28 Thermal denatura-tion curves for Ssb-gly and Tab-gly were obtainedby plotting the changes of the amide I0 bandwidth as afunction of the temperature29,30 (data not shown). Thetemperature of melting for Ssb-gly and Tab-gly resulted98.1 and 98.48C, respectively and was calculated asdescribed.18

This trend of thermal stability is confirmed also bykinetics of thermal inactivation. The activity of proteinsamples of Ssb-gly, incubated for increasing times at 90and 958C, showed half-lives of 38 and 3 min, respec-tively; under the same conditions Tab-gly showed a sta-bility 2.4-fold higher at both 90 and 958C (92 and 7 min,respectively). In striking contrast, Pfb-gly resulted muchmore stable and no inactivation was observed at 908Cwhile at 958C this enzyme showed an half-life of 225min, which is 75- and 32-fold higher than those of Ssb-gly and Tab-gly, respectively, at the same temperature.It is worth noting that the thermal inactivation iscoupled to protein aggregation for all the proteins tested.The thermal denaturation of Pfb-gly could be followed

only in the presence of SDS. Figure 6 shows the secondderivative spectra of Pfb-gly in the absence and presenceof 1.2 or 5% SDS, and at different temperatures. In theabsence of detergent the enzyme retains its secondarystructure at all temperatures while the spectra show

only a decrease in intensity of the residual amide IIband confirming the data reported in Figure 5(C). In thepresence of 1.2% SDS the amide I0 component bands arewell visible at 208C although they display differentintensities with respect to the control (see also Fig. 3),indicating that the detergent affects the secondary struc-ture of the protein. With the increase in temperature theamide I0 component bands decrease significantly in in-tensity starting from 908C while at 958C the bands areno longer visible. At 99.58C, the spectrum is character-ized by a broad band, centered at 1650 cm�1, whichappears after the loss of the secondary structural ele-ments of the protein. The presence of this band is un-usual for a completely denatured protein since its posi-tion is characteristic for a-helices. However, this bandwas previously observed in the presence of high SDSconcentration and at high temperatures in b-galactosi-dase31 and also in Ssb-gly.26 It was assigned to a-heliceswith the possible contribution of unordered structures.In the case of Ssb-gly, the assignment of the 1650 cm�1

band to a-helices was supported also by the CD meas-urements that showed a high a-helix content in the pres-ence of 5.0% SDS at 958C. 26. In the presence of 5% SDSthe spectrum of the protein at 208C does not display thesecondary structural elements as in the case of the con-trol spectrum, indicating that a high SDS concentrationinduces a partial loss of the secondary structure. Withthe increase in temperature, the spectrum contourchanges reflecting denaturation. However, the changesare less visible as compared to the control or to the spec-trum in the presence of 1.2% SDS. Indeed, the disap-pearance of the 1638 cm�1 band seems to occur at about55–608C. In the case of the 1650.1 cm�1 band the identi-fication of the temperature of its disappearance is lesscertain since at 99.58C a 1650 cm�1 band, most likelydue to unordered structures and a-helix induced by theSDS binding, is also present.

Fig. 6. Temperature-dependent changes in the second derivative infrared spectra of Pfb-gly in the ab-sence (A) and in the presence of 1.2% SDS (B) or 5% SDS (C). In each panel, 16 spectra in the 20–958Cinterval are shown, starting from 208C (bottom) to 958C (top) with 58C increments. Spectra recorded at 98,99, and 99.58C are also shown.



Figure 6 also shows that at high temperatures and inthe presence of SDS the two bands characteristic ofintermolecular interactions (aggregation) brought aboutby protein denaturation [see Fig. 5(A,B)] are lacking.This phenomenon may be explained by the fact that thebinding of SDS to protein segments endows the polypep-tide with additional negative charges that keep the poly-peptide molecules apart, thereby avoiding intermolecularaggregation.26

Analysis of the 3D-Structures

In an effort to find a rational to explain the differentstabilities of the glycosidases studied here, we analyzedthe 3D-structures available for these enzymes. The 3D-structure of Ssb-gly has recently been resolved at 1.95A32 and it is deposited in the Brookheaven Protein DataBank as 1UWQ while the structure of Tab-gly (1QVB) isavailable at 2.4 A.8 Finally, the structure of Pfb-gly(CelB) was a 3.3 A structural model33 obtained by molec-ular replacement from the structure of Ssb-gly previ-ously obtained at 2.6 A.7 It must be pointed out that themid-level resolution at 3.3 A of the 3D-structure of the P.furiosus enzyme does not allow to drive conclusiveremarks on the structural details of this enzyme, there-fore, the inspection of the structure of this enzymereported below was performed to find a structural basisto the experimental data.Preliminary analyses strongly indicated that the three

enzymes had similar functional features summarized inTable II; in particular, the differences in terms of thesurface and the volume of the tetrameric functionalunits were below 5%. This observation is also confirmedby the superimposition of the tetramers that for the cou-ples 1UWQ-1QVB and 1UWQ-CelB produced r.m.s. of1.64 and 0.69 A, respectively, suggesting that the globalfolding of the three enzymes was almost identical. Theonly striking difference clearly observable is the pres-ence of loops in the b-glycosidases from S. solfataricusand T. aggregans, which are missing in Pfb-gly. Thisalso explains the different length of the two former pro-teins in comparison with the b-glycosidase from P. furio-sus (Table II).The inspection of most of the structural elements that

are reported as possible protein stabilizing featuresrevealed interesting differences among the three b-glyco-sidases (Table III). In fact, Tab-gly is the only enzymeshowing a S-S bridge involving the residues Cys343 andCys353 in each subunit while Ssb-gly showed the high-est number of salt bridges per functional unit andbetween surfaces. These findings are not surprising. Thepresence of large networks of ion-pairs has been recog-nized as one of the main determinants of the stability ofenzymes from hyperthermophiles34 and in Ssb-gly thiswas experimentally proved.19 Furthermore, the presenceof an intrasubunit cystein bridge has been proposed as apossible reason for the increased stability of Tab-gly overSsb-gly.8 In addition, these enzymes showed larger con-tact surfaces between the two different monomer–mono-

TABLE

II.Comparison

ofStructu

ralFeatu

resAmongHyperth

ermophilic

GH1b-G

lycosidase

s

Enzy

me

Number

ofresidues

Functional

unit

Number

ofa-helices

aNumber

ofstrandsa

Number

ofturn

sa

Total

surface

(A2)a

Total

volume

(A3)a

Mon

omer

average

surface

(A2)a

Mon

omer

average

volume(A

3)a

Ssb

-gly

489

Tetramer

96

118

190

56,981

272,129

15,705�

117

66,955�

177

Tab-gly

481

Tetramer

100

116

188

58,422

266,663

15,637�

60

65,845�

39

Pfb-gly

472

Tetramer

108

120

172

59,920

259,922

15,801�

78

64,339�

93

aData

werecalculatedbyusingtheprogram

SwissPDB

Viewer.20



TABLE

III.

Comparison

ofth

ePuta

tiveSta

bilizin

gIn

teractionsin

theStructu

resofHyperth

ermophilic

GH1b-G

lycosidase

s

Enzy

me

Symmetries

inthe

functional

unita

Number

ofprolines

Number

ofS-S

Number

ofintersubunit

H-bon

ds

Number

ofsa

lt-bridges

inthe

mon

omer

b

Number

salt-bridges

inthe

functional

unitb

Number

ofintersubunit

salt-bridges

b

Surface

betwee

nnon

-crystallog

raphic

mon

omers(A

2)c

Number

ofintern

al

cavitiesper

functional

unitc ,d

Average

size

oftheintern

al

cavities

(A2)c,d

Ssb

-gly

26

014atinterface

ACe

33

138

14

1,390atinterface

ACe

13

178�

83

14atinterface

AD

f1,118atinterface

AD

f

2atinterface

AB

Tab-gly

30

113atinterface

ABg

25

114

12

871atinterface

AD

1,194atinterface

CD

14

186�

68

5atinterface

AD

h

non

eatinterface

AC

Pfb-gly

24

06atinterface

ACe

21

92

8945atinterface

ACe

5153�

34

7atinterface

AD

f658atinterface

AD

f

non

eatinterface

AB

aThecrystallog

raphic

dim

ersare

show

nin

black

;figureswerepreparedwiththeprogram

RasM

ol.39

bData


Protein

Explorer.21

c Data


SwissPDB

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mer interfaces and an increased number of hydrogenbonds if compared to the enzyme from P. furiosus (TableIII). This confirms the observation that oligomerizationcan play a relevant role in protein stabilization.13

The analysis of the 3D-structure suggested that thehigher stability of Pfb-gly could be obtained by means ofdifferent molecular mechanisms, since this enzymeshowed few intersubunit contacts in terms of H-bonds,ion-pairs, and surfaces between adjacent monomers (Ta-ble III). In addition, in Pfb-gly the ion-pairs per subunitare reduced by 1.5- and 1.2-fold if compared to Ssb-glyand Tab-gly, respectively (see Fig. 7). However, we founda striking reduction in the number of internal cavities of

Pfb-gly vs Ssb-gly and Tab-gly, though the average sizeof these cavities is only slightly different (Table III andFig. 7). This observation would indicate that the hydro-phobic core of Pfb-gly is much more compact than thatof the enzymes from S. solfataricus and T. aggregans.Unfortunately, the limited resolution of the Pfb-gly 3D-structure did not allowed us to conclude that the higherstability of this enzyme unequivocally result from thereduced number of ion-pairs and internal cavities. Infact, we cannot exclude that the 3.3 A resolution affectedthese features. However, it is worth noting that theseobservations go in agreement with the thermal denatu-ration IR data (Fig. 5), which suggested a particular

Fig. 7. Structural comparison of archaeal b-glycosidases. A: The monomers of the three enzymes showcationic and anionic atoms in blue and red respectively. The catalytic diads are shown as sticks in cyan.Images were prepared with the program Protein Explorer.21 B: The tetramers of the three enzymes show in-ternal cavities larger than 100 A2. Images were prepared with the program Swiss PDB Viewer.20



compact structure and deeply buried protein segments.Protein interiors are tightly packed and buried apolarside chains influence protein stability through the hydro-phobic effect that favors the shielding of apolar residuesfrom water; in addition, packing interactions are thoughtto give rise to van der Waals stabilization of the nativestate.35 Protein engineering studies demonstrated thatcavities-creating mutations are generally destabiliz-ing36,37; this suggests that the reduction in both thenumber and size of the internal cavities in the hydropho-bic core of Pfb-gly greatly contributed to its stability.The experimental observation that the thermal stabil-

ity of Pfb-gly was reduced in the presence of SDS (seeabove) further supports the hypothesis that the stabilityof this enzyme is mainly driven by the compactness ofthe hydrophobic core and strikingly contrasts with theincreased stability of Ssb-gly in the presence of SDS.26

Presumably, the alkyl chains of SDS have a markeddestabilizing effect on the hydrophobic core of theenzyme from P. furiosus increasing its rate of thermaldenaturation. It is worth noting that SDS had a similareffect on a protein disulfide oxidoreductase from thesame source38 and it has been reported that the extremestability of a rubredoxin from the same organism is pro-vided by the increased compactness of its structure.4

Remarkably, high-throughput analyses and computa-tional studies from these authors demonstrated that thisis a general method of stabilization of proteins from P.furiosus, and they propose that the increment in packag-ing of the protein core could be the result of an evolu-tionary strategy of stabilization occurring in archaea.This mechanisms of stabilization, named ‘‘structure-based’’ counterpoises to a ‘‘sequence-based’’ mechanismsencountered in hyperthermophilic bacteria, in which asmall number of point mutations (especially leading toan increase number of ion-pairs) are, apparently, respon-sible of the stability of the proteins from this source.4

The different strategy of stabilization would result fromthe evolutionary history of the two groups of organisms:hyperthermophilic archaea originated in extreme envi-ronments while hyperthermophilic bacteria recolonizedhot environments. Therefore, archaea enzymes were denovo designed in an hot abitat and were subjected to abias toward certain structures and sequences that haveto fold and be stable at high temperatures. Instead, theadaptation of hyperthermophilic enzymes from bacteriawould be acquired by enhancing the thermostability ofexisting proteins of mesophilic origin. In these respects,our data, though referring to a limited number of pro-teins, confirmed the observation of Berezovsky andShakhnovich experimentally demonstrating these mech-anisms of stabilization. Nevertheless, our results indi-cate that this picture might be even more complicatedsince different stabilization methods could have beenadopted among archaea. In fact, Ssb-gly and Tab-glyshowed a ‘‘sequence-based’’ stabilization strategy whilePfb-gly apparently followed a ‘‘structure-based’’ mecha-nism; further genomic and proteomic studies might helpin shedding light on this intriguing issue.

ACKNOWLEDGMENTS

We thank Thijs Kaper for the structural coordinates ofthe P. furiosus b-glycosidase. The IBP-CNR belongs tothe Centro Regionale di Competenza in Applicazioni Tec-nologico-Industriali di Biomolecole e Biosistemi.

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A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures