Tertiary structure in 7.9 M guanidinium chloride ) the role of Glu53 and Asp287 in Pyrococcus furiosus endo-b-1,3-glucanase Roberta Chiaraluce1, Rita Florio1, Sebastiana Angelaccio1, Giulio Gianese2, Johan F T van Lieshout3, John van der Oost3 and Valerio Consalvi1 ` Dipartimento di Scienze Biochimiche ‘A Rossi Fanelli’ Sapienza Universita di Roma, Italy Ylichron Srl c ⁄ o ENEA Casaccia Research Center, S Maria di Galeria, Italy Laboratory of Microbiology, Wageningen University, the Netherlands Keywords double mutant; glycoside hydrolases; laminarinase; protein stability; thermodynamic stability Correspondence V Consalvi, Dipartimento di Scienze ` Biochimiche ‘A Rossi Fanelli’ Universita ‘La Sapienza’, P.le A Moro 5, 00185 Rome, Italy Fax: +39 06 4440062 Tel: +39 06 49910939 E-mail: consalvi@caspur.it (Received 26 July 2007, revised October 2007, accepted 10 October 2007) doi:10.1111/j.1742-4658.2007.06137.x The thermodynamic stability of family 16 endo-b-1,3-glucanase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A ⁄ D287A) mutation of Asp287 and Glu53 In accordance with the homology model prediction, both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator The present study shows that, in P furiosus, endo-b-1,3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 m guanidinium chloride The persistence of tertiary structure in 7.9 m guanidinium chloride, a feature of the wild-type enzyme, is observed also for the three variant proteins The DGH2O values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximatelty 50% lower than that of the wild-type The destabilizing effect of the combined mutations of the double mutant is non-additive, with an energy of interaction of 24.2 kJỈmol)1, suggesting a communication between the two mutated residues The decrease in the thermodynamic stability of D287A, E53A and E53A ⁄ D287A is contained almost exclusively in the m-values, a parameter which reflects the solventexposed surface area upon unfolding The decrease in m-value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non-native state in 7.9 m guanidinium chloride comparable to that induced by the presence of calcium on the wildtype These results suggest that the stabilization of a compact non-native state may be a strategy for P furiosus endo-b-1,3-glucanase to thrive under adverse environmental conditions Endo-b-1,3-glucanase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus (pfLamA) is a laminarinase that displays considerable residual tertiary structure in 7.9 m guanidinium chloride (GdmCl) [1] A high DGH2O value of 61.5 kJỈmol)1 is associated with the partial unfolding of pfLamA which, in 7.9 GdmCl, maintains the ability to bind calcium with substantial recovery of native tertiary structure, a unique property of this enzyme [2] pfLamA belongs to family 16 glycoside hydrolases [3], a family composed by 748 enzymes (http://www cazy.org/) According to their substrate specificity, the enzymes of this family can be assigned to different subgroups [4] (http://www.ghdb.uni-stuttgart.de ⁄ ) Abbreviations ANS, 8-anilinonaphthalene-1-sulfonate; BAPTA, 5,5¢-Br2-1,2-bis(O-aminophenoxy)ethan-N,N,N¢,N¢-tetraacetic acid; GdmCl, guanidinium chloride; pfLamA, endo-b-1,3-glucanase from Pyrococcus furiosus; SVD, singular value decomposition FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6167 Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R Chiaraluce et al which share the same b-jelly roll fold but display notable differences in their primary structure Twenty-three crystal structures of members of this family have been solved (http://www.cazy.org/); however, the crystal structure of the laminarinase subfamily is still missing [4,5] Seventeen out of the 23 available crystal structures demonstrate the presence of at least one metal binding site (http://www.cazy.org/http://www.ghdb uni-stuttgart.de ⁄ ) [4,5] Sequence alignments of pfLamA from different sources suggested that Asp287 [2,6], a residue conserved in most family 16 glycoside hydrolases, may be part of a calcium binding site (http://www.ghdb.uni-stuttgart.de ⁄ ) According to this hypothesis, a 3D homology model of pfLamA has predicted the presence of one or two potential binding sites for metals and two pairs of negatively charged amino acid residues have been assumed to be involved in calcium binding: Glu53 and Asp287, and Glu239 and Glu246 [2] In the present work, and on the basis of pfLamA homology modeling prediction [2], residues Glu53 and Asp287 were replaced with alanine residues by sitedirected mutagenesis, either individually (E53A, D287A) or simultaneously (E53A ⁄ D287A), in order to demonstrate their involvement in calcium binding in native conditions and in the presence of 7.9 m GdmCl The thermodynamic stability of pfLamA variant proteins has been studied by GdmCl-induced unfolding equilibrium experiments The thermodynamic characterization of the double mutant provided more information than a study of single mutants, especially with respect to the direct or indirect involvement of residues Glu53 and Asp287 either in electrostatic interactions with other protein residues or in metal binding [7] Glu53 and Asp287 are negatively charged at neutral pH and contribute to the optimization of electrostatic charges balance of pfLamA in the native state, independently of their interaction with calcium The role of electrostatic interactions in protein stability has been widely investigated and the stabilizing effect of salt bridge networks on the native state of hyperthermophilic proteins has been proposed on the basis of several computational and experimental studies [8–12] Studies of proteins from hyperthermophiles have provided an array of hypotheses on the structural determinants responsible for their resistance to denaturation [13,14]; however, a unifying description remains elusive [15] Investigations on protein stability are also necessary to advance our skills in designing new catalysts resistant to temperature and extreme solvent conditions In addition to their role in the stabilization of pfLamA native state, Glu53 and Asp287 could also contribute to the persistence of residual tertiary struc6168 ture in the non-native state in 7.9 m GdmCl [1] The study of the properties of non-native states of proteins has received considerable attention in the last 10 years because the residual structure within the unfolded state may play an important role in a protein’s energetics and function [16–19] Changes in the denatured states induced by mutations can therefore affect protein stability [20,21] and, in thermophilic proteins, the persistence of residual structure in non-native states may contribute toward avoiding irreversible denaturation under extreme environmental conditions [22] Under denaturing conditions, the spectroscopic characterization of the residual structure in proteins is very difficult, although it has been reported in several studies [18,23–25] A measure of the residual structure in the unfolded form of a protein is the thermodynamic parameter m, as obtained in equilibrium unfolding studies [26–28] This parameter represents the rate of change of the free energy of unfolding as a function of denaturant concentration and is proportional to the amount of additional surface area exposed upon unfolding [26] The m-value may provide information about the residual structure present in the denatured states of mutants in comparison to that of the wild-type protein [26,29] Mutations affecting m-values are more likely to change the accessible surface area of the unfolded form rather than that of the native state; thus, a change in m-value is generally considered to reflect a change in the compactness of the denatured state [26] The present study reports on the thermodynamic stability of pfLamA single mutants E53A, D287A and double mutant E53A ⁄ D287A, as well as the binding of calcium to the mutant proteins in native conditions The single and combined mutations dramatically decrease the thermodynamic stability of the proteins with a significant decrease of m-values relative to the GdmCl-induced unfolding equilibrium A double-mutant thermodynamic cycle reveals a non-additive effect of the mutations on the thermodynamic parameters [30,31] The effect of the mutations indicates a key role for Glu53 and Asp287 in the interactions responsible for the residual structure in the non-native state, as well as for calcium binding of pfLamA in the native state and in 7.9 m GdmCl Results Spectroscopic characterization of the mutants in native conditions and in 7.9 M GdmCl In native conditions, the near-UV CD spectra of the three mutants E53A, D287A and E53A ⁄ D287A are FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS R Chiaraluce et al very similar to those of the wild-type enzyme, except for minor differences in the ellipticity signals all centered around the same main aromatic bands of the native wild-type (Fig 1A) The fluorescence emission spectra of native wild-type and mutant enzymes are all centered at the same maximum emission wavelength of 342 nm and have similar emission fluorescence intensities (Fig 1B) Analogously, the far-UV CD spectra are virtually superimposable (data not shown) These results indicate that the mutations had no effect on the secondary and tertiary structure arrangements of the protein and suggest that, in the native state, the effect of the mutations are directed and localized to the mutated residue In the presence of 7.9 m GdmCl, and analogous to that observed with the wild-type pfLamA [1,2], the near-UV CD spectra of the three mutants indicate the presence of substantial residual tertiary structure Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl (Fig 1C) Minor differences in the near-UV CD spectra of the mutants, in comparison to that of the wildtype, are evident in the 275–290 nm region, where the positive ellipticity is progressively reduced in the three mutants from the D287A to the double mutant, and in the 260–270 nm region where the negative ellipticity of the E53A ⁄ D287A mutant is somewhat decreased (Fig 1C) The changes observed in the near-UV CD spectra of the three mutants indicate that, in 7.9 m GdmCl, the environment of the aromatic residues is slightly perturbed In particular, for the double mutant, the decrease in the dichroic activity around 260 nm suggests that Phe residues are locked differently in their tertiary contacts compared to the wildtype (Fig 1C) In 7.9 m GdmCl, the maximum fluorescence emission wavelength of the three mutants is shifted to 357 nm, similar to the wild-type in the same condi- Fig Spectral properties of pfLamA wild-type and mutants: effect of GdmCl in the presence and absence of CaCl2 Near-UV CD (A, C, E) and fluorescence (B, D, F) spectra of pfLamA wild-type (– Ỉ Ỉ –), D287A (—–), E53A ( ) and E53A ⁄ D287A (– ) –) were recorded at 20 °C after 20 h of incubation of the protein in native conditions (20 mM Tris ⁄ HCl, pH 7.4) (A, B) and in 7.9 M GdmCl, pH 7.4, in the absence (C, D) and presence (E, F) of 40 mM CaCl2 The spectral properties of all the proteins under native conditions are unchanged upon addition of 40 mM CaCl2 (data not shown) Near-UV CD spectra (A, C, E) were recorded in a 1-cm quartz cuvette at 0.6 mgỈmL)1 protein concentration Fluorescence spectra (B, D, F) were recorded at 40 lgỈmL)1 protein concentration (290 nm excitation wavelength) FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6169 Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R Chiaraluce et al tions, but the relative fluorescence intensities are increased to a different extent (Fig 1D) The increase in relative fluorescence intensity emission at 342 nm is approximately two-fold for the wild-type and 2.5, 2.6 and 2.8-fold for the D287A, E53A and the double mutant, respectively (Fig 1D) Noteworthy, similar to that reported for the wild-type enzyme [1,2], the farUV CD spectra of the three mutants are not affected by equilibrium incubation at increasing concentrations of GdmCl up to 7.9 m (data not shown) Equilibrium transition studies in GdmCl The effect of increasing GdmCl concentrations (0–8 m) on the structure of the three mutants was analyzed in comparison to the effect exerted on the wild-type pfLamA in 20 mm Tris ⁄ HCl, pH 7.4, containing 100 lm dithiothreitol and 100 lm EDTA The intrinsic fluorescence emission intensity of the three mutants increases after 20 h of incubation at increasing GdmCl concentrations (Fig 2) and, in 7.9 m GdmCl, the maximal fluorescence emission wavelength shifts to 357 nm (Fig 1D) The changes in relative intrinsic fluorescence emission intensity of the mutants show a sigmoidal Fig GdmCl-induced fluorescence changes of pfLamA wild-type (r, e), D287A (m, n), E53A (d, s) and E53A ⁄ D287A (j, h) Continuous lines are the nonlinear regression to Eqn (3) of the fluorescence data at varying denaturant concentrations, as described in the Experimental Procedures The reversibility points (empty symbols) were not included in the nonlinear regression analysis All spectra were recorded at 20 °C after 20 h of incubation at the indicated GdmCl concentrations 6170 Table Thermodynamic parameters for GdmCl-induced unfolding equilibrium of pfLamA wild-type and mutants All data were obtained at 20 °C in 20 mM Tris ⁄ HCl, pH 7.4, containing 100 lM dithiothreitol and 100 lM EDTA DGH2O and mg values were obtained from Eqn (3); [GdmCl]0.5 was calculated from Eqn (4) Data are reported as the mean ± SE of the fit DDGH2O ¼ DGH2O mutant ) DGH2O wild-type The SE value relative to DDGH2O was calculated according to: [SE(DDGH2O)]2 ¼ [SE(DGH2O wild-type)]2 + [SE(DGH2O mutant)]2 Protein [GdmCl]0.5 DGH2O (M) (kJỈmol)1) mg DDGH2O )1 )1 (kJỈmol ỈM ) (kJỈmol)1) Wild-type D287A E53A E53A ⁄ D287A 6.7 6.2 6.0 6.2 9.2 5.9 5.6 5.3 ± ± ± ± 0.13 0.15 0.12 0.15 61.5 36.5 33.9 33.1 ± ± ± ± 1.23 0.91 0.68 0.83 ± ± ± ± 0.18 0.15 0.11 0.13 )25.0 ± 1.53 )27.6 ± 1.40 )28.4 ± 1.48 dependence on GdmCl concentration and follow a two-state denaturation process without any detectable intermediate, similar to that reported for the wild-type pfLamA (Fig 2) [2] The changes are fully reversible upon dilution of the denaturant, and the transition midpoints are at 6.2 ± 0.15 m for D287A and the double mutant, and at 6.0 ± 0.12 m for E53A (Fig 2), with the values being slightly lower than that of the wild-type pfLamA, which is at 6.7 ± 0.13 m GdmCl (Fig 2) [2] Table shows the thermodynamic parameters values obtained for wild-type and mutant forms of pfLamA The mg value of 9.2 kJỈmol)1ỈM)1 of the wild-type is approximately 30% lower than the value predicted from the number of the aminoacid residues (approximately 13 kJỈmol)1ỈM)1 for 263 amino acid residues) [28], in accordance with the persistence of residual structure in 7.9 m GdmCl The mutants are thermodynamically less stable than the wild-type, with a significant decrease of DGH2O and mg values, suggesting that the mutations considerably affect the stability of pfLamA The double mutant shows a slightly smaller stability than either the single mutants and the similarity between the DDGH2O values of the variant proteins (Table 1) indicates that the double mutant E53A ⁄ D287A is more stable than expected from the sum of the stability change from single mutants E53A and D287A, and hence the effect of the mutations is non-additive Calculation of the energy of interaction between two mutated residues, DDGint, according to Eqn (5), yields a value of 24.2 ± 1.87 kJỈmol)1 Effect of calcium on pfLamA mutants in 7.9 GdmCl and in native conditions M The addition of 40 mm CaCl2 to calcium-depleted samples of D287A and E53A in 7.9 m GdmCl causes changes in their tertiary structure, but these are much FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS R Chiaraluce et al less pronounced than those observed for the wild-type, indicating the involvement of Glu53 and Asp287 in the interaction with the cation (Fig 1E,F) The regain of aromatic chirality at 295 nm and in the 260–270 nm region for D287A is similar to that observed for the wild-type enzyme, whereas it is much less evident for E53A (Fig 1E) With the double mutant, the near-UV CD spectrum in 7.9 m GdmCl shows very minor changes upon addition of CaCl2 (Fig 1E) The intrinsic fluorescence emission spectra of the mutants in 7.9 m GdmCl are affected by the presence of 40 mm CaCl2 to different extents (Fig 1F) With D287A, the intrinsic fluorescence emission intensity at 342 nm is 1.6-fold decreased, similar to the wild-type enzyme, and the maximum emission wavelength is shifted to 347 nm, nm higher than with the wild-type [2] (Fig 1F) For E53A and the double mutant, the changes of intrinsic emission fluorescence are less evident: the relative intensities are decreased 1.3-fold and 1.2-fold and the maximum emission wavelengths are shifted to 350 nm with E53A and to 356 nm with the double mutant, nm and 13 nm more red-shifted than that observed with the wild-type [2] (Fig 1F) The farUV CD spectra of the three mutants in 7.9 m GdmCl, which are the same as those measured in the absence of denaturant, are not affected by the addition of CaCl2 (data not shown) Changes in both near-UV CD and fluorescence spectra occur by titration in 7.9 m GdmCl with CaCl2, from 0.2 nm to 150 mm unchelated Ca2+, although with remarkably different amplitudes, depending on the protein form (Fig 3) The amplitude of the ellipticity changes at 295 nm decreases from the wild-type to D287A and E53A and, above 200 lm Ca2+, no further changes are observed (Fig 3A) For the double mutant, only minor changes in the dichroic activity at 295 nm are detected over the whole range of the cation concentration (Fig 3A) Nonlinear regression analysis of the [Q]295 data for the wild-type pfLamA was used to define two limiting slopes, intersecting at a value which suggests that mol of Ca2+ per mol of enzyme are required to reach an apparent saturation effect (Fig 3A) [2] The changes in the fluorescence properties in 7.9 m GdmCl induced by CaCl2, represented by a blue-shift of the maximum emission wavelength and by a quenching of the fluorescence intensities (Fig 1F), are reported in Fig 3B as the intensity-averaged emis sion wavelength (k) calculated according to Eqn (1) and follow a hyperbolic dependence on CaCl2, similar to that observed for [Q]295 In native conditions, the addition of 40 mm CaCl2 to the mutants did not affect the near-UV, the far-UV CD and the fluorescence properties (data not shown), Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl Fig Interaction of calcium with pfLamA wild-type and mutant forms in 7.9 M GdmCl (A) Left axis: [Q]295 of wild-type (e) and D287A (n); right axis: [Q]295 of E53A (s) and E53A ⁄ D287A (h) measured from near UV-CD spectra at 22 lM protein concentration  (B) Intensity-averaged emission wavelength k of wild-type (e), D287A (n), E53A (s) and E53A ⁄ D287A (h) measured at 1.2 lM protein concentration from fluorescence spectra (290 nm excitation wavelength) All spectra were recorded at 20 °C, after each Ca2+ addition [Q]295 is reported after removal of the high-frequency noise and the low-frequency random error by the singular value decomposition algorithm (SVD) [2] in the spectral region 250–  310 nm k was calculated according to Eqn (1) The two limiting slopes, calculated by nonlinear regression analysis to the [Q]295 and  to k data, intersect at a point corresponding to [Ca2+unchelated] ⁄ [protein] ¼ The reported unchelated Ca2+ concentrations intervals, calculated according to [49], are 0.2 nM to 140 mM and 0.2 nM to 7.6 mM for [Q]295 and fluorescence changes, respectively similar to that reported for the pfLamA wild-type [2] The interaction of the mutants with calcium, in native conditions, was studied by titration with CaCl2 in the FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6171 Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R Chiaraluce et al Table Calcium binding constants for pfLamA wild-type and mutants determined in the presence of the chromophoric chelator BAPTA v2 represents the best fit of the absorbance data Replicate determinations indicate a standard deviation for the calcium binding constants K1 and K2 less than 5% Ca2+ binding sites Protein K1 (M)1) Wild-type D287A E53A E53A ⁄ D287A 5.0 2.7 1.4 4.6 · · · · 107 105 106 105 Ca2+ binding site K2 (M)1) 2.6 2.6 1.5 2.5 · · · · 105 104 105 101 presence of the chromophoric chelator 5,5¢-Br2-1,2bis(O-aminophenoxy)ethan-N,N,N¢,N¢-tetraacetic acid (BAPTA) and compared with the results obtained with the wild-type enzyme [2] The fitting of the titration data by caligator software [32] allows a quantitative determination of the corresponding binding constants, as reported in Table All the mutants bind calcium with a significantly lower affinity compared to the wild-type Similar to that observed for the wild-type, the v2 obtained by fitting the data of E53A and D287A titration to a two calcium binding sites model are both lower than that obtained by fitting the same data to a one calcium binding site model (Table 2) In the case of the double mutant, the v2 value relative to the fitting to a one calcium binding site model is lower than that relative to the fitting to a two calcium binding sites model Furthermore, the higher value of the second binding constant suggests that only one of the two binding sites may be functional in E53A ⁄ D287A (Table 2) Effect of calcium on the equilibrium transitions in GdmCl Incubation of pfLamA mutants at increasing GdmCl concentrations (0–8 m) in 20 mm Tris ⁄ HCl, pH 7.4, containing 100 lm dithiothreitol, 100 lm EDTA and 40 mm CaCl2 for 20 h at 20 °C results in changes in the intrinsic fluorescence emission (Fig 4) The GdmCl-induced unfolding process in the presence of 40 mm CaCl2 is reversible and, by contrast to the wildtype (Fig 4A), does not follow a simple two-state mechanism, as suggested by the lack of coincidence of  the changes in relative fluorescence intensity and in k and by the hysteresis of the reversibility process (Fig 4) At the end of the transition, the intrinsic fluorescence emission intensity at 342 nm is increased 1.7-fold for D287A, 2.1-fold for E53A and 2.4-fold for the double mutant and the fluorescence maximum emission wavelength is shifted to 347 nm for D287A, 350 nm for E53A and to 356 nm for the double 6172 K1 (M)1) v2 9.8 2.5 9.6 3.0 · · · · 10)5 10)4 10)4 10)3 3.3 3.4 1.1 4.4 · · · · 107 105 106 105 v 2.9 3.1 1.7 3.1 · · · · 10)4 10)4 10)3 10)4 mutant (Fig 4, insets) Notably, in 7.9 m GdmCl and 40 mm CaCl2, the maximum fluorescence emission wavelength of the wild-type was still centred at 342 nm [2] The fluorescence emission spectra of the three variants measured after incubation in 7.9 m GdmCl and 40 mm CaCl2 are comparable with those resulting from the progressive addition of CaCl2 to the proteins in 7.9 m GdmCl (Fig 1F, Fig 4, insets) 8-Anilinonaphthalene-1-sulfonic acid ammonium salt (ANS) fluorescence and acrylamide quenching The amphiphilic dye ANS has affinity for hydrophobic clusters present in tertiary structure elements, which are not tightly packed within a fully folded structure The accessibility of hydrophobic residues of pfLamA wild-type and variant proteins in 7.9 m GdmCl was compared with that in native conditions, by analysis with the fluorescent probe ANS The fluorescence emission spectrum of ANS in the presence of all the variant proteins in 7.9 m GdmCl shows a modest, twofold increase in intensity compared to that in native conditions, without any change in the maximum fluorescence emission wavelength (results not shown) This suggests that, in 7.9 m GdmCl, the hydrophobic surface area of the mutants is not significantly exposed, similar to that observed for the wild-type The uncharged fluorescence quencher acrylamide was used to probe the accessibility of the hydrophobic core and the dynamic properties of the three mutants in comparison to the wild-type in native conditions and in 7.9 m GdmCl Effective acrylamide quenching constants from the modified Stern–Vollmer plots for the proteins in the native state were 7.9 m)1, 8.6 m)1, 8.4 m)1 and 9.4 m)1 for the wild-type and D287A, E53A and the double mutant, respectively In 7.9 m GdmCl, the acrylamide quenching constants were 13.0 m)1, 9.9 m)1, 11.5 m)1 and 10.3 m)1 for the wild-type, D287A, E53A and the double mutant, respectively A quantitative analysis of the data is not FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS R Chiaraluce et al Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl Fig GdmCl-induced fluorescence changes of pfLamA mutant forms in the presence of CaCl2 (A) D287A, (B) E53A and (C) E53A ⁄ D287A Fluorescence changes are reported as relative fluorescence intensity at 342 nm (left axis: j, h) and as intensity-averaged  emission wavelength k (right axis: d, s) calculated according to Eqn (1) The wild-type reversible transition in the presence of 40 mM CaCl2 monitored by relative fluorescence intensity at 342 nm (left axis: e, r) is also shown in (A) for comparison [2] The solid lines through the mutants unfolding data points (filled symbols) are intended to guide the eye of the reader and not represent the fitting of the data Reversibility points are indicated by empty symbols All spectra were recorded at 20 °C after 20 h of incubation at the indicated GdmCl concentrations at 40 lgỈmL)1 protein concentration Insets show intrinsic fluorescence emission spectra of the mutants measured after 20 h of incubation in 7.9 M GdmCl and 40 mM CaCl2 (continuous), the spectra resulting from the addition of CaCl2 to the mutants after 20 h of incubation in 7.9 M GdmCl (dotted) and the spectra of the native mutants in 20 mM Tris ⁄ HCl pH 7.4 (dashed) All spectra were recorded at 20 °C (290 nm excitation wavelength) possible because pfLamA wild-type and variant proteins are heterogeneously emitting systems; however, the results indicate that the fluorophores accessibility of the protein variants to the uncharged quencher, in comparison with the wild-type, decreases in 7.9 m GdmCl and increases in native conditions Discussion The results obtained with pfLamA mutant forms indicate that residues Glu53 and Asp287 are involved in calcium binding, in accordance to the homology modelling The pfLamA single (D287A and E53A) and double (E53A ⁄ D287A) mutants in 7.9 m GdmCl show a residual tertiary structure comparable to that of the wild-type; however, the integrity of the calcium binding site formed by Asp287 and Glu53 is essential for interaction with the cation in 7.9 m GdmCl An interesting finding of the present study is the significant decrease in the thermodynamic stability of the three pfLamA mutants in comparison to the wild-type, which shows a high DGH2O value of 61.5 kJỈmol)1 associated with its partial unfolding [2] The DGH2O associated with the reversible fluorescence changes at increasing GdmCl concentration is decreased from 1.7-, 1.8- to 1.9-fold with respect to the wild-type pfLamA, going from D287A, to E53A and to the double mutant, respectively Notably, the transition midpoints for the fluorescence changes of the three mutants are not significantly changed with respect to pfLamA wild-type; hence, the decrease of DGH2O values is mainly due to a decrease in mg values The decrease in mg value observed in all the pfLamA variant proteins is significant (1.6-fold) and not unprecedented for other single [22,33,34] and double-mutant proteins [7] The mecha- nism responsible for m– mutant proteins, which display a mg value lower than that of the wild-type, is usually referred to a decrease in the solvent-exposed surface area upon unfolding This is more frequently ascribed to an increase in the compactness of the residual structure in the non-native state ensemble, rather than to an FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6173 Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R Chiaraluce et al increase of the accessible surface area of the native state [26,27,29] Similar to that reported for most m– mutants [16], the spectral properties of the pfLamA variants in 7.9 m GdmCl not indicate to a significant increase in the structure of the non-native state to support the significant decrease in mg Consistent with these results and similar to that observed for the wild-type, the ANS binding experiments indicate that, in 7.9 m GdmCl, the hydrophobic surface area of pfLamA mutants is not significantly exposed An increase in compactness of the non-native state ensemble of the variants is suggested by the decreased fluorophores accessibility to the uncharged quencher acrylamide in 7.9 m GdmCl compared to that of the wild-type In native conditions, the spectral properties of the three variants point to tertiary structures almost identical to that of the wild-type; however, the higher fluorophores accessibility to the uncharged quencher suggests a less compact native state for the three mutant proteins A decrease in mg value upon single mutation has been also referred, in some cases, to the population of a third intermediate state during chemical unfolding [35]; however, in our experimental conditions, the presence of an intermediate state was not observed for any of the pfLamA variants Thus, the decrease of mg value may be ascribed to an increase in the compactness of the non-native state in 7.9 m GdmCl The effect of the double mutation on the thermodynamic parameters was non-additive, being lower than the sum of the effects of the two single mutations Non-additivity is generally observed when two mutated residues communicate directly or indirectly through electrostatic interactions or structural perturbation, so that they not behave independently [7,36] Therefore, the comparison of the thermodymamic parameters of the pfLamA double mutant with those of single mutants may provide information about any direct or indirect interconnection between the two mutated residues The non-additivity of the stability change can be expressed by the free energy coupling DDGint, a parameter calculated from a double-mutant cycle (Scheme 1) that reflects the interaction energy between the two mutated residues, Asp287 and Glu53 The positive value of the interaction free energy between the two mutated residues in pfLamA indicates that the double mutant is more stable than predicted, on the assumption that the effects of the two single mutants would be additive A significant DDGint (above 20 kJỈmol)1) has been related either to a direct communication between two residues or a short range steric interaction involving a mediating residue or a ligand [37] The large interaction free energy between the two pfLamA mutated residues (DDGint ¼ 24.2 ± 1.87 kJỈmol)1) is 6174 Scheme in accordance with the calcium binding results described in the present study The analysis of electrostatic interactions in pfLamA model, including histidine residues and considering a distance threshold of ˚ A, reveals that Glu53 may be involved in a large salt bridge network of seven ion pairs, three of which are ˚ strong ion pairs (distance threshold of A) whereas the Asp287 is involved in three ion pairs, only one of which is a strong ion pair (Fig 5) The putative calcium binding site is localized in proximity of the well conserved Asp287 residue in family 16 [2,6] The homology model suggests ionic interactions between Fig Model structure of the calcium binding site of wild-type pfLamA The pfLamA model is represented in teal blue cartoons The two acidic residues binding calcium and those with which they form ion pair interactions are shown as stick models with superimposed violet CPK space-filling models Carbon, oxygen and nitrogen atoms are displayed with green, red and blue colors, respectively Calcium ion is represented as an Nb sphere model with yellow color and a superimposed violet CPK space-filling model Orange dashes indicate ion pair interactions This figure was rendered using PYMOL [50] FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS R Chiaraluce et al the cation and the carboxylate moiety of both Glu53 and Asp287 (Fig 5) In native conditions, the spectroscopic studies of pfLamA variant proteins with the chromophoric chelator BAPTA show the involvement of Glu53 and Asp287 in the interaction with calcium and indicate that a second binding site might be present, as predicted by the homology model [2] In 7.9 m GdmCl, the capability to interact with calcium with a consistent recovery of tertiary structure is still observed, to a lesser extent than the wild-type, for D287A and E53A, but not for D287A ⁄ E53A Hence, the integrity of the calcium binding site formed by Asp287 and Glu53 is essential for interaction with Ca2+ in 7.9 m GdmCl The second calcium binding site revealed in native conditions by the chromophoric chelator BAPTA and located by modeling between Glu239 and Glu246 [2], either does not bind calcium in 7.9 m GdmCl or it interacts with the cation without affecting the enzyme tertiary structure In the presence of calcium, the GdmCl equilibrium transitions are complex and hysteretic for all the three mutants, indicating that the cation may stabilize some refolding intermediate(s) and ⁄ or increase the population of some states that are not evident under calcium depletion The comparison with the simple two state GdmCl transition of the wild-type in the presence of calcium [2] suggests that the integrity of the cation binding site formed by Glu53 and Asp287 prevents the population of folding intermediates The single or double substitution of Glu53 and Asp287 by alanine decreases the capability of pfLamA to interact with calcium as well as its thermodynamic stability but not its intrinsic resistance to denaturation, as indicated by the minor differences in the transition midpoints The destabilizing effect appears to be mainly realized through a stabilization of the nonnative state in 7.9 m GdmCl, rather than a destabilization of the native state, as suggested by the decrease of mg and of fluorophores accessibility to acrylamide for all the variants compared to the wild-type The replacement by Ala of any of the negatively charged residues shown to be involved in the composition of a calcium binding site induces a stabilization of the nonnative state in 7.9 m GdmCl comparable, but not identical, to that exerted by calcium on the wild-type [2] Ion pair networks play an important role in protein stability [8], and their involvement in interactions with cations offers new perspective in the analysis of the contribution of ions as cofactors in protein folding [38] and in the design of variants of proteins with enhanced stability [14] Changes in the denatured states induced by mutation affect protein stability [20,21] and, for thermophilic proteins, the persistence of residual Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl structure in non-native states may contribute to avoid irreversible denaturation under extreme environmental conditions [22] The stabilization of a compact nonnative state may represent a strategy for P furiosus endo-b-1,3-glucanase to thrive under the most adverse environmental conditions Experimental procedures Site-directed mutagenesis The pfLamA D287A mutant was prepared by overlap extension PCR [39], using the wild-type construct pET9d::LamA as template [6], with the primers 5¢-GCAAAG (sense) ATGGTGGTGGCATATGTAAGGGTTTAC-3¢ and 5¢-GTAAACCCTTACATATGCCACCACCATCT TTGC-3¢ (antisense) The E53A mutant and the double mutant (E53A ⁄ D287A) of pfLamA were produced using as primers 5¢-GCACGATGCGTTTGAAGG-3¢, and its complementary oligonucleotide The mutated bases are underlined The mutant forms of pfLamA E53A and E53A ⁄ D287A were produced using as template the wild-type construct pET9d::LamA [6] and the mutant construct pET24d::LamA D287A, respectively, using the QuikChangeTM site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA) The kit employs double-stranded DNA as template, two complementary oligonucleotide primers containing the desired mutation, and DpnI endonuclease to digest the parental DNA template Oligonucleotides were synthesized by MWG-Biotech AG (Anzinger, Germany) Escherichia coli strain DH5a cells were transformed The coding regions of the mutated pfLamA gene were sequenced to confirm the mutations and then E coli strain HMS174 (DE3) cells were transformed and used for expression Enzyme preparation and assay pf LamA wild-type was functionally produced in E coli BL21(DE3) strain, and the three mutant forms were functionally produced in E coli HMS174(DE3) strain and purified according to Kaper et al [40] The conditions used for expression and purification of the mutant proteins in E coli were as described for the wild-type enzyme The protein concentration was determined for wild-type and mutant forms at 280 nm using e280 ¼ 83070 m)1 · cm)1 calculated according to Gill and von Hippel [41] The enzyme activity was determined by measuring the amount of reducing sugars released upon incubation in 0.1 m sodium phosphate buffer, pH 6.5, containing mgỈmL)1 of laminarin, at 60 or 80 °C for 10 min, as described previously [40] Calciumdepleted protein was obtained by extensive dialysis with 100 lm EDTA and 100 lm EGTA in 10 mm Tris ⁄ HCl, pH 7.4 All the precautions required to prevent Ca2+ FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6175 Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R Chiaraluce et al contamination were followed during the preparation and storage of protein and buffer solutions [32] Calcium-loaded protein refers to the protein in the presence of 40 mm CaCl2 Chemicals and buffers ANS, dithiothreitol, EDTA, GdmCl, and laminarin were from Fluka (Buchs, Switzerland) 3¢,5¢-dinitrosalicylic acid was purchased from Sigma (St Louis, MO, USA) BAPTA was from Molecular Probes Europe BV (Leiden, the Netherlands) Buffer solutions were filtered (0.22 lm) and carefully degassed All buffers and solutions were prepared with ultra-high quality water (ELGA UHQ, High Wycombe, UK) Buffers for calcium titrations were prepared as previously described [32] Spectroscopic techniques Intrinsic fluorescence emission and 90° light scattering measurements were carried out with a LS50B Perkin Elmer spectrofluorimeter (Perkin Elmer, Waltham, MA, USA) using a 1.0-cm pathlength quartz cuvette Fluorescence emission spectra were recorded at 300–450 nm (1 nm sampling interval) at 20 °C with the excitation wavelength set at 290 nm 90° light scattering was measured at 20 °C with both excitation and emission wavelength set at 480 nm to check for the presence of aggregated particles Far-UV (185–250 nm) and near-UV (250–320 nm) CD measurements were performed at 20 °C in a 0.1–0.2-cm and 1.0-cm pathlength quartz cuvette, respectively CD spectra were recorded on a Jasco J-720 spectropolarimeter (Jasco Inc., Easton, MD, USA) The results are expressed as the mean residue ellipticity [Q] assuming a mean residue weight of 110 per amino acid residue In all the spectroscopic measurements, 100–250 lm EDTA was always present unless otherwise stated Experiments with the fluorescent dye ANS were performed at 20 °C by incubating each protein sample, wildtype and variant proteins, with ANS at : 20 molar ratio After min, fluorescence emission spectra were recorded at 400–600 nm with the excitation wavelength set at 390 nm The maximum fluorescence emission wavelength and the intensity of the hydrophobic probe ANS depend on the environmental polarity (e.g on the hydrophobicity of the accessible surface of the protein) [42] Fluorescence quenching was carried out by adding increasing amounts of acrylamide (0–104 mm) to solutions containing wild-type or variant proteins (40 lgỈmL)1) in 20 mm Tris ⁄ HCl, pH 7.4, 100 lm dithiothreitol and 100 lm EDTA Emission spectra (300–400 nm) were recorded at 20 °C after each acrylamide addition with the excitation wavelength set at 290 nm The effective quenching constants were obtained from modified Stern– Vollmer plots by analyzing F0 ⁄ DF versus ⁄ [acrylamide] (23 data points) [43] 6176 GdmCl-induced unfolding and refolding For equilibrium transition studies, protein samples (final concentration 40–50 lgỈmL)1) were incubated at 20 °C at increasing concentrations of GdmCl (0–8 m) in 20 mm Tris ⁄ HCl, pH 7.4, containing 100 lm dithiothreitol and 100 lm EDTA and, when indicated, 40 mm CaCl2 After 20 h, the equilibrium was reached and intrinsic fluorescence emission and far-UV CD spectra (0.2-cm cuvette) were recorded in parallel at 20 °C To test the reversibility of the unfolding, protein samples were unfolded at 20 °C in 7.8 m GdmCl at 0.8 mgỈmL)1 protein concentration in 25 mm Tris ⁄ HCl, pH 7.4, containing 100 lm dithiothreitol and 100 lm EDTA, in the presence and absence of 40 mm CaCl2 After 20 h, refolding was started by 20-fold dilution of the unfolding mixture, at 20 °C, into solutions of the same buffer used for unfolding containing decreasing GdmCl concentrations The final protein concentration was 40 lgỈmL)1 After 24 h, which had been established as sufficient to reach equilibrium, intrinsic fluorescence emission and far-UV CD spectra were recorded at 20 °C Data analysis The changes in intrinsic fluorescence emission spectra at increasing GdmCl concentrations were quantified as the  intensity-averaged emission wavelength, k [44] calculated according to  k ¼ RðIi ki Þ=RðIi Þ ð1Þ where ki and Ii are the emission wavelength and its corresponding fluorescence intensity at that wavelength, respectively This quantity is an integral measurement, negligibly influenced by the noise, which reflects changes in the shape and position of the emission spectrum Far-UV CD and near-UV CD spectra from GdmCl and Ca2+ titrations were analyzed by the singular value decomposition (SVD) algorithm [1,45] using the software matlab (MathWorks, South Natick, MA, USA) SVD is useful to find the number of independent components in a set of spectra and to remove the high-frequency noise and the low-frequency random error CD spectra in the 210–250 nm region or in the 250–310 region (0.2 nm sampling interval) were placed in a rectangular matrix A of n columns, one column for each spectrum collected in the titration The A matrix is decomposed by SVD into the product of three matrices: A ¼ U*S*VT where U and V are orthogonal matrices and S is a diagonal matrix The columns of U matrix contain the basis spectra and the columns of the V matrix contain the denaturant or the Ca2+ dependence of each basis spectrum Both U and V columns are arranged in terms of their decreasing order of the relative weight of informa- FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS R Chiaraluce et al Role of Glu53 and Asp287 in the stability in 7.9 tion, as indicated by the magnitude of the singular values in S The diagonal S matrix contains the singular values that quantify the relative importance of each vector in U and V The signal-to-noise ratio is very high in the earliest columns of U and V and the random noise is mainly accumulated in the latest U and V columns The wavelength averaged spectral changes induced by increasing denaturant or Ca2+ concentrations are represented by the columns of matrix V; hence, the plot of the columns of V versus the denaturant or Ca2+ concentrations provides information about the observed transition GdmCl-induced equilibrium unfolding was analyzed by fitting baseline and transition region data to a two-state linear extrapolation model [46] according to: DGunfolding ẳ DGH2 O ỵ mg ẵGdmCl ẳ RT ln Kunfolding ð2Þ where DGunfolding is the free energy change for unfolding for a given denaturant concentration, DGH2O is the free energy change for unfolding in the absence of denaturant and mg is a slope term that quantitates the change in DGunfolding per unit concentration of denaturant, R is the gas constant, T is the temperature and Kunfolding is the equilibrium constant for unfolding The model expresses the signal as a function of denaturant concentration: yi ẳ yN ỵ mN ẵXi ỵ yD ỵ mD ẵXi ị expẵDGH2 O mg ẵXi ị=RT ỵ expẵDGH2 O mg ẵXi ị=RT 3ị where yi is the observed signal, yN and yD are the native and denatured baseline intercepts, mN and mD are the native and denatured baseline slopes, [X]i is the denaturant concentration after the ith addition, DGH2O is the extrapolated free energy of unfolding in the absence of denaturant, mg is the slope of a G unfolding versus [X] plot, R is the gas constant and T is the temperature [GdmCl]0.5 is the denaturant concentration at the midpoint of the transition and, according to Eqn (2), is calculated as: ẵGdmCl0:5 ẳ DGH2 O =mg ð4Þ The free energy coupling parameter DDGint, which reflects the interaction energy between the two mutated residues Asp287 and Glu53, is calculated from a double mutant cycle (Scheme 1) [47] where the changes in the free energy relative to the GdmCl unfolding are denoted by M GdmCl protein variants E53A, D287A and E53A ⁄ D287A Hence, the free energy coupling parameter DDGint is calculated by: DDGint ¼ DGE53AWT DGE53A=D287AD287A ẳ DGD287AWT DGE53A=D287AE53A 5ị The standard error (SE) for determination of DDGint was determined by: ðSEDDGint ị2 ẳ SEDGE53A=D287A ị2 ỵ SEDGE53A ị2 ỵ SEDGD287A ị2 þ ðSEDGWT Þ2 ð6Þ Calcium titrations and determination of binding constants Calcium-depleted wild-type and mutant forms of pfLamA (9–16 lm) were titrated with CaCl2 in the presence of 24 lm of the chromophoric chelator BAPTA [48] BAPTA concentration was determined by measuring the absorbance at 263 nm using e239.5 ¼ 1.6 · 104 m)1Ỉcm)1 [32] Titrations were performed at 20 °C in 10 mm Tris ⁄ HCl pH 7.5 by the addition of 1–2 lL of CaCl2 solutions ranging from 0.015 mm to 20.0 mm to a mL protein solution containing 24 lm BAPTA Absorbance spectra were monitored between 200–450 nm after each CaCl2 addition To determine protein binding constants and number of binding sites, the variation of BAPTA absorbance as a function of calcium addition was fitted by nonlinear analysis using the caligator software [32] The v2 value as calculated by the program was used as the measure of the goodness-of-fit All the precautions required to prevent Ca2+ contamination were followed during the preparation and storage of the proteins and buffer solutions [32] Calcium titration of calcium-depleted wild-type and mutant forms of pfLamA in 7.9 m GdmCl (in 25 mm Tris ⁄ HCl, pH 7.4 containing 200 lm dithiothreitol and 250 lm EDTA) was performed by addition of increasing CaCl2 concentrations (0–40 mm) under continuous stirring Five minutes after each CaCl2 addition, near-UV CD (240–320 nm, 22 lm protein concentration) and fluorescence (300–450 nm, 1.2 lm protein concentration) spectra were recorded at 20 °C The spectral changes observed after each CaCl2 addition were not affected by a longer incubation time The concentration of unchelated Ca2+ was calculated using the program WINMAXC, version 2.40 [49] (http://www.stanford.edu/$cpatton/maxc.html) DGD287ẦWT ¼ DGWT À DGD287A ; DGE53A=D287ẦE53A ¼ DGE53A À DGE53A=D287A ; DGE53ẦWT ¼ DGWT À DGE53A ; DGE53A=D287AÀD287A Acknowledgements ¼ DGD287A À DGE53A=D287A This work was supported by a Grant from ‘Progetti strategici MIUR Legge 499 ⁄ 97’, Project Genefun and from FIRB 2003 RBNE034XSW We thank Dr Roberto Contestabile for helpful discussion where DG is the free energy change between the denatured and the native state for pfLamA wild-type (WT) and the FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6177 Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R Chiaraluce et al References Chiaraluce R, Van Der Oost J, Lebbink JH, Kaper T & Consalvi V (2002) Persistence of tertiary structure in 7.9 M guanidinium chloride: the case of endo-beta-1,3-glucanase from Pyrococcus furiosus Biochemistry 41, 14624–14632 Chiaraluce R, Gianese G, Angelaccio S, Florio R, van Lieshout JF, van der Oost J & Consalvi V (2005) Calcium-induced tertiary structure modifications of endobeta-1,3-glucanase from Pyrococcus furiosus in 7.9 M guanidinium chloride Biochem J 386, 515–524 Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach In Recent Advances in Carbohydrate Bioengineering (Gilbert HJ, Davies GJ, Henrissat B & Svensson B, eds), pp 3–12 Royal Society of Chemistry, Cambridge Strohmeier M, Hrmova M, Fischer M, Harvey AJ, Fincher GB & Pleiss J (2004) Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases ⁄ hydrolases in cell wall modification in the poaceae Protein Sci 13, 3200– 3213 Allouch J, Jam M, Helbert W, Barbeyron T, Kloareg B, Henrissat B & Czjzek M (2003) The three-dimensional structures of two beta-agarases J Biol Chem 278, 47171–47180 Gueguen Y, Voorhorst WG, van der Oost J & de Vos WM (1997) Molecular and biochemical characterization of an endo-b-1,3-glucanase of the hyperthermophilic archaeon Pyrococcus furiosus J Biol Chem 272, 31258– 31264 Green SM & Shortle D (1993) Patterns of nonadditivity between pairs of stability mutations in staphylococcal nuclease Biochemistry 32, 10131–10139 Kumar S & Nussinov R (2002) Relationship between ion pair geometries and electrostatic strengths in proteins Biophys J 83, 1595–1612 Makhatadze GI, Loladze VV, Ermolenko DN, Chen X & Thomas ST (2003) Contribution of surface salt bridges to protein stability: guidelines for protein engineering J Mol Biol 327, 1135–1148 10 Dominy BN, Minoux H & Brooks CL III (2004) An electrostatic basis for the stability of thermophilic proteins Proteins 57, 128–141 11 Lebbink JH, Consalvi V, Chiaraluce R, Berndt KD & Ladenstein R (2002) Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: cooperativity and electrostatic contribution to stability Biochemistry 41, 15524–15535 12 Karshikoff A & Ladenstein R (2001) Ion pairs and the thermotolerance of proteins from hyperthermophiles: a ‘traffic rule’ for hot roads Trends Biochem Sci 26, 550– 556 6178 13 Razvi A & Scholtz JM (2006) A thermodynamic comparison of HPr proteins from extremophilic organisms Biochemistry 45, 4084–4092 14 Razvi A & Scholtz JM (2006) Lessons in stability from thermophilic proteins Protein Sci 15, 1569–1578 15 Petsko GA (2001) Structural basis of thermostability in hyperthermophilic proteins, or ‘there’s more than one way to skin a cat’ Methods Enzymol 334, 469– 478 16 Shortle D (1996) The denatured state (the other half of the folding equation) and its role in protein stability FASEB J 10, 27–34 17 Anil B, Craig-Schapiro R & Raleigh DP (2006) Design of a hyperstable protein by rational consideration of unfolded state interactions J Am Chem Soc 28, 3144– 3145 18 Reed MA, Jelinska C, Syson K, Cliff MJ, Splevins A, Alizadeh T, Hounslow AM, Staniforth RA, Clarke AR, Craven CJ et al (2006) The denatured state under native conditions: a non-native-like collapsed state of N-PGK J Mol Biol 357, 365–372 19 Bowler BE (2007) Thermodynamics of protein denatured states Mol Biosyst 3, 88–99 20 Cho JH, Sato S & Raleigh DP (2004) Thermodynamics and kinetics of non-native interactions in protein folding: a single point mutant significantly stabilizes the N-terminal domain of L9 by modulating non-native interactions in the denatured state J Mol Biol 338, 827–837 21 Pace CN, Alston RW & Shaw KL (2000) Charge– charge interactions influence the denatured state ensemble and contribute to protein stability Protein Sci 9, 1395–1398 22 Robic S, Guzman-Casado M, Sanchez-Ruiz JM & Marqusee S (2003) Role of residual structure in the unfolded state of a thermophilic protein Proc Natl Acad Sci USA 100, 11345–11349 23 Shortle D & Ackerman MS (2001) Persistence of nativelike topology in a denatured protein in M urea Science 293, 487–489 24 Choy WY, Mulder FA, Crowhurst KA, Muhandiram DR, Millett IS, Doniach S, Forman-Kay JD & Kay LE (2002) Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques J Mol Biol 316, 101–112 25 Religa TL, Markson JS, Mayor U, Freund SM & Fersht AR (2005) Solution structure of a protein denatured state and folding intermediate Nature 437, 1053–1056 26 Shortle D (1995) Staphylococcal nuclease: a showcase of m-value effects Adv Protein Chem 46, 217–247 27 Wrabl J & Shortle D (1999) A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease Nat Struct Biol 6, 876–883 FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS R Chiaraluce et al 28 Myers JK, Pace CN & Scholtz JM (1995) Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding Protein Sci 4, 2138–2148 29 Pradeep L & Udgaonkar JB (2004) Effect of salt on the urea-unfolded form of barstar probed by m value measurements Biochemistry 43, 11393–11402 30 Carter PJ, Winter G, Wilkinson AJ & Fersht AR (1984) The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus) Cell 38, 835–840 31 Mildvan AS, Weber DJ & Kuliopulos A (1992) Quantitative interpretations of double mutations of enzymes Arch Biochem Biophys 294, 327–340 32 Andre I & Linse S (2002) Measurement of Ca2+-binding constants of proteins and presentation of the CaLigator software Anal Biochem 305, 195–205 33 Hammack B, Attfield K, Clayton D, Dec E, Dong A, Sarisky C & Bowler BE (1998) The magnitude of changes in guanidine-HCl unfolding m-values in the protein, iso-1-cytochrome c, depends upon the substructure containing the mutation Protein Sci 7, 1789–1795 34 Hammack BN, Smith CR & Bowler BE (2001) Denatured state thermodynamics: residual structure, chain stiffness and scaling factors J Mol Biol 311, 1091–1104 35 Spudich G & Marqusee S (2000) A change in the apparent m value reveals a populated intermediate under equilibrium conditions in Escherichia coli ribonuclease HI Biochemistry 39, 11677–11683 36 Wells JA (1990) Additivity of mutational effects in proteins Biochemistry 29, 8509–8517 37 LiCata VJ & Ackers GK (1995) Long-range, small magnitude nonadditivity of mutational effects in proteins Biochemistry 34, 3133–3139 38 Bushmarina NA, Blanchet CE, Vernier G & Forge V (2006) Cofactor effects on the protein folding reaction: acceleration of alpha-lactalbumin refolding by metal ions Protein Sci 15, 659–671 39 Ho SN, Hunt HD, Horton RM, Pullen JK & Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction Gene 77, 51–59 Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl 40 Kaper T, Verhees CH, Lebbink JH, van Lieshout JF, Kluskens LD, Ward DE, Kengen SW, Beerthuyzen MM, de Vos WM & van der Oost J (2001) Characterization of beta-glycosylhydrolases from Pyrococcus furiosus Methods Enzymol 330, 329–346 41 Gill SC & von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal Biochem 182, 319–326 42 Semisotnov GV, Rodionova NA, Razgulyaev OI, Uversky VN, Gripas’ AF & Gilmanshin RI (1991) Study of the ‘molten globule’ intermediate state in protein folding by a hydrophobic fluorescent probe Biopolymers 31, 119–128 43 Lehrer SS (1971) Solute perturbation of protein fluorescence The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion Biochemistry 10, 254–3263 44 Royer CA, Mann CJ & Matthews CR (1993) Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants Protein Sci 2, 1844–1852 45 Ionescu RM, Smith VF, O’Neill JC Jr & Matthews CR (2000) Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles Biochemistry 39, 9540–9550 46 Santoro MM & Bolen DW (1988) Unfolding free energy changes determined by the linear extrapolation method Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants Biochemistry 27, 8063–8068 47 Horovitz A & Fersht AR (1990) Strategy for analysing the co-operativity of intramolecular interactions in peptides and proteins J Mol Biol 214, 613–617 48 Tsien RY (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures Biochemistry 19, 2396–2404 49 Patton C, Thompson S & Epel D (2004) Some precautions in using chelators to buffer metals in biological solutions Cell Calcium 35, 427–431 50 DeLano WL (2002) The PyMOL Molecular Graphics System DeLano Scientific, San Carlos, CA FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6179 ... 7.9 M GdmCl and 40 mM CaCl2 (continuous), the spectra resulting from the addition of CaCl2 to the mutants after 20 h of incubation in 7.9 M GdmCl (dotted) and the spectra of the native mutants in. .. and the fluorescence properties (data not shown), Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl Fig Interaction of calcium with pfLamA wild-type and mutant forms in 7.9 M GdmCl (A) Left... value of 24.2 ± 1.87 kJỈmol)1 Effect of calcium on pfLamA mutants in 7.9 GdmCl and in native conditions M The addition of 40 mm CaCl2 to calcium-depleted samples of D287A and E53A in 7.9 m GdmCl