Báo cáo khoa học: Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding ppt

Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding Hueih-Min Chen 1 , Siu-Chiu Chan 1 , King-Wong Leung 1 , Jiun-Ming Wu 1 , Huey-Jen Fang 1 and Tian-Yow Tsong 2,3 1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan 2 Institute of Physics, Academia Sinica, Taipei, Taiwan 3 Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul, MN, USA Staphylococcal nuclease (SNase) is a globular protein that consists of 149 amino acids with a molecular mass of about 16 kDa. This protein lacks disulfide bonds in its native structure. Because of the absence of covalent bonds, electrostatic interactions, hydrophobic forces, van der Waals’ forces and hydrogen bonds are the only significant forces acting during protein denaturation and renaturation. Thus SNase provides a simplified model for studying protein folding. In addition, this protein contains a large number of charged amino acids, including 20 negatively charged residues and 32 positively charged residues (more than 35% of the 149 amino acids). When SNase is unfolded by pH titration in acid (pH 7 to pH 2), the transition point during unfolding occurs around pH 4 [1], which corresponds to the pK a of glutamic acid or aspartic acid [2]. The electrostatic interactions between charged residues therefore make significant contributions to protein stability in unfolding. Because electrostatic force is inversely proportional to the distance between charges, we chose to examine the interaction of oppositely charged residues that were predicted to be in close proximity to each other. We hypothesized that only certain charged amino acids at particular locations in SNase are critical for stabilizing local protein structure. Recent developments in the cloning and expression of SNase and its mutants in Escherichia coli have allowed this protein to be used as a model for biophys- ical studies of protein folding kinetics [1–3], rotational mobility of tryptophan residues [4], the effects of mutations on its structure and stability [5–7] and calorimetric analysis [6–8]. Our previous studies indica- ted the presence of significant electrostatic interactions between charged amino acids in the protein’s native state. Specifically, we identified a local stable segment surrounding the glutamic acid at position 75 formed Keywords staphylococcal nuclease; local stability; key acidic amino acid; unfolding Correspondence H M. Chen, Institute of BioAgricultural Sciences Academia Sinica, Taipei, Taiwan Fax: +886 2 2788 8401 Tel: +886 2 2785 5696 extn 8030 E-mail: robell@gate.sinica.edu.tw (Received 3 May 2005, revised 3 June 2005, accepted 13 June 2005) doi:10.1111/j.1742-4658.2005.04816.x Staphylococcal nuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of pro- tein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in ther- mal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lower DH cal values than the wild-type protein. Furthermore, two mutants, E75Q and E129Q, were created and the results imply that substitution of the Gly resi- due has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability. Two local stable areas formed by the charge–charge interactions around E75 and E129 with particular positively charged amino acids are thus iden- tified as being significant in maintenance of the three-dimensional structure of the protein. Abbreviations SNase, staphylococcal nuclease; WT, wild-type. FEBS Journal 272 (2005) 3967–3974 ª 2005 FEBS 3967 by the interactions of E75 with H121 and K97. It is believed that a small number of key amino acid seg- ments play major roles in generating these forces that most likely support the protein’s structural frame. If one observes a native protein as it refolds from a ran- dom unfolded state, the efficiency of refolding indi- cates that some energetically interactive areas within the protein must form preferentially due to relatively strong forces of attraction. Based on this model, pro- tein refolding can be considered a process of thermo- dynamic nucleation that includes an essential step in which specific stable segments are formed. We have been trying to find these stable segments in SNase: one of these may be located in the domain among E75, K9, Y93 and H121. An area formed by these inter- actions can be imagined as a ‘hand’ clamped tightly around the ‘neck’ of the whole protein. This clamped area was first suggested by Privalov’s group [8] with two strong interactions in SNase: an electrostatic inter- action between E75 and H121 and a hydrogen bond between D77 and T120. These bonds play significant roles in the cooperation of the two subdomains: a C-terminal a-helix and a b-barrel [9,10]. In our E75G mutant, X-ray diffraction results had shown that this locally stable network is absent due to the loss of the hydrogen bonding among E75 and K9, Y93 and H121 [11]. Instead, one water molecule hydrogen bonds to Y93 and H121. Both G75 and K9 are completely out- side of this local bonding area. Thus, the two sub- domains are unable to interact cooperatively. This structural rearrangement may allow E75G mutant to unfold with only half the enthalpic energy of the wild- type protein. If E75G mutant was to be denatured by a change in pH, we further predicted that its C-ter- minal a-helix would unfold through the I state (inter- mediate state) [8,12] and the flexible b-barrel would be subsequently destroyed. In addition to E75 forming a local stable area in SNase, in this study we searched for the existence of other local stable segments that may play similar key roles in sustaining protein native structure. We used site-directed mutagenesis to create 20 distinct glycine substitution mutants of single negative charged resi- dues in order to dissect elements important for SNase stability. We found that E75 and E129 play the role for retaining SNase tertiary structure. In order to dis- cern whether it is due to the absence of an acidic resi- due (glutamic acid or aspartic acid), or the presence of glycine which destabilizes SNase secondary structure, both E75Q and E129Q were created. The results revealed that only a little influence on the stability of protein tertiary structure is due to the particular sub- stitution of glycine at positions 75 and 129. The charge–charge interactions around these two local areas (around E75 and E129) are the most significant factors for maintaining the whole protein stability. Results CD spectra Secondary structures of SNase and its mutants were determined by CD with spectrapolorimetry measure- ments. Figure 1 shows the superimposed far-UV CD curves of wild-type SNase and G substitute mutants. All mutants other than E129G had similar spectra to the wild-type protein with two separated negative peaks at 208 nm and 222 nm, consistent with the pres- ence of an a-helix [13,14]. In contrast, the E129G mutant exhibited less CD than the others. Thus, while most of the mutations of negatively charged glutamic acid or aspartic acid residues to uncharged glycine resi- dues do not disrupt the secondary structure of SNase, replacement of E129 with G may cause changes in pro- tein secondary structure. However, this replacement of G at E129 does not significantly influence its whole protein stability (see Results of thermal analysis sec- tion below). Tryptophan fluorescence spectra W140 is located near the flexible C terminus of SNase. Changes in the fluorescence intensity of W140 reflect a change in the surrounding hydrophobic environment of the tryptophan and thus indicate a change in the overall (tertiary) structure of the protein [15]. Figure 2 Fig. 1. CD spectra of wild-type and SNase mutants. CD spectra (far UV) of nine proteins (WT, E73G, E75G, E101G, E122G, E129G, D77G, D83G and D95G). All spectra are similar except the E129G spectrum (bold line). Protein concentration, 0.5 mgÆmL )1 . Local stability and key acidic amino acids in staphylococcal nuclease H M. Chen et al. 3968 FEBS Journal 272 (2005) 3967–3974 ª 2005 FEBS shows the fluorescence spectra of the wild-type and glycine mutants. Peak fluorescence intensities of the mutants at W140 were 21% to 45% lower than that of the wild-type protein. As shown in Fig. 2, the peak amplitude for the E129G mutant was exceptionally low, with a 45% decrease in fluorescent intensity relat- ive to the wild-type protein. The fluorescence changes of W140 were in agreement with the thermodynamic results obtained from thermal unfolding (DH cal ) (for data see the following section and Table 1). These data indicate that the mutants are much less stable than the wild-type protein. In the E129G mutant, the trypto- phan at residue 140 may be exposed to the water environment due to the mutant’s loosened tertiary structure. For those mutants exhibiting only small decreases in fluorescence intensity, those amino acids around the tryptophan are probably still in the form of their native conformation. This hypothesis is sup- ported by the CD results, showing that some secon- dary structure still exists in these mutants (Fig. 1). Thermal analysis of protein unfolding Thermal denaturation determined by DSC was used to measure the stability of the mutant proteins. Based on the measurements of heat capacity over a range of temperatures (DSC curves are shown in Fig. 3), parameters such as melting point (T m ), calorimetric enthalpy (DH cal ), difference of DH cal of mutant from wild-type (%), heat capacity (DC p ) and difference of DC p of mutant from wild-type (%) were determined and the results for the wild-type protein and 20 mutants are given in Table 1. The DH cal and T m of the wild-type protein were in good agreement with previ- ous studies [7]. The various mutations, however, exis- ted difference in their effects on the stability of SNase. Among them, eight mutants (E73G, E75G, E101G, E122G, E129G, D77G, D83G and D95G) showed decreases in DH cal of greater than 20% compared with the wild-type protein, indicating that these charged residues have a strong influence on the unfolding of SNase. The heat denaturation curves for the wild-type protein and eight mutants are shown in Fig. 3, demon- strating that the mutants have heat denaturation pro- perties distinct from the wild-type protein. These mutants had lower DH cal and T m values, and their DSC curves were shifted toward lower temperatures than the wild-type protein. D77G, D83G and D95G mutants, in which aspartic acid residues were replaced by glycine, had T m values of about 44.14 °C, 37.21 °C and 37.38 °C, respectively, compared with the wild- type T m of 50.98 °C. As listed in Table 1, the DH cal values for D77G, D83G and D95G were about 64.35 kcalÆmol )1 , 57.50 kcalÆmol )1 and 64.35 kcalÆ mol )1 , respectively, 26% to 33% lower than that of the wild-type protein. The thermodynamic parameters of the glycine susti- tution mutants also differed greatly from the wild-type protein. The T m values of these mutants ranged from 35 °Cto55°C. Among them, the E75G and E129G mutants showed the largest decreases in thermal dynamic quantities. The T m values of E75G (37.0 °C) and E129G (34.39 °C) were much lower than that of wild-type protein (50.98 °C). E129G had the lowest DH cal of the mutants (47.6 kcalÆmol )1 ) and E75G had the second lowest (48.2 kcalÆmol )1 ). These DH cal values are more than 44.13% (E129G) and 43.43% (E75G) lower than that of the wild-type protein, suggesting that Glu residues at positions 75 and 129 play key roles in maintaining the native structure of the protein. Furthermore, two mutants, E75Q and E129Q, were created for consideration. The results of thermal dena- turation show that E75Q (DH cal ¼ 51.4 ± 2.0 kcalÆ mol )1 ) and E129Q (DH cal ¼ 54.2 ± 0.8 kcalÆmol )1 ) have approximately more than 40% (vs. 43% of E75G) and 37% (vs. 44% of E129G), respectively, lower DH cal values than the wild-type protein. These outcomes indicate that little influence is exerted by Gly Fig. 2. Steady-state fluorescent spectra of wild-type and mutants of SNase. Spectra of nine proteins (WT, E73G, E75G, E101G, E122G, E129G, D77G, D83G and D95G). The spectrum of E129G (bold line) is much lower in intensity than the others. Protein con- centration, 0.4 mgÆmL )1 . H M. Chen et al. Local stability and key acidic amino acids in staphylococcal nuclease FEBS Journal 272 (2005) 3967–3974 ª 2005 FEBS 3969 residue substitution on the destabilization of secondary structure that leads to the large perturbation of the stability of the protein tertiary structure (although there is about 7% difference of thermal energy change between E129Q and E129G). Discussion Protein folding and unfolding are driven by the free energy of stabilization when a peptide chain in ran- dom configuration (denatured state, D) folds into the compact three-dimensional structure of the native state (N) [16,17]. For SNase, we previously reported the conversion of these two states by the kinetic scheme [18–22] N ¢ D 1 ¢ D 2 ¢ D 3 , where D i denotes the denatured states of protein and the conversion energy between each state less than 5 kcalÆmol [1]. Therefore, the measured energy of thermal unfolding mainly contributes to the conversion of N to D. For wild-type SNase, the unfolding energy needed is about 85.9 kcalÆmol )1 (Table 1). This energy is used to destroy all network forces in the native protein. Some significant local forces play roles to help this network frame [7] resist unfolding. In the scheme above, the structure and dynamics of the N state of SNase has been elucidated, but relatively little is known about the D state [23]. Zhou used the Gaus- sian chain model [24] to point out that an unfolded protein could be viewed as a collection of peptide Table 1. Comparison of thermodynamic parameters for wild-type SNase and 20 SNase mutants in which negative charged D and E residues were mutated to G. Phosphate buffer (25 m M Na 2 HPO 4 ,25mM NaH 2 PO 4 ,100mM NaCl, pH adjusted to 7.0) was used. All proteins were used at a concentration of 2 mgÆmL )1 . WT, wild-type. Difference of DH from WT (%) is calculated by [(DH mutant –DH WT ) ⁄DH WT] ] · 100. Differ- ence of DC p from WT (%) is calculated by[(DC pmutant –DC pWT ) ⁄DC p ( WT ) · 100. Average T m (°C) DH (kcalÆmol )1 ) Difference of DH from WT (%) DC p (kalÆmol )1 ÆK )1 ) DDC p (kcalÆmol )1 ÆK )1 ) Difference of DC p from WT (%) Wild-type 50.98 ± 0.28 85.90 ± 1.40 2.47 ± 0.10 E10G 43.80 ± 0.10 68.90 ± 0.10 )19.13 0.52 ± 0.08 )1.95 )78.95 E43G 54.99 ± 0.07 91.55 ± 0.05 7.45 2.81 ± 0.10 0.34 13.77 E52G 52.10 ± 0.07 82.15 ± 0.05 )3.58 2.28 ± 0.08 )0.19 )7.69 E57G 46.60 ± 0.08 75.35 ± 0.35 )11.56 1.62 ± 0.10 )0.85 )34.41 E67G 46.53 ± 0.62 71.40 ± 1.00 )16.20 4.14 ± 0.12 1.67 67.61 E73G 37.00 ± 0.06 67.35 ± 0.35 )20.95 0.22 ± 0.04 )2.25 )91.09 E75G 36.99 ± 0.06 48.20 ± 0.50 )43.43 1.49 ± 0.03 )0.98 )39.68 E101G 43.04 ± 0.02 61.20 ± 0.50 )28.17 0.55 ± 0.07 )1.92 )77.73 E122G 44.12 ± 0.13 62.95 ± 0.25 )26.12 0.30 ± 0.05 )2.17 )87.85 E129G 34.59 ± 0.09 47.60 ± 0.50 )44.13 0.59 ± 0.04 )1.88 )76.11 E135G 44.54 ± 0.08 69.55 ± 0.50 )18.37 1.92 ± 0.06 )0.55 )22.27 E142G 49.41 ± 0.15 82.25 ± 0.15 )3.46 0.75 ± 0.08 )1.72 )69.64 D19G 52.06 ± 0.07 81.45 ± 0.75 )5.18 1.06 ± 0.03 )1.41 )57.09 D21G 53.74 ± 0.20 82.75 ± 1.55 )3.67 3.41 ± 0.07 0.94 38.06 D40G 50.44 ± 0.11 84.55 ± 0.85 )1.57 1.95 ± 0.10 )0.52 )21.05 D77G 44.14 ± 0.12 64.35 ± 0.35 )25.09 0.45 ± 0.03 )2.02 )81.78 D83G 37.21 ± 0.20 57.50 ± 0.70 )33.06 )0.48 ± 0.03 )2.95 )119.43 D95G 37.38 ± 0.19 64.35 ± 0.65 )25.09 )0.72 ± 0.07 )3.19 )129.15 D143G 50.53 ± 0.01 86.85 ± 0.25 1.11 0.12 ± 0.03 )2.35 )95.14 D146G 50.99 ± 0.03 84.50 ± 0.40 )1.63 2.6 ± 0.04 0.13 5.26 Fig. 3. Calometric melting curves of wild-type and mutants of SNase. DSC curves of nine proteins (WT, E73G, E75G, E101G, E122G, E129G, D77G, D83G and D95G). Lines 3 and 6 represent E75G and E129G, respectively; the curves are lower in intensity than the others. Protein concentration, 2 mgÆmL )1 . Thermodynamic parameters such as DH cal were calculated based on the description of Privalov and Potekhin [6]. Local stability and key acidic amino acids in staphylococcal nuclease H M. Chen et al. 3970 FEBS Journal 272 (2005) 3967–3974 ª 2005 FEBS fragments that dynamically vary their conformation and relative distances. For SNase pH denaturation, the D i states (scheme shown above) can be considered as many different stages of an assembly with local structural elements capturing each other. However, we do not favour the author’s theoretical conclusion [24] that residual electrostatic effects on protein re- folding are not important due to the nonrandom interactions between charged amino acids. Experi- mentally, our results based on thermal unfolding and pH titration [22] indicate that the local electrostatic interactions at specific positions (E75 and E129, Table 1) play important roles in stabilizing protein structure [25,26]. Fink and his coworkers [27] have demonstrated that SNase is only marginally stable at pH 7 due to a combination of high net protein charge and low hydrophobicity. Anderson et al. used NMR as a tool to measure protein apparent pK a and emphasized that the electrostatic contribution of each ionizable group may play a role in the stability of the folded SNase [28]. Schwehm et al. also reported that electrostatic interactions are the most important net stabilizing factor in SNase for single site mutations that reverse or neutralize a surface charge [29]. On the contrary, Shortle and coworkers [30] argued that ionizable amino acids do not contribute greatly to the stability of SNase. Their viewpoint was based on the similar calculated free energy differences, DDG (DG mut,H2O –DG wt,H2O ) and fluorescence titration melt- ing points, m GuHCl , of total 104 mutants. It should be noted that Shortle et al. used indirect measure- ments and much theoretical calculation to determine protein stability, which may account for their hypo- thesis that ionizalbe residues do not significantly affect SNase stability. Our study on 20 mutants using thermal analysis is more accurate and we believe this refects the acutal stability shifts due to single charged amino acid mutations. In our pH titration study (pH 7.0–2.0), we found that SNase protein could be denatured by the addition of only 2.5 protons (data not shown). Therefore, we hypothesize that pH unfolding SNase is mainly processed by the addition of 2.5 protons and both E75 and E129 are the targets of protonation. It is of interest to determine which other amino acids these glutamic acid residues at positions 75 and 129 might be interacting with to stabilize SNase struc- ture. Based on the X-ray structure (1EYD) [31] of SNase, the positively charged amino acids expected to have the most significant interactions with E75 (OE1 ⁄ OE2) – are H121 (NE2) + (¼ 3.06 A ˚ ) and K9 (Nz) + (¼ 5.25 A ˚ ). Similarly, the amino acids expected to have the strongest interactions with E129 (OE1 ⁄ OE2) – are K110 (Nz) + (¼ 6.29 A ˚ ) and K133 (Nz) + (¼ 6.36 A ˚ ). These two regions with predicted strong local interactions are circled in Fig. 4. Within the circled areas, other charged amino acids such as D77, D83 and E101 may provide supportive forces to the areas surrounding the interactions with E75 and E129. Privalov and coworkers reported the presence of noncovalent bonding, including an electrostatic bond between E75 and H121, and a hydrogen bond between D77 and T1208. Thus interactions between D77 and E75 and surrounding positively charged amino acids may result in the cooperative formation of two sub- domains: a C terminus comprised of an a-helix and a b-barrel. This hypothesis is supported by our thermal DSC data, indicating decreases of 25.09% (D77G) to 43.43% (E75G) in the enthalpic energy of these mutants compared to wild-type SNase. Furthermore, D83G, which results in a 33.06% decrease in DH com- pared to wild-type SNase, is also located between the two subdomains. These results imply that E75, D77 and D83 are part of the same local stable area. From this study, E129G and E101G mutations resulted in decreases of 44.13% and 28.17%, respectively, in ther- mal stability as compared with wild-type protein. These residues are located in two adjacent helices of SNase, namely helix 2 (V99 to Q106) and helix 3 (E122 to K134). This finding implies that E129 and E101 form another local stable area through inter- actions with K110 and K133 (Fig. 4) and is in agree- ment with the fluorescence measurements in which the Fig. 4. Local stable segments in SNase. The X-ray crystal structure of wild-type SNase was obtained from the Protein Data Bank. The circled areas show local stable segments: interactions of G75 with H121 and K9, and of G129 with K110 and K133. Other charged amino acids such as D73, D83 and E101 reinforce the interactions of E75 and E129. H M. Chen et al. Local stability and key acidic amino acids in staphylococcal nuclease FEBS Journal 272 (2005) 3967–3974 ª 2005 FEBS 3971 amplitude of E129G was decreased by about 44% compared with the wild-type protein. Conclusions Comparisons of thermal stability and protein unfold- ing for wild-type SNase and 20 glycine mutants suggest that the negatively charged aspartic acid and glutamic acid residues in the protein play unequal roles in main- taining the protein’s native structure. More than one local stable segment is present: E75 with K9 and H121 form one local stable area and E129 with K110 and K133, located in two adjacent a-helices (helix 3 and helix 2, respectively) form another in SNase. These two areas must be significantly perturbed before the protein can be unfolded. Experimental procedures Materials Luria–Bertani (LB) broth and isopropyl thio-b-d-galactoside were from USB (Cleveland, OH). Salmon testes DNA and some analytical grade chemicals such as EDTA, Tris ⁄ HCl, CaCl 2 , NaCl and mineral oil were from Sigma (St Louis, MO, USA). Salmon testes DNA applied for the enzyme activity test was used without further purification. Guani- dine hydrochloride and dNTPs were from Boehringer (Mannheim, Germany). Ethanol (> 99%) was from Pan- reac (Barcelona, Spain). Urea was a product of Acros, USA. The Stratagene Quickchange TM kit containing Pfu DNA polymerase, 10 · reaction buffer and DpnI restriction enzyme was from Stratagene (La Jolla, CA, USA). Water used for these experiments was deionized and distilled. PCR site-directed mutagenesis The wild-type SNase nuc gene (originally obtained from D. Shortle, Johns Hopkins University, Baltimore, MD, USA) was cloned into pTrc-99 A and used to transform Escherichia coli strain JM105. Plasmid DNA was purified by the alkaline lysis method (Gibco-BRL, Gaithersburg, MD, USA; GFX TM kit), and stored at )20 °C before being subject to mutagenesis. Twenty complementary 33-mer oligonucleotides were synthesized to each introduce one gly- cine at positions 10, 19, 21, 40, 43, 52, 57, 67, 73, 75, 77, 83, 95, 101, 122, 129, 135, 142, 143 and 146 of wild-type SNase (Life Technologies, Rockville, MD, USA). Similarly, two complementary 33-mer oligonucleotides were also syn- thesized to each introduce one glutamine at positions 75 and 129 of the SNase. For site-directed mutagenesis, a 10 · reaction buffer (Stratagene, QuickChange TM kit) was mixed with 1.5 lL double-stranded DNA template, 1.2 lLofa pair of complementary oligonucleotides, 1 lL10mm each dNTP and double-distilled H 2 O to a final volume at 50 lL. One microlitre Pfu DNA polymerase (2.5 UÆlL )1 ) was added to the solution, and the mixture was overlaid with 30 lL mineral oil. A PCR consisting of 16 cycles of 50 °C (1.5 min), 68 °C (14 min), and 94 °C (1 min) was per- formed using a PerkinElmer 480 thermal cycler (Foster City, CA, USA). The wild-type DNA template was then digested by adding 1 lLofDpnI restriction enzyme (10 UÆlL )1 ) to the PCR mixture and incubating at 37 °C for 1 h. Ten microlitres of the reaction (containing undi- gested mutant plasmid) were used to transform 100 lLof competent JM105 cells. The mixture was incubated on ice for 1 h and at 42 °C for 2 min, followed by a further 2 min incubation on ice. After transformation, 800 lLofLB medium were added and incubated was at 37 °C for 1 h. Transformed cells were selected on plates containing ampi- cillin (100 lgÆmL) and mutant DNA was isolated from the resulting colonies. Mutant plasmids were identified by BamHI and NcoI restriction digestion and sequences were confirmed by DNA sequencing. DNA sequencing Plasmid DNA was isolated using a GFX TM Micro Plasmid Prep Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and the resulting double-stranded DNA was mixed with 8 lL of BigDye TM master mix (BigDye TM Terminator Ready Reaction Kit, Applied Biosystems) and 3.2 pmol sequencing primer. The final solution was mixed with de- ionized water to a final volume of 20 lL in a 0.5-mL thin- walled PCR tube and overlaid with 40 lL of light mineral oil. DNA sequencing was performed by cycle sequencing using 25 cycles of 96 °C for 30 s, 50 °C for 15 s, and 60 °C for 4 min in a Perkin-Elmer 480 thermal cycler. The exten- sion products were purified by Centri-Sep TM spin column chromatography (Princeton Separation, Adelphia, NJ, USA) to remove unincorporated dye terminators. Five microlitres of template suppression reagent (PE Applied Biosystems) was mixed with the purified extension prod- ucts. The samples were heated at 95 °C for 2 min and then chilled on ice. Capillary electrophoresis was performed using an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems) fitted with a 47 cm capillary containing POP-6 polymer. The mutant sequences (positions 1–149 for mutant proteins or 1–139 and 1–141 for truncated proteins) were compared to that of wide-type and were confirmed to have the correct mutant sequence. Protein purification Escherichia coli JM105 carrying recombinant plasmids were grown in LB broth containing 100 lgÆmL )1 ampicillin at 37 °C. Protein expression was induced by adding isopropyl thio-b-d-galactoside. The cells were harvested after 4 h of incubation and suspended in chilled buffer A (6 m urea, Local stability and key acidic amino acids in staphylococcal nuclease H M. Chen et al. 3972 FEBS Journal 272 (2005) 3967–3974 ª 2005 FEBS 0.05 m Tris ⁄ HCl, 0.2 m NaCl, pH 9.2, filtered through a 0.45 lm membrane). Proteins were collected after two alco- hol precipitations and stored in buffer B (6 m urea, 0.05 m Tris ⁄ HCl, pH 9.2, filtered through a 0.45 lm membrane). The recombinant proteins were purified by cation exchange chromatography (WASHED CM-25 ion-exchange gel col- umn). The proteins were dialysed after purification for 2 days at 4 °C against phosphate buffer (0.025 m NaH 2 PO 4 , 0.025 m NaHPO 4 , 0.1 m NaCl, pH adjusted to 7.0) and were then lyophilized. The average yields of the recombinant proteins were about 30Æmg L )1 . SNase purity was investigated by SDS ⁄ PAGE, gels were stained with Coomassie blue and analysed by a densitometer, revealing protein purity of greater than 90%. Protein concentration was determined by measuring the extinction coefficient with Gill and von Hippel’s method [32]. Circular dichroism Circular dichroism was performed on wild-type protein and mutants using a Jasco Model J-720 spectropolarime- ter. The spectra were measured between 200 and 320 nm. Wild-type and mutant proteins were dissolved in phos- phate buffer (0.025 m NaH 2 PO 4 , 0.025 m NaHPO 4 , 0.1 m NaCl, pH adjusted to 7.0) at a concentration of 0.5 mgÆmL )1 . Spectra were obtained as the average of five successive scans with a bandwidth of 1.0 nm and a scan speed of 20 nmÆmin )1 . Steady-state tryptophan fluorescence measurements Measurements were made with a LS-50B Spectrometer (PerkinElmer). Samples were dissolved in phosphate buffer at 0.4 mgÆmL )1 . Excitation was set at 298 nm and emis- sions were observed at 350 nm. The fluorescence spectra were measured between 300 and 550 nm with a scanning speed of 150 nmÆs )1 and an excitation slit of 5.0 nm. Calorimetric measurements Thermal analysis of protein denaturation was performed by nano DSC (Model 6100 Nano II; Calorimetry Sciences Corp., Provo, UT, USA). Lyophilized wild-type and mutant SNase were dissolved in phosphate buffer (25 mm NaH 2 PO 4 ,25mm NaHPO 4 , 100 mm NaCl, pH adjusted to 7.0) at a concentration of 2 mgÆmL )1 . Samples were soni- cated for 15 min, and then 1 mL of buffer or sample was loaded into a clean reference or sample cell, respectively, ensuring that the samples were free of air bubbles. Samples were heated from 20 °Cto75°C under 3 atm at a heating rate of 1 °CÆmin )1 . The melting point (T m ) was obtained directly from the DSC curve. The enthalpy change (DH cal ) was calculated by the integration of the curve covering area (T m was taken as the curve peak point) using origin soft- ware. Acknowledgements This work wass partially supported by a grant (NSC- 92-2311-B-001) from National Science Council, Tai- wan, R.O.C and the theme project of Academia Sinica, Taipei, Taiwan, R.O.C. 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