Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding Zhengding Su 3 , Jiun-Ming Wu 1 , Huey-Jen Fang 1 , Tian-Yow Tsong 2,3 and Hueih-Min Chen 1 1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC 2 Institute of Physics, Academia Sinica, Taipei, Taiwan, ROC 3 Department of Biochemistry, University of Minnesota College of Biological Sciences, St Paul, MN, USA Staphylococcal nuclease (SNase) is a single-domain protein of 149 amino acids. Its 3D structure has been examined by NMR [1–3] and X-ray crystallography [4,5]. However, only part of the structure (positions 1– 141) can be confirmed. The segment 142–149 has not been defined with certainty because of its apparent flexibility. Structural observations made with NMR or X-ray have led to the prediction that certain amino acid(s) in the flexible segment stabilize the rest of the structure, in particular the key amino acid(s) located close to this flexible segment. The tryptophan at posi- tion 140, for example, may play an important role in maintaining the protein structure formed by amino acids 1–141. In this study, we used site-directed muta- genesis to generate point mutations and truncations around this position to explore the above prediction. As shown by Chen and colleagues [6], SNase protein can be unfolded by lowering its pH (for example, from pH 7 to pH 2). About 2.5 protons are associated with the key glutamic amino acid residues at positions 75 and 129. This association between protons and key amino acids leads the protein to unfold. However, the refolding process may be different and more complex because the transition of refolding is from many unfolded states to a single native state. Based on previous kinetic experi- ments using single-jump and double-jump stopped-flow for refolding [7,8], SNase protein can be refolded in vitro to its active 3D conformation in milliseconds. The sequence of equilibrium reactions between the three denatured states and one native state can be shown as [9] N « D 1 « D 2 « D 3 , where N is the protein in its native state and D i (i ¼ 1–3) indicates the protein in its unfolded state. This scheme has been used to solve puz- zles such as accumulated intermediates and to conduct random searches among ‘microscopic states’ [8]. The early stages of refolding (D i fi N) occur via key amino acid(s) which act as nucleation centres before proton dissociation. Subsequently, these centres trigger the condensation of random polypeptide chains into the compact form of the native state. In this study, the effects of mutating W140 [10] on SNase protein conformation and stability were Keywords aromatic amino acid; refolding; stability; staphylococcal nuclease Correspondence H-M. Chen, Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan 115, R.O.C. Fax: +886 2 2788 8401 Tel: +886 2 2785 5696 ext. 8030 E-mail: robell@gate.sinica.edu.tw (Received 3 May 2005, revised 3 June 2005, accepted 13 June 2005) doi:10.1111/j.1742-4658.2005.04814.x Staphylococcal nuclease (SNase) is a model protein that contains one domain and no disulfide bonds. Its stability in the native state may be maintained mainly by key amino acids. In this study, two point-mutated proteins each with a single base substitution [alanine for tryptophan (W140A) and alanine for lysine (K133A)] and two truncated fragment proteins {positions 1–139 [SNase(1–139) or W140O] and positions 1–141 [SNase(1–141) or E142O]} were generated. Differential scanning micro- calorimetry in thermal denaturation experiments showed that K133A and E142O have nearly unchanged DH cal relative to the wild-type, whereas W140A and W140O display zero enthalpy change (DH cal  0). Far-UV CD measurements indicate secondary structure in W140A but not W140O, and near-UV CD measurements indicate no tertiary structure in either W140 mutant. These observations indicate an unusually large contribution of W140 to the stability and structural integrity of SNase. Abbreviations DSC, differential scanning calorimetry; SNase, staphylococcal nuclease. 3960 FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS investigated. Two point-mutated proteins, with a single base substitution of alanine for tryptophan (W140A) and alanine for lysine (K133A), and two truncated fragment proteins, with fragments 1–139 [SNase(1– 139) or W140O] and 1–141 [SNase(1–141) or E142O], were generated. The effect of these C-terminal trunca- tions and point mutations to C-terminal residues on SNase stability and conformation integrity was exam- ined by subjecting these mutants and wild-type pro- teins to CD [11,12] and differential scanning calorimetry (DSC) [13,14]. The preliminary refolding process in terms of assembly of fragments is discussed. Results CD spectra Secondary structure of SNase and its mutants was deter- mined by CD with spectrapolorimetry measurements. The approximate fraction of secondary structure type that is present in any protein can be determined by its far-UV CD spectrum as the sum of fractional multiples of the reference spectra for each structural type. Figure 1A shows the CD spectra of W140A, E142O and K133A. There are no significant differences from that of the wild-type protein. In contrast, W140O has a completely different CD spectrum. There are no peak minima at 222 nm and 208 nm, but instead a peak mini- mum at 200 nm, indicating that it has no secondary structure only random coil. We can therefore postulate that, when the segment beyond W140 was removed, the secondary structure of the protein disintegrated. Using the peak at 222 nm of the wild-type protein as an index of helical conformational stability, this CD spectrum of W140O suggests a decrease in helicity on removal of all amino acids beyond position 140. In CD spectra in the near-UV region (Fig. 1B), the wild-type protein and mutants E142O and K133A show strong intensity at  277 nm (h  )77 degreesÆcm )2 Æ dmol )1 ), revealing an intact tertiary conformation. In contrast, W140A and W140O both lacked tertiary struc- ture, as their intensities at 277 nm were only h  )22 and )13 degreesÆcm )2 Ædmol )1 (Fig. 1B). However, W140A at 295 nm had a similar spectrum to that of the wild-type protein, but W140O did not (h  0). This may indicate that the aromatic F or Y in the W140A mutant are more ordered and compact than in the W140O protein. 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 hydrophobic environment surrounding W140 and thus indicate a change in the overall (ter- tiary) structure of the protein. Figure 2 shows the fluorescence spectra of wild-type SNase and the four mutants E142O, K133A, W140A and W140O. The fluorescence spectra of E142O and K133A are similar to that of the wild-type protein. However, the fluores- cence in the mutants without tryptophan at 140 (W140A and W140O) was much lower than both the wild-type protein and mutants E142O and K133A. Thermal analysis of protein unfolding The DSC curves of the wild-type protein and the mutants K133A, E142O, W140A and W140O are shown in Fig. 3, with their thermodynamic parameters summarized in Table 1. The calorimetric DH cal values Fig. 1. CD spectra of wild- type and SNase mutants. (A) CD spectra (far-UV) of five proteins [wild-type (WT), W140A, E140O, W140O and K133A]. All spectra are similar except the W140O spectrum (bold line). (B) CD spectra (near-UV) of the same proteins. Protein concentration was 0.5 mgÆmL )1 . Z. Su et al. Staphylococcal nuclease refolding FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS 3961 of the wild-type protein and K133A and E142O were 88.45, 84.70, and 83.60 kcalÆmol )1 , respectively. The melting point and DSC profile of E142O (52.8 °C) was almost identical with that of the wild-type protein (52.32 °C). K133A had a similar DH cal but a slightly different melting point (47.70 °C) compared with the wild-type protein. These results suggest that lysine at position 133 and the residues that follow glutamic acid at position 142 play negligible roles in maintaining the native structure of SNase. In contrast, W140A and W140O revealed no distinct maxima in their DSC profiles. This is very different from the wild-type protein and K133A and E142O, which showed a significant enthalpy change. This implies that either the protein has poor stability or no secondary ⁄ tertiary structure results from either repla- cing the tryptophan at position 140 with alanine or the removal of a fragment from positions 140–149. This is supported by the melting points of the proteins (52.32 °C for the wild-type and ‘not detectable’ for W140A and W140O). Flanagan et al. [15] reported that multiple mutations can cause large changes in the average conformation of denatured proteins. Here we show that a specific single mutation or removal of a specific fragment can cause large changes in the native state of SNase. Discussion In addition to its local electrostatic interactions, which contribute largely to SNase stability via specific charged amino acids (Results shown in the preceding paper), other nonelectrostatic interactions at even more specific positions play a significant role in maintaining SNase tertiary structure. Our CD and DSC data show that the W140 in SNase is the amino acid responsible for the stability of the whole protein. However, in comparison with the wild-type protein, the mutant W140A retains signifi- cant secondary structure (Fig. 1A). Yin & Jing [16] reported that W140 plays an important role in the native-like SNase conformation and the enrichment of an ordered secondary structure. If tryptophan is relocated from position 140 to 34, the mutant (F34W ⁄ W140F) [17] retains its secondary structure, but its tertiary structure is lost. This implies that tryp- tophan plays a significant role in maintaining the 3D structure only at 140 (wild-type SNase has only one W). Without W140, the secondary structure may be maintained by other interactions such as local stable segments interacting around E75 and E129, areas with oppositely charged amino acids. The truncated protein W140O in this study (deletion of residues 1–140) shows zero enthalpy on thermal unfolding (Table 1) and has no secondary structure (Fig. 1A). This indicates that, without tryptophan and residues 140–149, the protein Fig. 2. Steady-state fluorescent spectra of wild-type and mutant SNase. Spectra of five proteins [wild-type (WT), W140A, E140O, W140O and K133A]. Protein concentration was 0.4 mgÆmL )1 . Fig. 3. Calometric melting curves of wild-type and mutants of SNase. DSC curves of five proteins [wild-type (WT), W140A, E140O, W140O and K133A]. The curves of W140A and W140O are nearly linear in terms of intensity. All protein concentrations were 2 mgÆmL )1 . Staphylococcal nuclease refolding Z. Su et al. 3962 FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS cannot maintain its tertiary conformation or even the secondary motif. However, if residues 142–149 are removed, the protein largely retains its 3D structure (Table 1 and Fig. 2). Related experiments have been performed previously. Parker et al. [18] showed that the apparent association constant of SNase(1–126) bound to pdTp in the presence of Ca 2+ is approxi- mately threefold lower than that of the wild-type pro- tein. Therefore, some or all of the residues in the 127– 149 segment are critical for maintenance of the native conformation of the protein. In addition, Griko and coworkers [19], studying SNase(1–136), showed that this fragment (residues 1–136) has no secondary struc- ture but retains the tertiary conformation. They thus proved that SNase(1–136) is not a ‘molten globule’ protein, although its unfolding process was a first- order phase transition. This situation contrasts with our W140O [SNase(1–139)] mutant, which maintains its secondary structure to a certain extent but has no tertirary conformation (Fig. 1A). As to the point of fragment assembly during protein folding, Anfinsen and coworkers [20,21] originally studied the enzymic effect using overlapping structure- less fragments [for instance, SNase(99–149) interacting with SNase(1–126)] and found that the reconstructed complex was active and exhibited much of the ordered structure. Anfinsen consequently predicted that this protein was organized from its polypeptide chain into N-terminal and C-terminal centers during refolding [21]. Since then, many scientists have been interested in demonstrating this concept by using N-terminal and C-terminal fragments of SNase as models for the study of folding and unfolding pathways. For example, Choy & Kay [22] using NMR 15 N and 2 H spin relaxation techniques illustrated that the fragment D131D (dele- tion of wild-type residues 3–12 and 141–149) of SNase was completely unfolded under nondenaturing condi- tions (pH  5 and low salt concentration). This is because they observed much more vibrational motion in the unfolded protein than in folded ones on the picosecond time scale. Taniuchi et al. [23] used both SNase(1–126) and SNase(50–149) as typical fragments to form type I and type II (in equilibrium) complex proteins. They reported that these two overlapping fragments could form enzymically active complement- ary structures, and their folding rates were not related to any decrease in energy from the unfolded to the folded state. Feng et al. [24] created the SNase frag- ments SNase(1–110), SNase(1–121) and SNase(1–135) and used them in NMR spectroscopy experiments to study the folding process. They concluded that the conformation of these fragments could be considered as native-like partially folded and unfolded states. Recently, they further used the short N-terminal frag- ments SNase(1–20), SNase(1–28) and SNase(1–36) to show that the folding nucleation sites of SNase may start from the N-terminus [25]. However, we used the C-terminal fragments SNase(1–140) and SNase(1–142) to show that SNase(1–140) plays a role in the assembly of the protein during refolding. Our results also show that SNase(1–140), without Trp at position 140, does not have any structure, whereas the fragment including Trp at position 140, i.e. SNase(1–142), has a similar structure to the wild-type enzyme. Our experiments show that tryptophan at position 140 plays an important role in maintaining protein ter- tiary integrity. Figure 4A shows how tryptophan (loop 1) may interact with loop 2 and loop 3. These inter- actions seem to form a ‘lower neck’ in the protein as compared with the ‘upper neck’ which is formed by E75 with H121 and K915. The forces responsible for the interactions between these loops are not yet clear. One possibility is that the loops together with W140 constitute a nucleation center. The indole ring [26] of W140 may be the main target for the interaction with both loop 2 and loop 3. Furthermore, from the protein folding scheme discussed previously [9], N « D 1 « D 2 « D 3 , the energy needed for transfor- mation among the D i states of protein unfolding ⁄ refolding is negligible (4.57 kcalÆmol )1 for D 1 « D 2 and 4.32 kcalÆmol )1 for D 2 « D 3 ) and a possible intermediate state such as SNase(1–139) exists Table 1. DSC results of SNase and its mutants. Phosphate buffer (25 mM Na 2 HPO 4 ⁄ 50 mM NaH 2 PO 4 ⁄ 200 mM NaCl, pH adjusted to 7.0) was used in the experiments. All proteins were used at a concentration of 2 mgÆmL )1 . Difference from DH of WT (%) calculated by [(DH mutant ) DH WT ) ⁄DH WT ] · 100. Difference from DC p of WT (%) calculated by [(DC p mutant ) DC pWT ) ⁄DC pWT ] · 100. WT, Wild-type; ND, not detectable. Average T m (°C) DH (kcalÆmol )1 ) Difference of DH from WT (%) DC p (kcalÆmol )1 ÆK )1 ) DDC p (kcalÆmol )1 ÆK )1 ) Difference of DC p from WT (%) Wild-type 52.32 ± 2.5 88.45 ± 1.40 2.40 ± 0.20 K133A 47.70 ± 0.35 84.70 ± 2.5 )1.40% 2.38 ± 0.05 )0.02 )0.8% E142O 52.80 ± 0.55 83.60 ± 3.5 )2.68% 2.41 ± 0.04 0.01 0.4% W140A ND ND )100% ND ND )100% W140O ND ND )100% ND ND )100% Z. Su et al. Staphylococcal nuclease refolding FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS 3963 (Fig. 4B, State 2). When tryptophan occupies position 140, the whole protein is folded into its native confor- mation (Fig. 4B, State 3). Summary Trp140 located in the C-terminal loop of SNase plays an important role in protein stability and conforma- tional integrity. During protein unfolding ⁄ refolding, the addition of 2.5 protons (both E75 and E129 are the targets of protonation) results in SNase unfolding, with refolding at an early stage through the formation of a SNase(1–139) fragment in which the tryptophan at position 140 is needed for formation of the native structure. Experimental procedures Materials Luria–Bertani broth and isopropyl thio-b-d-galactoside were purchased from USB (Cleveland, OH). Salmon testes DNA and some analytical grade chemicals such as EDTA, Tris ⁄ HCl, CaCl 2 , NaCl and mineral oil were obtained from Sigma (St Louis, MO, USA). Salmon testes DNA for the enzyme activity test was used without further purification. Guanidine hydrochloride and dNTPs were purchased from Boehringer (Mannheim, Germany). Ethanol (> 99%) was obtained from Panreac (Barcelona, Spain). Urea was a product of Acros (Pittsburgh, PA, USA). The Quick- change TM kit containing Pfu DNA polymerase, 10 · reac- tion buffer and DpnI restriction enzyme was purchased 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, the Johns Hopkins University, Baltimore, MD, USA) was cloned into pTrc-99A, which was used to trans- form Escherichia coli strain JM105. Plasmid DNA was purified by the alkaline lysis method (Gibco-BRL, Gaithers- burg, MD, USA; GFX TM kit), and stored at )20 °C before being subjected to mutagenesis. Two complementary 33-mer primers that included the alanine codon at positions 140 or 133 were designed and synthesized (Life Technologies, Rockville, MD, USA). Single-point mutations were made by site-directed mutagenesis to generate W140A and K133A. Truncated proteins W140O (the segment between positions 140 and 149 of the wild-type protein was removed) and E142O (the segment between positions 142 and 149 of the wild-type protein was removed) were generated by using suitable complementary primers. For site-directed mutagen- esis, a 10 · reaction buffer (Stratagene; QuickChange TM kit) was mixed with 1.5 lL dsDNA template, 1.2 lL of a pair of complementary oligonucleotides, 1 lL10mm each dNTP and double-distilled water to a final volume of 50 lL. Then 1 lL Pfu DNA polymerase 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 performed using a PerkinElmer 480 thermal cycler (Wellesley, MA, USA). The wild-type DNA template was then digested by adding 1 lL DpnI restriction enzyme to the PCR mixture and incubating at 37 °C for 1 h. Then 10 lL of the reaction mixture (containing undi- gested mutant plasmid) was used to transform 100 lL B A Fig. 4. Global segment interactions and the folding profiles in wild- type SNase. (A) W140, in loop 1 interacts with loop 2 and loop 3 which forms a ‘lower neck’ network area in maintaining protein ter- tiary structure. State 1 denotes the nascent polypeptide fragment. (B) The fragment folding pathway induced via the SNase(1–139) fragment and formed to its minimum energy native state via the addition of either tryptophan at position 140 or fragment 140–149. Staphylococcal nuclease refolding Z. Su et al. 3964 FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS competent JM105 cells. The mixture was incubated on ice for 1 h, and at 42 °C for 2 min, followed by 2 min incuba- tion on ice. After transformation, 800 lL Luria–Bertani medium was added, and the mixture incubated at 37 °C for 1 h. Transformed cells were selected on ampicillin plates, and mutant DNA was isolated from the resulting colonies. Mutant plasmids were then identified by BamHI and NcoI restriction digestion, and their sequences confirmed by DNA sequencing. DNA sequencing Plasmid DNA was isolated with the GFX TM Micro Plas- mid Prep Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and the resulting dsDNA was mixed with 8 lL BigDye TM master mix (BigDye TM Terminator Ready Reac- tion Kit; Applied Biosystems, Foster City, CA, USA) and 3.2 pmol sequencing primer. The final solution was mixed with deionized water to a final volume of 20 lLina 0.5-mL thin-walled PCR tube and overlaid with 40 lL 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 PerkinElmer 480 thermal cycler. The extension products were purified by a Centri- Sep TM spin column chromatography (Princeton Separa- tions, Adelphia, NJ, USA) to remove unincorporated dye terminators. Template suppression reagent (5 lL; PE Applied Biosystems, Foster City, CA, USA) was mixed with purified extension products. The samples were heated at 95 °C for 2 min and then chilled on ice. Capillary elec- trophoresis was performed using an ABI PRISM 310 Gen- etic 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 with that of the wild-type and confirmed to have the correct mutant sequences. Protein purification Escherichia coli JM105 carrying recombinant plasmids were grown in Luria–Bertani broth containing 100 lg ÆmL )1 ampicillin at 37 °C. Protein expression was induced by adding isopropyl thio-b-d-galactoside. The cells were har- vested after 4 h of incubation and suspended in chilled buffer A (6 m urea, 0.05 m Tris, 0.2 m NaCl, pH 9.2, fil- tered through a 0.45 lm membrane). Proteins were collec- ted after two alcohol precipitations and stored in buffer B (6 m urea, 0.05 m Tris, 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 column). The proteins were dialysed after purification for 2 days at 4 °C against phosphate buffer (25 mm NaH 2 PO 4 ⁄ 50 mm NaHPO 4 ⁄ 200 mm NaCl, pH adjusted to 7.0) and were then lyophilized. The average yield of recombinant proteins was  25 mgÆL )1 . SNase purity was investigated by SDS ⁄ PAGE. The gel was stained with Coomassie blue and analyzed by densitome- try, revealing protein purity of greater than 85%. Protein concentration was determined by measuring the absorption coefficient of each mutant by the method of Gill & von Hippel [27]. CD measurements CD was performed on wild-type protein and mutants using a Jasco model J-720 spectropolarimeter. The spectra were measured between 200 and 320 nm. Wild-type and mutant proteins were dissolved in phosphate buffer (25 mm NaH 2 PO 4 ⁄ 50 mm NaHPO 4 ⁄ 200 mm NaCl, pH adjusted to 7.0) at a concentration of 0.5 mgÆmL )1 . Spec- tra 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 a concentration of 0.4 mgÆmL )1 . Excitation was set at 298 nm, and emissions were observed at 350 nm. The fluor- escence 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 with DSC (a model 6100 Nano II; Calorimetry Sciences Corp., Provo, UT, USA). Lyophilized wild-type and mutant SNase were dissolved in phosphate buffer at a concentration of 2 mg ÆmL )1 . Samples were first sonicated for 15 min. Then 1 mL 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 ) of protein tested was directly obtained from the DSC curve. The enthalpy change (DH cal ) of each protein was calculated by integra- tion of the curve covering area (T m was taken as the curve peak point) using origin software. Acknowledgements This work is partially supported by a grant (NSC- 92-2311-B-001) from the National Science Council, Taiwan, R.O.C. and the theme project of Academia Sinica, Taipei, Taiwan, R.O.C. Z. Su et al. 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