Probing the subtle conformational state of N138ND2-Q106O hydrogen bonding deletion mutant (Asn138Asp) of staphylococcal nuclease using time of flight mass spectrometry with limited proteolysis

Recent studies indicate that the N138ND2-Q106O hydrogen bonding deletion in staphylococcal nuclease significantly alters the conformational integrity and stability of the nuclease. To find out the structural basis of the changes, mass spectrometry and limited proteolysis methods were combined to probe the subtle conformational changes in the SNaseN138D mutant and SNaseN138D-Ca2+-pdTp complex. The results reveal that the N138ND2-Q106O hydrogen bonding deletion makes the C-terminal part of alpha-helix 1 and alpha-helix 2 in the C-terminal subdomain of SNaseN138D unfold to some extent, but does not have much effect on the N-terminal part of alpha-helix 1, alpha-helix 3, and the N-terminal beta-barrel subdomain of SNaseN138D. Binding of ligands makes the alpha-helices 1 and 2 more resistant to protease Glu-C attack and converts the partially unfolded state to a native-like state. This study also demonstrates how mass spectrometry can be combined with limited proteolysis to observe conformational changes induced by ligand binding.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Role of C-terminal region of Staphylococcal nuclease for foldability,stability, and activity

The role of the C-terminal region of Staphylococcal nuclease (SNase) was examined by deletion mutation. Deletions up to eight residues do not affect the structure and function. The structure and enzymatic activity were partially lost by deleting Ser141-Asn149 (Delta141-149), and deletion of Trp140-Asn149 (Delta140-149) resulted in further loss of structure and activity. A 13-residue deletion showed the same effect as the 10-residue deletion. Both Ser141Gln and Ser141Ala mutations for an eight-residue deletion mutant did not alter properties as well as Ser141A1a for full-length SNase. In contrast, Trp140Ala mutation for Delta141-149 shows the same effect as the deletion of Trp140. Trp140Ala mutation for full-length SNase causes the loss of native structure. These observations indicate the significance of the 140th and the 141st residues. The side-chain of the 140th residue is required to be tryptophan; however, the backbone of the 141st residue is solely critical for foldability, but the side-chain information is not crucial. All of the mutants that take a non-native conformation show enzymatic activity and inhibitor-induced folding, suggesting that foldability is required for the activity.     

Deletion of the omega-loop in the active site of staphylococcal nuclease. 2. Effects on protein structure and dynamics

It has been shown (Poole et al., 1991) that deletion of residues 44-49 from the sequence of staphylococcal nuclease (E43 SNase) results in an enzyme (E43 delta SNase) that is significantly more active than D43 SNase, an enzyme that differs from the wild-type enzyme by deletion of a single methylene group. In addition, both E43 delta SNase and D43 delta SNase are significantly more stable than their respective parent enzymes. Herein we use high-resolution 2D and 3D NMR spectroscopy to characterize the solution conformations of the four enzymes in order to better understand their differences in stability and activity. The backbone assignments of E43 SNase were extended to the three mutant proteins (uniformly 15N-enriched) by using 2D HSQC, 3D HOHAHA-HMQC, and 3D NOESY-HMQC spectra. The NOE patterns observed for E43 and D43 SNase in solution are consistent with the crystal structures of these proteins. The NOESY data further show that the intact and deleted proteins have essentially the same structures except that (a) the disordered omega-loops in the intact proteins are replaced by tight type II' turns, formed by residues 43-50-51-52, in the deleted proteins and (b) the orientation of the D43 side chain in crystalline D43 SNase differs from that found for D43 delta SNase in solution. Except for regions neighboring the omega-loops, the intact and deleted proteins show nearly identical amide 15N and 1H chemical shifts. In contrast, there are widespread, small and similar, chemical shift differences (a) between E43 SNase and D43 SNase and (b) between E43 delta SNase and D43 delta SNase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding

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 microcalorimetry in thermal denaturation experiments showed that K133A and E142O have nearly unchanged DeltaH(cal) relative to the wild-type, whereas W140A and W140O display zero enthalpy change (DeltaH(cal) approximately 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.     

Molecular Dynamics Studies of the Inhibitor C34 Binding to the Wild-Type and Mutant HIV-1 gp41: Inhibitory and Drug Resistant Mechanism

Mutations on NHR (N-terminal heptad repeat) associated with resistance to fusion inhibitor were observed. In addition, mutations on CHR (C-terminal heptad repeat) accompanied NHR mutations of gp41 are noted in many cases, like N43D/S138A double mutation. In this work, we explored the drug resistant mechanism of N43D mutation and the role of S138A second mutation in drug resistance. The binding modes of the wild type gp41 and the two mutants, N43D and N43D/S138A, with the HIV-1 fusion inhibitor C34, a 34-residue peptide mimicking CHR of gp41, were carried out by using molecular dynamics simulations. Based on the MD simulations, N43D mutation affects not only the stability of C34 binding, but also the binding energy of the inhibitor C34. Because N43D mutation may also affect the stable conformation of 6-HB, we introduced S138A second mutation into CHR of gp41 and determined the impact of this mutation. Through the comparative analysis of MD results of the N43D mutant and the N43D/S138A mutant, we found that CHR with S138A mutation shown more favorable affinity to NHR. Compelling differences in structures have been observed for these two mutants, particularly in the binding modes and in the hydrophobic interactions of the CHR (C34) located near the hydrophobic groove of the NHR. Because the conformational stability of 6-HB is important to HIV-1 infection, we suggested a hypothetical mechanism for the drug resistance: N43D single mutation not only impact the binding of inhibitor, but also affect the affinity between NHR and CHR of gp41, thus may reduce the rate of membrane fusion; compensatory mutation S138A would induce greater hydrophobic interactions between NHR and CHR, and render the CHR more compatible to NHR than inhibitors.     

Searching for folding initiation sites of staphylococcal nuclease: a study of N-terminal short fragments

The N-terminal short fragments of staphylococcal nuclease (SNase), SNase20, SNase28, and SNase36, corresponding to the sequence regions, Ala1-Gly20, Ala1-Lys28, and Ala1-Leu36, respectively, as well as an 8-residue peptide (Ala17-Ile18-Asp19-Gly20-Asp21-Thr22-Val23-Lys24) have been synthesized. The conformational states of these fragments were investigated using CD and NMR spectroscopy in aqueous solution and in trifluoroethanol (TFE)-H(2)O mixture. SNase20 containing a sequence corresponding to a bent peptide in native SNase shows a transient population of bend-like conformation around Ala12-Thr13-Leu14 in TFE-H(2)O mixture. The sequence region of Ala17-Thr22 of SNase28 displays a localized propensity for turn-like conformation in both aqueous solution and TFE-H(2)O mixture. The conformational ensemble of SNase36 in aqueous solution includes populated turn-like conformations localized in sequence regions Ala17-Thr22 and Tyr27-Gln30. The analysis suggests that these sequence regions, which form the regular secondary structures in native protein, may serve as the folding nucleation sites of SNase fragments of different chain lengths starting from the N-terminal end. Thus, the formation of bend- and turn-like conformations of these sequence regions may be involved in the early folding events of the SNase polypeptide chain in vitro.     

Monoclonal antibody, a novel probe for protein folding

Ｈｙｂｒｉｄｏｍａａｎｔｉｂｏｄｉｅｓａｒｅａｐｏｗｅｒｆｕｌｔｏｏｌｆｏｒｓｔｕｄｙｉｎｇｓｔｒｕｃｔｕｒｅａｎｄｆｕｎｃｔｉｏｎｏｆｐｒｏｔｅｉｎｓｏｗｉｎｇｔｏｔｈｅｉｒａｂｉｌｉｔｙｔｏｒｅｃｏｇｎｉｚｅａｎｄｂｉｎｄｔｈｅｉｒｃｏｒｒｅｓｐｏｎｄｉｎｇａｎｔｉｇｅｎｓａｔｓｐｅｃｉａｌｒｅｇｉｏｎｓ(ｉ.ｅ.ｅｐｉｔｏｐｅ)ｗｉｔｈｈｉｇｈｅｆｆｉｃａｃｙａｎｄｓｐｅｃｉｆｉｃｉｔｙ.Ｍｏｎｏｃｌｏｎａｌａｎｔｉｂｏｄｉｅｓ,ｅｓｐｅｃｉａｌｌｙｔｈｅｃｏｎｆｏｒｍａｔｉｏｎｄｅｐｅｎｄｅｎ…     

Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability

The high-resolution X-ray structure of wild-type staphylococcal nuclease (E43 SNase) suggests that Glu 43 acts a general basic catalyst to assist the attack of water on a phosphodiester substrate [Loll, P., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183]. Glu 43 is located at the base of the solvent-exposed and conformationally mobile omega-loop in the active site of E43 SNase having the sequence Glu43-Thr44-Lys45-His46-Pro47-Lys48- Lys49-Gly50-Val51-Glu52, where the gamma-carboxylate of Glu 52 is hydrogen bonded to the amide hydrogen of Glu 43. With a metabolic selection for SNase activity produced in an Escherichia coli host, we detected an unexpected deletion of residues 44-49 of the omega-loop of E43 SNase in cassette mutagenesis experiments designed to randomize codons 44 and 45 in the omega-loop and increase the activity of the previously described E43D mutation (D43 SNase). A high-resolution X-ray structure of D43 SNase has revealed that the E43D substitution significantly changes the structure of the omega-loop, reduces the interaction of the essential Ca2+ ion with its active-site ligands, and diminishes the network of hydrogen-bonded water molecules in the active site [Loll, P., & Lattman, E. E. (1990) Biochemistry 29, 6866]. This deletion of six amino acids from the omega-loop generates a protein (E43 delta SNase) having a partially solvent-exposed, surface beta-turn with the sequence Glu43-Gly50-Val51-Glu52; the structure of this beta-turn is addressed in the following article [Baldisseri et al. (1991) Biochemistry (following paper in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of conformational features of staphylococcal nuclease in ternary complexes with pdTp, pdGp, and nitrophenyl-pdTp.

The conformations of wild-type staphylococcal nuclease (SNase) in the ternary complexes with thymidine 3',5'-bisphosphate (pdTp), 2'-deoxyguanine 3',5'-bisphosphate (pdGp), and thymidine 3'-phosphate 5'-(p-nitrophenylphosphate) (NpdTp) with Ca2+ were examined by two-dimensional NMR NOESY and ROESY experiments. The results of these experiments indicate that the conformational features of the SNase are quite similar in the three ternary complexes. This suggests that the conformational features of SNase, in these ternary complexes, are not strongly dependent on whether the 5'-phosphate is a mono- or diester. This is in contrast to our prior studies on substitutions of active site charged amino acids which indicated that the conformational features of SNase in the ternary complex are quite sensitive to substitutions for active site charged amino acids (Hibler et al., 1987; Wilde et al., 1988; Pourmotabbed et al., 1990). The similarity of the SNase conformational features in the ternary complexes with pdTp and pdGp indicates that the features of the nucleotide bound at the active site are not strong determinants of the enzyme conformation in the ternary complexes. These conclusions are in general agreement with the results on pdApdT ternary complexes with SNase which suggested that it is the conformational features of the bound nucleic acid which determine the differences in catalysis observed for SNase with different substrates (Weber et al., 1991), more so than the conformational features of the enzyme.     

Conformational adjustments of SNase R and its N-terminal fragments probed by monoclonal antibodies

Two monoclonal antibodies specific for staphylococcal nuclease R (SNase R) (McAb2C9 and McAb1B8) were prepared and used to probe protein folding during peptide elongation, by measuring antibody binding to seven N-terminal fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) of SNase R. Comparative studies of the conformations of the N-terminal fragments have shown that all seven fragments of SNase R have a certain amount of residual structure, indicating that folding may occur during elongation of the nascent peptide chain. We show that the binding abilities of the intact enzyme and its seven fragments to the monoclonal antibodies are not simply proportional to the length of the peptide chain, suggesting that there may be continuous conformational adjustment in the nascent peptide chain as new C-terminal amino acids are added. A folding intermediate close in structure to the native state but with structural features in common with SNR121 is highly populated in 0.6 M GuHCl, and is also formed transiently during folding.     

Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum.

The gene for staphylococcal nuclease (SNase), an extracellular enzyme of Staphylococcus aureus, was introduced into Corynebacterium glutamicum. The heterologous gene was expressed in this host organism, and SNase was efficiently exported to the culture medium. Amino-terminal sequencing of SNase secreted by C. glutamicum revealed that the signal peptide was apparently cleaved off at precisely the same position as in the original host, S. aureus. As with S. aureus, a second smaller form of SNase (A form), whose appearance is presumably the result of a secondary processing step, was found in the culture medium of the recombinant C. glutamicum strain. The A form was one residue shorter than the mature nuclease A produced by S. aureus. Variation of the sodium chloride concentration in the growth medium had a marked influence on the location and the processing of SNase by C. glutamicum. In a complex growth medium containing 4% sodium chloride, SNase was exclusively located in the supernatant, but a significant amount of the enzyme remained cell associated if the strain was grown in a low-salt medium. Also, high salt concentrations seemed to inhibit processing of the high-molecular-weight form of SNase (B form) to the smaller A form. Similarities and differences in the export and modes of processing of SNase by three different, nonrelated gram-positive host organisms are discussed. Finally, a versatile Escherichia coli-C. glutamicum tac-lacIq expression shuttle vector was constructed. With this vector, it was possible to achieve isopropyl-beta-D-galactopyranoside (IPTG)-inducible overexpression and secretion of SNase in C. glutamicum, whereby the expression level was dependent on the concentration of the inducer.     

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.     

Folding of the C-terminal fragment V111-D143 of staphylococcal nuclease in aqueous solution

Studies of conformational features of fragments SNase(111-143) and SNase(118-143) and segment E122-K136 in 1-139 fragment (SNase139) suggest that the high intrinsic helical propensity can drive segment E122-K136 fold into a stable helix only when the segments V111-H121 and L137-D143 flanked on segment E122-K136 in staphylococcal nuclease (SNase) have stable folding.     

金黄色葡萄球菌核酸酶C末端去9肽对酶蛋白溶液构象的影响

通过多维异核核磁共振方法,结合运用荧光和圆二色等光谱方法,比较研究了V8菌株金黄色葡萄球菌核酸酶（含149个氨基酸残基）,酶蛋白1－140片段（SNase140)以及在TMP（thymidine 5′-monophosphate)和Ca^2 存在下的SNase140的溶液构象状态。探讨了酶蛋白C末端去9肽后对酶蛋白构象和活力的影响。研究指出,远离酶蛋白活性部位残基间相互作用的变化,将通过酶蛋白两个亚结构域之间所形成的氢键,影响酶蛋白活性部位的空间构象,从而影响酶蛋白的活力。     

13C-, 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins.

Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with [U-(13)C(17),U-(15)N(4)]FAD and analysed, in their oxidized and reduced states, by (13)C-, (15)N- and (31)P-NMR spectroscopy. The enzymes studied showed very similar (31)P-NMR spectra in the oxidized state, consisting of two peaks at -9.8 and -11.5 p.p.m. In the reduced state, the two peaks merge into one apparent peak (at -9.8 p.p.m.). The data are compared with published (31)P-NMR data of enzymes closely related to TrxR. (13)C and (15)N-NMR chemical shifts of TrxR and the mutant proteins in the oxidized state provided information about the electronic structure of the protein-bound cofactor and its interactions with the apoproteins. Strong hydrogen bonds exist between protein-bound flavin and the apoproteins at C(2)O, C(4)O, N(1) and N(5). The N(10) atoms in the enzymes are slightly out of the molecular plane of the flavin. Of the ribityl carbon atoms C(10alpha,gamma,delta) are the most affected upon binding to the apoprotein and the large downfield shift of the C(10gamma) atom indicates strong hydrogen bonding with the apoprotein. The hydrogen bonding pattern observed is in excellent agreement with X-ray data, except for the N(1) and the N(3) atoms where a reversed situation was observed. Some chemical shifts observed in C138S deviate considerably from those of the other enzymes. From this it is concluded that C138S is in the FO conformation and the others are in the FR conformation, supporting published data. In the reduced state, strong hydrogen bonding interactions are observed between C(2)O and C(4)O and the apoprotein. As revealed by the (15)N chemical shifts and the N(5)H coupling constant the N(5) and the N(10) atom are highly sp(3) hybridized. The calculation of the endocyclic angles for the N(5) and the N(10) atoms shows the angles to be approximately 109 degrees, in perfect agreement with X-ray data showing that the flavin assumes a bent conformation along the N(10)/N(5) axis of the flavin. In contrast, the N(1) is highly sp(2) hybridized and is protonated, i.e. in the neutral state. Upon reduction of the enzymes, the (13)C chemical shifts of some atoms of the ribityl side chain undergo considerable changes also indicating conformational rearrangements of the side-chain interactions with the apoproteins. The chemical shifts between native TrxR and C138S are now rather similar and differ from those of the two other mutant proteins. This strongly indicates that the former enzymes are in the FO conformation and the other two are in the FR conformation. The data are discussed briefly in the context of published NMR data obtained with a variety of flavoproteins.     

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study

A continuum electrostatics model is used to calculate the relative stabilities of 117 mutants of staphylococcal nuclease (SNase) involving the mutation of a charged residue to an uncharged residue. The calculations are based on the crystallographic structure of the wild-type protein and attempt to take implicitly into account the effect of the mutations in the denatured state by assuming a linear relationship between the free energy changes caused by the mutation in the native and denatured states. A good correlation (linear correlation coefficient of approximately 0.8) is found with published experimental relative stabilities of these mutants. The results suggest that in the case of SNase (i) charged residues contribute to the stability of the native state mainly through electrostatic interactions, and (ii) native-like electrostatic interactions may persist in the denatured state. The continuum electrostatics method is only moderately sensitive to model parameters and leads to quasi-predictive results for the relative mutant stabilities (error of 2-3 kJ mol(-1) or of the order of k(B)T), except for mutants in which a charged residue is mutated to glycine.     

Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.