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)     

Effect of salts on the stability and folding of staphylococcal nuclease

The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.     

The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first beta-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20A相似文献   

Diverse interactions between the individual mutations in a double mutant at the active site of staphylococcal nuclease.

In principle, the quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on the kinetic or thermodynamic parameter measured, the D21E and R87G mutations of staphylococcal nuclease exhibit four of these five categories of interaction in the double mutant. While Vmax of the R87G single mutant of staphylococcal nuclease is 10(4.8)-fold lower than that of the wild-type enzyme and the Vmax of the D21E single mutant is 10(3.0)-fold below that of wild type, the double mutant D21E + R87G was found to lose a factor of only 10(4.1) in Vmax relative to wild type, rather than the product of the two single mutations (10(7.8)). These results suggest antagonistic structural effects of the individual R87G and D21E mutations. An alternative explanation for the nonadditivity of effects, namely, the separate functioning of these residues in a stepwise mechanism involving the prior attack of water on phosphorus followed by protonation of the leaving group by Arg-87, is unlikely since no enzyme-bound phosphorane intermediate (less than 1% of [enzyme]) was found under steady-state conditions on the R87G mutant by 31P NMR at 242.9 MHz. Like the effects on Vmax, quantitatively similar antagonistic effects of the two mutations were detected on the binding of divalent cations in binary enzyme-Ca2+ and enzyme-Mn2+ complexes and in the ternary enzyme-Ca2(+)-5'-pdTdA complex, suggesting that the effects on Vmax result from antagonistic structural changes at the Ca2+ binding site. Simple additive weakening effects of the two mutations were found on the binding of the substrate 5'-pdTdA, in both the absence and the presence of the divalent cations, Mn2+ and Ca2+. However, synergistic effects of the two mutations were found on the binding of the substrate analogue 3',5'-pdTp, profoundly weakening its binding to the double mutant in both the absence and the presence of divalent cations. Such synergistic effects of the two mutations may result from negative cooperativity or strain in the binding of 3',5'-pdTp to the wild-type enzyme. It is concluded that the quantitative interactions of two active-site mutations of an enzyme can vary greatly depending on which parameter of the enzyme is measured. When the two mutations interact in the same way on several parameters, a common underlying mechanism is suggested.     

Phage display of catalytically active staphylococcal nuclease

Staphylococcal nuclease (SNase), a 14 kD enzyme that catalyzes the hydrolysis of single- and double-stranded nucleic acid, was fused to the N-terminus of the gene III (pIII) protein of filamentous phage fdtet. The SNase-pIII protein is infective and the catalyzes DNA hydrolysis, demonstrating that functional SNase can be displayed on the phage surface.     

The role of tryptophan in staphylococcal nuclease stability

Staphylococcal nuclease (SNase) has a single Trp residue at position 140. Circular dichroism, intrinsic and ANS-binding fluorescence, chemical titrations and enzymatic assays were used to measure the changes of its structure, stability and activities as the Trp was mutated or replaced to other positions. The results show that W140 is critical to SNase structure, stability, and function. Mutants such as W140A, F61W/W140A, and Y93W/W140A have unfolding, corrupted secondary and tertiary structures, diminished structural stability and attenuated catalytic activity as compared to the wild type. The deleterious effects of W140 substitution cannot be compensated by concurrent changes at topographical locations of position 61 or 93. Local hydrophobicity defined as a sum of hydrophobicity around a given residue within a distance is found to be a relevant property to SNase folding and stability.     

Effect of N-terminal deletions on the foldability, stability, and activity of staphylococcal nuclease

The effect of N-terminally successive deletions on the foldability, stability, and activity of staphylococcal nuclease was examined. The structural changes in the nuclease caused by the deletions follow a hierarchical pattern: N-terminal truncation of the nuclease by up to nine residues clearly perturbs the conformation of the N-terminal beta-subdomain but does not affect the C-terminal alpha-subdomain; deletion of 11 or 12 residues perturbs the C-terminal alpha-subdomain, resulting in formation of a molten globule state; deletion of 13 residues causes the nuclease to become highly unfolded. N-terminally deleted nuclease delta11 retains the ability to fold but delta12 is not able to fold into an enzymatically active conformation, suggesting that 11 residues is the maximum length that can be deleted from the N-terminus while still retaining the folding competence of the nuclease. Further, the results suggest that proper folding of the C-terminal alpha-subdomain probably relies on the integrity of the N-terminal beta-subdomain.     

Inhibitor binding increases the mechanical stability of staphylococcal nuclease

Staphylococcal nuclease (SNase) catalyzes the hydrolysis of DNA and RNA in a calcium-dependent fashion. We used AFM-based single-molecule force spectroscopy to investigate the mechanical stability of SNase alone and in its complex with an SNase inhibitor, deoxythymidine 3′,5′-bisphosphate. We found that the enzyme unfolds in an all-or-none fashion at ∼26 pN. Upon binding to the inhibitor, the mechanical unfolding forces of the enzyme-inhibitor complex increase to ∼50 pN. This inhibitor-induced increase in the mechanical stability of the enzyme is consistent with the increased thermodynamical stability of the complex over that of SNase. Because of its strong mechanical response to inhibitor binding, SNase, a model protein folding system, offers a unique opportunity for studying the relationship between enzyme mechanics and catalysis.     

Effect of active site residues in barnase on activity and stability.

We have mutated residues in the active site of the ribonuclease, barnase, in order to determine their effects on both enzyme activity and protein stability. Mutation of several of the positively charged residues that interact with the negatively charged RNA substrate (Lys27----Ala, Arg59----Ala and His102----Ala) causes large decreases in activity. This is accompanied, however, by an increase in stability. There is presumably electrostatic strain in the active site where positively charged side-chains are clustered. Mutation of several residues that make hydrogen bonds (Ser57----Ala, Asn58----Asp and Tyr103----Phe) causes smaller decreases in activity, but increases or has no effect on stability. Deletion of hydrogen bonding groups elsewhere in proteins has been found previously to decrease stability by 0.5 to 1.5 kcal mol-1. Conversely, we find that two mutations (Asp54----Asn and Gln104----Ala) decrease stability and increase activity. Another mutation (Glu73----Ala) decreases both activity and stability. It is clear that many residues in the active site do not contribute to stability and that for some, but not all, of the residues there is a compromise between activity and stability. This suggests that certain types of local instability may be necessary for substrate binding and catalysis by barnase. This has implications for the understanding of enzyme activity and the design of enzymes.     

Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of staphylococcal nuclease

The high-resolution X-ray crystal structure of staphylococcal nuclease (SNase) suggests that the guanidinium groups of Arg 35 and Arg 87 participate as electrophilic catalysts in the attack of water on the substrate phosphodiester. Both arginine residues have been replaced with "conservative" lysine residues so that both the importance of these residues in catalysis and the effect of changes in electrostatic interactions on active site conformation can be assessed. The catalytic efficiencies of R35K and R87K are decreased by factors of 10(4) and 10(5) relative to wild-type SNase, with R87K showing a very significant reduction in its affinity for both DNA substrate and the competitive inhibitor thymidine 3',5'-bisphosphate (pdTp). The thermal denaturation behavior of both mutant enzymes differs from that of wild type both in the absence and in the presence of the active site ligands Ca2+ and pdTp. Both the 1H NMR chemical shifts and interresidue nuclear Overhauser effects (NOEs) of residues previously assigned to be in the hydrophobic core of SNase are altered in R35K and R87K. These observations, similar to those recently reported by our laboratories for substitutions for Glu 43 [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A., Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278; Wilde, J. A., Bolton, P. H., Dell'Acqua, M., Hibler, D. W., Pourmotabbed, T., & Gerlt, J. A. (1988) Biochemistry 27, 4127], suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.     

Effect of glycosylation of bacterial amylase on stability and active site conformation.

With a view to understand the changes in the conformation of bacterial amylase, the enzyme preparation was conjugated to dextran. Glycosylation of purified bacterial amylase resulted in increased stability against heat, proteolytic enzymes and denaturing agents. Several group specific inhibitors exhibited dose-dependent inhibition and the extent of inhibition was same for native as well as for the glycosylated enzyme. The pH optima of native and glycosylated enzyme remained the same indicating that the ionization at the active site is not greatly influenced as a result of glycosylation. Although the native as well as the glycosylated enzyme bind to the substrate with the same affinity, the rate of reaction differed greatly at 90 and 100 degrees C. At 70 degrees C, the rate of reaction was similar for the conjugated as well as the unconjugated amylase. Thermostability at different temperatures clearly showed that the glycosylated enzyme had greater stability compared to the native enzyme. The divalent cation binding site in the amylase also appears to be unaltered upon glycosylation since EDTA inhibited both enzymes to the same extent and addition of calcium ion restored the activity to almost the same level. These studies showed that conjugating the amylase enzyme with a bulky molecule like dextran does not affect the conformation at the active site.     

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.     

Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

Association of partially-folded intermediates of staphylococcal nuclease induces structure and stability

Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (< or =0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less-ordered partially-folded intermediates (A1 and A2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small-angle X-ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.