Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis

Evans, Philip A.; Dobson, Christopher M.; Kautz, Roger A.; Hatfull, Graham F.; Fox, Robert O.

doi:10.1038/329266a0

Cited by 185 publications

(162 citation statements)

References 9 publications

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“…This behavior has been observed both in nuclease (Evans et al, 1987(Evans et al, , 1989 and in calbindin (Wendt et al, 1988;Skelton et al, 1990). In these instances, the Gibbs energy difference between the two folded conformations is small and a substituted amino acid is therefore likely to form a trans peptide bond.…”

Section: Decreased Stability Of Cis Proline Mutantsmentioning

confidence: 77%

Cis proline mutants of ribonuclease A. I. thermal stability

Schultz

Baldwin

1992

Protein Science

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A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give AT,,, e 10 "C and AAGo (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis H trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.

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Section: Decreased Stability Of Cis Proline Mutantsmentioning

confidence: 77%

Cis proline mutants of ribonuclease A. I. thermal stability

Schultz

Baldwin

1992

Protein Science

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“…As mentioned in the Introduction, the 'H" resonances of His', His'", and Hisiz4 in the NMR spectrum of nuclease report on the Lys""-Pro'17 peptide bond trandcis equilibrium (Evans et al, 1987;Alexandrescu et al, 1988), and the 'H" resonances of His46 report on the peptide bond trandcis equilibrium (Loh et a]., 1991). Figure 1 compares the histidine 'H" region of the 'H NMR spectra of WT, H124L, and H124L+P117G.…”

Section: Peptide Bond Trandcis Ratiosmentioning

confidence: 99%

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

Truckses¹,

Somoza²,

Prehoda³

et al. 1996

Protein Science

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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 HisIz4, is 1.9 kcal.molp' less stable (at pH 5.5 and 20 "C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains LeuIZ4. In addition, the population of the trans conformer at the Ly~'"-Pro''~ peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease HI 24L. 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 HI241 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 Lys'"-Pro'17 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys"h-Pro"7. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro' l7 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H 124L, and compared it with the published high-resolution X-ray structure of the cis form of the least 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 transkis equilibrium of the Lys'16-Pro'17 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination techniques. 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 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 kc,, affected more than Km, and their effects are additive. The PI 17G mutation in nuclease H124L leads to the same local conformational rearrangement described for the PI 17G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 335021-5030). In both P117G mutants, the loop formed by residues 112-1 17 is located closer to the adjacent loop formed by residues 77-...

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“…Unfolded nuclease and a peptide analogue of this segment favor the trans isomer of the Lys 116-Pro 117 peptide bond (Evans et al, 1987;Raleigh et al, 1992). Thus,…”

mentioning

confidence: 99%

Stress and strain in staphylococcal nuclease

et al. 1993

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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 @-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 116-1 17 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 112-1 17), 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 @-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-1 17 loop only allow trans conformations where the local backbone interactions associated with the Q and $ torsion angles are strained. When the 116-1 17 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.

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Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis

Cited by 185 publications

References 9 publications

Cis proline mutants of ribonuclease A. I. thermal stability

Cis proline mutants of ribonuclease A. I. thermal stability

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

Stress and strain in staphylococcal nuclease

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