STRESS AND STRAIN IN STAPHYLOCOCCAL NUCLEASE

Protein molecules generally adopt a tertiary structure in which all ba ckbone and side chain conformations are arranged in local energy minim a; however, in several well-refined protein structures examples of loc ally strained geometries, such as cis peptide bonds, have been observe d. Staphylococcal nuclease A contains a single cis peptide bond betwee n residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternativ e native folded forms of nuclease A have been detected by NMR spectros copy and attributed to a mixture of cis and trans isomers at the Lys 1 16-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. Th e structure of K116A is indistinguishable from that of nuclease A, inc luding a cis 116-117 peptide bond (92% populated in solution). The ove rall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a p redominantly trans Gly 116-Pro 117 peptide bond (80% populated in solu tion). 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 th at the position of the ends of the residue 112-117 loop only allow tra ns 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 conforma tion compensates for the energetically unfavorable cis X-Pro peptide b ond. With the removal of the side chain from residue 116 (Kl 16G), the backbone strain of the trans conformation is reduced to the point tha t the conformation associated with the cis peptide bond is no longer f avorable.