Residual Structure in Large Fragments of Staphylococcal Nuclease: Effects of Amino Acid Substitutions

In an attempt to develop a model of the denatured state of staphylococcal nuclease that can be analyzed experimentally under physiological conditions, a series of four large fragments of this small protein which extend from residues 1 to 103, 1 to 112, 1 to 128, and 1 to 136 have been generated through the overexpression of nuclease genes containing stop codons at defined positions. Large amounts of protein fragments were accumulated in induced cells and were purified by carrying out all fractionation steps in the presence of 6 M urea. The far-ultraviolet circular dichroism spectra of all four fragments suggested the presence of small to moderate amounts of residual structure. When the CD spectra were monitored as a function of concentrations of the tight-binding ligands Ca²⁺ and thymidine 3',5'-bisphosphate and the known affinity constants for wild-type nuclease (1–149) were used, apparent equilibrium constants of 160 and 2000 for the reversible denaturation reaction for fragments 1–136 and 1–128,respectively, were estimated. Four single and two double mutations, all of which exhibit unusual behavior in the full-length protein on solvent denaturation [Shortle, D., & Meeker, A.K. (1986) Proteins: Struct., Funct., Genet. 1, 81-89] and thermal denaturation [Shortle, D., Meeker, A. K., & Freire, E. (1988) Biochemistry 27, 4761–4768],were re-combined into the 1–136 and 1–128 fragment expression vectors, and purified mutant fragments were characterized. The CD spectra of the class I mutants (V66L, G88V, and V66L+G88V) showed evidence of an increase in residual structure relative to the wild-type fragment, whereas the spectra of the class II mutants (A69T, A90S, and A69T+A90S) showed evidence of a decrease in residual structure. Analysis of the apparent hydrodynamic Raman of these fragments by high-performance gel filtration provided independent support for large changes in residual structure on addition of these amino acid substitutions. When the ellipticity at 222 nm and the elution volume were monitored as a function of guanidine hydrochloride concentration, fragment 1–128 containing class I mutations exhibited a sigmoidal transition suggestive of a cooperative breakdown in the residual structure. From these observations, the existence of two alternative denatured states of staphylococcal nuclease is inferred: D1 representing a compact, structured, but normative state, and D2 representing an expanded, less structured state. The behavior of the two classes of mutations on reversible denaturation can be explained as resulting from a relative stabilization of the D1 state (class I) or a relative stabilization of the D2 state (class II).