Self-association reaction of denatured staphylococcal nuclease fragments characterized by heteronuclear NMR

Abstract

The self-association reaction of denatured staphylococcal nuclease fragments, urea-denatured G88W110, containing residues 1–110 and mutation G88W, and physiologically denatured 131-residue Δ131Δ, have been characterized by NMR at close to neutral pH. The two fragments differ in the extent and degree of association due to the different sequence and experimental conditions. Residues 13–39, which show significant exchange line broadening, constitute the main association interface in both fragments. A second weak association region was identified involving residues 79–105 only in the case of urea-denatured G88W110. For residues involved in the association reaction, significant suppression of the line broadening and small but systematic chemical shift variation of the amide protons were observed as the protein concentration decreased. The direction of chemical shift change suggests that the associated state adopts mainly β-sheet-like conformation, and the β-hairpin formed by strands β2 and β3 is native-like. The apparent molecular size obtained by diffusion coefficient measurements shows a weak degree of association for Δ131Δ below 0.4 mM protein concentration and for G88W110 in 4 M urea. In both cases the fragments are predominantly in the monomeric state. However, the weak association reaction can significantly influence the transverse relaxation of residues involved in the association reaction. The degree of association abruptly increases for Δ131Δ above 0.4 mM concentration, and it is estimated to form a 4 to 8 mer at 2 mM. It is proposed that the main region involved in association forms the core structure, with the remainder of residues largely disordered in the associated state. Despite the obvious influence of the association reaction on the slow motion of the backbone, the restricted mobility on the nanosecond timescale around the region of strand β5 is essentially unaffected by the association reaction and degree of denaturation.