Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: Energetics of peptide association and folding

Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (K-assn approximate to 1.4 x 10(5) M -1 at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimat es of stability from NMR indicating a Delta C(p)degrees for fragment comple xation in good agreement with that determined for native ubiquitin, suggest ing that fragment association results in the burial of a similar hydrophobi c surface area. The stability of the complex shows appreciable pH dependenc e, suggesting that ionic interactions on the surface of the protein contrib ute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn . HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptid e fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical pr opensity in the helical fragment (residues 21-35). In contrast, the C-termi nal peptide (36-76) shows evidence in aqueous solution, from some Hot chemi cal shifts, for nonnative phi and psi angles; nonnative alpha-helical struc ture is readily induced in the presence of organic cosolvents, indicating t hat tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that t he N-terminal fragment (1-35), where interaction between the helix and hair pin requires the minimum loss of conformational entropy, may provide the nu cleation site for fragment complexation.