A COMPARISON OF THE PH, UREA, AND TEMPERATURE-DENATURED STATES OF BARNASE BY HETERONUCLEAR NMR - IMPLICATIONS FOR THE INITIATION OF PROTEIN-FOLDING

The denatured states of barnase that are induced by urea, acid, and hi gh temperature and acid have been assigned and characterised by high r esolution heteronuclear NMR. The assignment was completed using a comb ination of triple-resonance and magnetisation-transfer methods. The la tter was facilitated by selecting a suitable mutant of barnase (Ile -- > Val51) which has an appropriate rate of interconversion between nati ve and denatured states in urea. (3)J NH-(CH)-H-alpha coupling constan ts were determined for pH and urea-denatured barnase and intrinsic ''r andom coil'' coupling constants are shown to be different for differen t residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and co upling constant data provides evidence that the acid-denatured state h as some residual structure in regions that form the first and second h elices and the central strands of beta-sheet in the native protein. Th e acid/temperature-denatured states has less structure in these region s, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase fro m kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the beta-sh eet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this r egion of the protein is non-productive. These data fit a model of prot ein folding in which local nucleation sites are latent in the denature d state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding process. Thus, studies of the structure of denatured states pinpoint where nucleation sites m ay be, and the kinetic and protein engineering studies show which ones are productive. (C) 1995 Academic Press Limited