Structure and dynamics of an acid-denatured protein G mutant

NMR studies of protein denatured states provide insights into potential ini tiation sites for folding that may be too transient to be observed kinetica lly. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of G(B1) which is on the margin of stability. At 5 degrees C, F30H-G(B1) is greater than 95% folded at pH 7 .0 and is greater than 95% unfolded at pH 4.0. This range of stability is u seful because the denatured state can be examined under relatively mild con ditions which are optimal for folding G(B1) We have assigned almost all bac kbone N-15, H-N, and H-alpha resonances in the acid-denatured state. Chemic al shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild con ditions than in the presence of chemical denaturants. The acid-denatured st ate populates nativelike conformations with both alpha-helical and beta-hai rpin characteristics. To our knowledge, this is the first example of a dena tured state with NOE and coupling constant evidence for beta-hairpin charac ter. A number of non-native turn structures are also detected, particularly in the region corresponding to the beta 1-beta 2 hairpin of the folded sta te. Steady-state {H-1-N-15} NOE results demonstrate restricted backbone fle xibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the beta 3-beta 4 hairpin, and t he beta 1-beta 2 turn may serve as potential initiation sites for folding o f G(B). Furthermore, residual structure in acid-denatured F30H-G(B1) is mor e extensive than in peptide fragments corresponding to the beta 1-beta 2, a lpha-helix, and beta 3-beta 4 regions, suggesting additional medium-to-long -range interactions in the full-length polypeptide chain.