Complex Folding Landscape of Apomyoglobin at Acidic pH Revealed by Ultrafast Kinetic Analysis of Core Mutants.

ABSTRACT: Under mildly acidic conditions (pH 4-4.5) apomyoglobin (apoMb) adopts a partially structured equilibrium state ( M-state) that structurally resembles a kinetic intermediate encountered at a late stage of folding to the native structure at neutral pH. We have previously reported that the M-state is formed rapidly (<1 ms) via a multistate process and thus offers a unique opportunity for exploring early stages of folding by both experimental and computational techniques. In order to gain structural insight into intermediates and barriers at the residue level, we studied the folding/unfolding kinetics of 12 apoMb mutants at pH 4.2 using fluorescence-detected ultrafast mixing techniques. Global analysis of the submillisecond folding/unfolding kinetics vs urea concentration for each variant, based on a sequential four-state mechanism ( U ? I ? L ? M), allowed us to determine elementary rate constants and their dependence on urea concentration for most transitions. Comparison of the free energy diagrams constructed from the kinetic data of the mutants with that of wild-type apoMb yielded quantitative information on the effects of mutations on the free energy (?? G) of both intermediates and the first two kinetic barriers encountered during folding. Truncation of conserved aliphatic side chains on helices A, G, and H gives rise to a stepwise increase in ?? G as the protein advances from U toward M, consistent with progressive stabilization of native-like contacts within the primary core of apoMb. Helix-helix contacts in the primary core contribute little to the first folding barrier ( U ? I) and thus are not required for folding initiation but are critical for the stability of the late intermediate, L, and the M-state. Alanine substitution of hydrophobic residues at more peripheral helix-helix contact sites of the native structure, which are still absent or unstable in the M-state, shows both positive (destabilizing) and negative (stabilizing) ?? G, indicating that non-native contacts are formed initially and weakened or lost as a result of subsequent structural rearrangement steps.

SUBMITTER: Mizukami T 

PROVIDER: S-EPMC6395567 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature