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Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.

ABSTRACT: The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.

SUBMITTER: Uzawa T

PROVIDER: S-EPMC337025 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.

Uzawa Takanori T Akiyama Shuji S Kimura Tetsunari T Takahashi Satoshi S Ishimori Koichiro K Morishima Isao I Fujisawa Tetsuro T

Proceedings of the National Academy of Sciences of the United States of America 20040107 5

The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined w ...[more]

PMID: 14711991

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Project description: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 (ΔΔ 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 ΔΔ 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) ΔΔ G, indicating that non-native contacts are formed initially and weakened or lost as a result of subsequent structural rearrangement steps.

Dataset Information

Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.

Publications

Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.

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