Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments.

Takuya Mizukami,Kosuke Maki,Yukiko Abe,Ligia O Martins

doi:10.1371/journal.pone.0134238

Abstract

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.

Highlights

Kinetic intermediates are often observed during the folding of proteins with more than ~100 amino acid residues
The results indicated that transient and equilibrium intermediates of h-apoMb are formed by a shared kinetic mechanism and that the transient intermediate is converted into a non-native equilibrium state as urea concentrations increase
H/D exchange NMR studies on sw-apoMb showed that the equilibrium state(s) populated at pH 4 and a transient kinetic intermediate formed in ~10 ms of refolding at pH 6 exhibited similar patterns of protection, i.e., both intermediates contained native-like secondary structures in the A, G- H, and part of the B, helices [5,6,7,29,34]

Summary

Introduction

Kinetic intermediates are often observed during the folding of proteins with more than ~100 amino acid residues. Some proteins accumulate equilibrium intermediates that are structurally and thermodynamically similar to kinetic intermediates [1] These equilibrium states have been investigated as potential analogues of short-lived kinetic intermediates in order to overcome the difficulty in characterizing the latter in detail [3,4,5,6,7,8,9,10,11]. This made it possible to elucidate properties of equilibrium intermediates such as topology (fold) [12] and structural cooperativity [13,14], which would be difficult to determine by kinetic experiments alone. There are, only a few studies in which the equivalence of transient and stable intermediates has been confirmed by directly following the kinetics of their formation

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