Quench‐flow experiments combined with mass spectrometry show apomyoglobin folds through an obligatory intermediate

Abstract

Folding of apomyoglobin is characterized by formation of a compact intermediate that contains substantial helicity. To determine whether this intermediate is obligatory or whether the protein can fold directly into the native state via an alternate parallel pathway, we have combined quench-flow hydrogen-exchange pulse labeling techniques with electrospray ionization mass spectrometry. The mass spectra of apomyoglobin obtained at various refolding times suggest that apomyoglobin indeed folds through a single pathway containing an obligatory intermediate with a significant hydrogenbonded secondary structure content.

Molecular dynamics simulation exploration of unfolding and refolding of a ten-amino acid miniprotein

“…Hydrogen-exchange mass-spectrometry can track the formation of secondary structures during folding by measuring how easily protons (H ? ions) are exchanged between water and amino acids in the proteins (Tsui et al 1999;Eyles and Kaltashov 2004;Pan et al 2010;Chen et al 2010;Gruebele 2010). However, a detailed understanding of folding is still missing due to the high dimensionality of the protein conformational spaces and by the wide range of relevant time scales (Daniel et al 2003).…”

Identification of Native and Non-native Structure in Kinetic Folding Intermediates of Apomyoglobin

“…all folding molecules pass through it, 26 and is on the folding pathway. 27 The kinetic intermediate is nearly as compact as the native apoprotein 28 and its rate of formation has been estimated to be w4000 s K1 at 0 M urea.…”

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

“…The structure of apomyoglobin is similar to that of the holoprotein except that residues in the F helix and the C terminus of the H helix are disordered (8-10). During refolding, apomyoglobin forms an on pathway kinetic intermediate, in which major portions of the A, G, and H helices and part of the B helix are folded, within the 6-ms burst phase of conventional quench-flow H/D exchange experiments (11)(12)(13)(14). These same regions adopt stable secondary structure in the equilibrium molten globule intermediate formed at pH 4.2 (10,(15)(16)(17).…”