The Apomyoglobin Folding Pathway Revisited: Structural Heterogeneity in the Kinetic Burst Phase Intermediate

Nishimura, Chiaki; Dyson, H. Jane; Wright, Peter E.

doi:10.1016/s0022-2836(02)00810-0

Cited by 84 publications

(130 citation statements)

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“…In contrast, at 0. (Table S1) (11,12). The present studies extend kinetic pulse-labeling experiments on apomyoglobin refolding into the submillisecond time regime and provide insights into the early folding events.…”

mentioning

confidence: 60%

“…Because holomyoglobin is more stable to exchange than apomyoglobin, heme was added to the exchange-labeled apomyoglobin and the CO complex was formed by a modification of previous procedures (12). The pulse-labeled samples were mixed with a 6-fold molar excess of hemin (Sigma) in a 2 H2O buffer containing 250 mM Hepes and 0.1-mg/ml KCN adjusted to pH* 8.2.…”

Section: Methodsmentioning

confidence: 99%

“…These same regions adopt stable secondary structure in the equilibrium molten globule intermediate formed at pH 4.2 (10,(15)(16)(17). Recent H/D exchange experiments under a variety of conditions detected heterogeneity in the apomyoglobin kinetic intermediate, with the E helix partly folded in a subset of molecules in the conformational ensemble (12).…”

mentioning

confidence: 97%

“…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).…”

mentioning

confidence: 99%

“…Data were obtained by using a threesolution mixer that can be applied for pulsed H/D exchange experiments in the submillisecond time domain (21,22). In contrast to previous pulse-labeling experiments (11)(12)(13), in which amides were exchanged by a single high pH pulse in the EX1 regime (k ex Ͼ Ͼ k cl ), our protocol uses a wide range of labeling pulse strengths in both the EX1 and EX2 regimes to measure the pH dependence of the proton occupancy (20). Because the pH dependence of k ex is known (23), the proton occupancy curves can be analyzed to give the values of k op and k cl without assuming EX1 or EX2 exchange mechanisms.…”

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confidence: 99%

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

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et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to timeresolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.protein folding ͉ pulse labeling ͉ rapid mixing M ost proteins fold rapidly from the highly heterogeneous conformational ensemble of the unfolded state into their well defined native conformations. For proteins with Ͼ100 residues, collapsed, partially folded intermediates are formed within hundreds of microseconds after the initiation of folding (1-4). Quench-flow pulse-labeling experiments have yielded considerable information about the development of secondary structure in such intermediates, on a time scale of milliseconds (5, 6). However, little is known about the processes that occur within the dead time (Ϸ6 ms) of the conventional quench flow apparatus, nor about the dynamic behavior of the kinetic folding intermediates. To gain insights into the early folding processes for sperm whale apomyoglobin, we have performed pulsed hydrogen-deuterium (H/D) exchange experiments with submillisecond time resolution.Apomyoglobin has been studied extensively by kinetic and equilibrium methods as a paradigm for understanding protein folding pathways and the structure of folding intermediates (7). 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). Recent H/D exchange experiments under a variety of conditions detected heterogeneity in the apomyogl...

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mentioning

confidence: 60%

Section: Methodsmentioning

confidence: 99%