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

Abstract

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...

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

“…For example, the Ia intermediate has a stable core consisting of helices A, G, and H, giving a total helical content of about 30%, the same as that found by CD for the pH 4.1, 2-kbar state within experimental error. Moreover, the EPR results under these conditions reveal an increase in the population of disordered states for R1 residues in noncore helices, consistent with the lower protection factors for hydrogen exchange in these helices in Ia compared with Ib (43). Note that the Ib intermediate is distinct from Ia in having greater helical content (48%) due to partial folding of the noncore B, C, and E helices (42).…”

“…The intermediate Ia is formed within 0.4 ms after initiation of folding from an acid denatured state, and relaxes to form Ib within 6 ms (43). In principle, these states are in equilibrium under all conditions, but at pH 4.1 and atmospheric pressure, at which the acid MG is formed, the dominant species is Ib (39,69).…”

“…Kinetic studies of apoMb folding near neutral pH suggested the mechanism U ↔ Ia ↔ Ib ↔ N, where U is the unfolded state and Ia and Ib are transient intermediates (43,69). The intermediate Ia is formed within 0.4 ms after initiation of folding from an acid denatured state, and relaxes to form Ib within 6 ms (43).…”

“…Relaxation dispersion NMR showed the MG to be in pH-dependent equilibrium with other partially folded forms, indicating that the MG state also will be present in an equilibrium mixture at neutral pH, although with a small population relative to the native structure (i.e., an excited state) (39). Furthermore, this equilibrium MG was found to be structurally similar to the second of two intermediates identified during kinetic refolding by ultrafast hydrogen/deuterium (H/D) exchange coupled with 2D NMR (43).…”

Protein Structural Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry with Top-down Electron Capture Dissociation

“…Pulse labeling is very effective for the characterization of folding intermediates. [6][7][8][9][10] In continuous labeling method, protein is directly exposed to D 2 O. At a series of time points, an aliquot of the labeled protein is removed and then quenched by reducing pH and temperature.…”