Two forms of the pH 4 folding intermediate of apomyoglobin

172

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...

Section: C-terminal Halvessupporting

confidence: 92%

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

Uzawa

Nishimura

Akiyama

et al. 2008

172

“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Lerch

Horwitz²,

McCoy³

et al. 2013

Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF 2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.P roteins in solution are dynamic molecules, exhibiting conformational flexibility across a range of time and length scales (1). In addition to a well-ordered native state, conformational excursions to low-lying "excited states" may be required in protein function (2). For example, on a funnel-shaped energy landscape (3, 4), excited states have increased configurational entropy that may give rise to the promiscuous protein-protein interactions that define a protein interactome (5, 6). Structural changes involved in formation of the excited state may take a variety of forms, from rigid body motions of helices (7) to local unfolding of secondary structural elements (8). Despite their functional relevance, excited states are sparsely populated and may escape detection by standard spectroscopic techniques. Hydrostatic pressure apparently offers a solution to this problem by reversibly populating excited states of proteins, allowing for spectroscopic characterization (9-14). Pressure application is particularly useful because it shifts the relative population of preexisting conformational states while minimally affecting the conformational landscape itself (15).Assuming that pressure can populate excited states for study, the increased configurational entropy of such states presents a challenge to spectroscopic methods that aim to describe structure; depending on the intrinsic time scale of the method, either a population-weighted average structure or a heterogeneous ensemble is observed. The intrinsic time scale of con...

“…Its structure is similar to that of the heme-bound protein except for enhanced flexibility and partial unfolding of the F-helix (25). At pH 4.2, apoMb exists as a mixture of two partially folded forms, I a and I b (26), which are classic molten globules in that they contain extensive secondary structure but loosely packed cores (27,28). Structure in I a is limited to helices A-G-H, and to a lesser extent, helix B; I b is similar except that helix B is more structured (29).…”

mentioning

confidence: 92%

Dynamics of equilibrium structural fluctuations of apomyoglobin measured by fluorescence correlation spectroscopy

Chen

Rhoades

Butler

et al. 2007

101

The spectra of equilibrium chain conformation fluctuations of apomyoglobin (apoMb) as a function of folding, from the aciddenatured state at pH 2.6 through the stable molten globule state pH Ϸ 4.1 to the folded state at pH 6.3, are reported, as measured by fluorescence correlation spectroscopy. The conformational fluctuations, which are detected by quenching of an N-terminal fluorescent label by contact with various amino acids, can be represented by superpositions of decaying exponentials with time scales ranging from Ϸ3 to Ϸ200 s. Both the time scales and amplitudes of the fluctuations increase with the degree of acid denaturation, with principal shifts associated with the transition across the molten globule state. Measurements of the diffusion of apoMb confirm theoretical values showing a Ϸ40% increase in the hydrodynamic radius upon acid denaturation. This study uses the model protein apoMb to illustrate the complex scope of foldingassociated structural dynamics.