Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments

Abstract

Hydrogen/deuterium exchange followed by trapping of the labeled species in the aprotic solvent DMSO has been used to elucidate structure in both the burst-phase molten globule-folding intermediate of apomyoglobin and in an equilibrium intermediate that models the kinetic intermediate. Precise estimates can be made of exchange times in an interrupted exchange-out experiment at pH 4 followed by analysis in DMSO solution, giving extensive sequence-specific information about the structure of the equilibrium intermediate. In addition, the use of DMSO as a solvent for NMR measurements after quench-flow pH-pulse labeling experiments gives a greatly increased data set for the elucidation of the kinetic folding pathway. Interestingly, differences are observed in some regions of apomyoglobin between the equilibrium and kinetic intermediates. These differences are quantitative rather than qualitative; that is, the overall patterns of labeling and secondary structure formation remain similar between the two species. However, local differences are observed, which probably reflect the difference in the solution conditions for the equilibrium experiment (pH 4) vs. the kinetic experiment (pH 6) and the change in the status of the stabilizing hydrogen bond between the side chains of His-24 and His-119.apomyoglobin ͉ NMR ͉ molten globule E lucidation of the detailed mechanism of protein folding remains one of the major challenges in structural biology. Information on the kinetic folding process can be obtained by various biophysical techniques, but the only method that simultaneously provides information on folding at individual sites throughout the protein is the well known quench-flow hydrogen exchange technique coupled with NMR spectroscopy (1, 2). In this method, amides are labeled by hydrogen͞deuterium (H͞D) exchange at multiple time points during the folding process; exchange is then quenched, the refolding is allowed to go to completion, and the H͞D population at individual amides is determined from NMR spectra of the folded protein. A major shortcoming is that kinetic information on the folding process is limited to those residues for which amide proton exchange rate in the native folded form of the protein is slow. For various reasons, including rapid exchange rates in folding intermediates, the number of useful amide proton ''probes'' for quench-flow experiments may be even further reduced. Information on the extent and rate of folding is therefore unavailable for a substantial portion of the polypeptide chain. In practical terms, the exchange rates of amide protons to be used as probes in the standard quench-flow experiment must be slower than Ϸ10 Ϫ4 s Ϫ1 (3). A few additional amides can sometimes be observed by the careful use of lowered pH and temperature. However, for most proteins studied by the quench-flow method, only 30-50% of the backbone amides in the molecule can reliably be used as probes in standard quench-flow folding experiments.Here, we describe a previously unreported method employing the aprotic organi...

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

“…2). This finding contrasts with the native apoprotein and the pH 4 equilibrium molten globule intermediate, where the protection factors for exchange of amides in the H helix are comparable to those of the A and G helices (10). , which implies that the primary contact for the B helix in the intermediate ensemble is to helix G. These observations are consistent with previous studies of the L32A mutant (32), in which the substitution caused a reduction in the observed proton occupancies at G-helix residues I107 and E109, which contact L32 in the folded state.…”

“…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)(9)(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).…”

“…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 apomyoglobin kinetic intermediate, with the E helix partly folded in a subset of molecules in the conformational ensemble (12).…”

How strong are side chain interactions in the folding intermediate?

“…In Figure 5, the color intensity is indicative of the extent to which the studied side chains are involved in stabilizing the apoMb intermediate state. 13 ), is lower than that in the native state even for the conserved residues: it amounts to 15-50% (of the native state strength) for the conserved residues forming the A, G, H-helical complex and is equal practically to 0% for the nonconserved residues from the B, C, D, and E helices. However, it is shown (unpublished results) that the situation in the folding nucleus for the ratelimiting I$N transition is quite the opposite to what we found in the folding intermediate: contribution of the conserved residues from the A, G, H helices to stabilization of the folding nucleus in this transition is minor, while contribution of the residues from the B, D, E helices is significant.…”

“…It is a single-domain protein of 17 kDa formed by seven helices (by eight ones for holomyoglobin). Its structure differs from that of holomyoglobin by the position of its loops and helices, with one of the latter, F-helix, being completely destroyed, [12][13][14] and the total helicity being reduced from 78% to 65%. 15 Nevertheless, apoMb holds its hydrophobic core 16 and a peculiar holomyoglobin-like tertiary structure.…”