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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Background-A myocardial bridge (MB) that partially covers the course of the left anterior descending coronary artery (LAD) sometimes causes myocardial ischemia, primarily because of hemodynamic deterioration, but without atherosclerosis. However, the mechanism of occurrence of myocardial infarction (MI) as a result of an MB in patients with spontaneously developing atherosclerosis is unclear. Methods and Results-One hundred consecutive autopsied MI hearts either with MBs [MI(ϩ)MB(ϩ) group; nϭ46] orwithout MBs (nϭ54) were obtained, as were 200 normal hearts, 100 with MBs [MI(Ϫ)MB(ϩ) group] and 100 without MBs. By microscopy on LADs that were consecutively cross-sectioned at 5-mm intervals, the extent and distribution of LAD atherosclerosis were investigated histomorphometrically in conjunction with the anatomic properties of the MB, such as its thickness, length, and location and the MB muscle index (MB thickness multiplied by MB length), according to MI and MB status. In the MI(ϩ)MB(ϩ) group, the MB showed a significantly greater thickness and greater MB muscle index (PϽ0.05) than in the MI(Ϫ)MB(ϩ) group. The intima-media ratio (intimal area/medial area) within 1.0 cm of the left coronary ostium was also greater (PϽ0.05) in the MI(ϩ)MB(ϩ) group than in the other groups. In addition, in the MI(ϩ)MB(ϩ) group, the location of the segment that exhibited the greatest intima-media ratio in the LAD proximal to the MB correlated significantly (PϽ0.001) with the location of the MB entrance, and furthermore, atherosclerosis progression in the LAD proximal to the MB was largest at 2.0 cm from the MB entrance. Conclusions-In the proximal LAD with an MB, MB muscle index is associated with a shift of coronary disease more proximally, an effect that may increase the risk of MI. (Circulation. 2009;120:376-383.)Key Words: myocardium Ⅲ myocardial infarction Ⅲ anatomy Ⅲ atherosclerosis T he coronary artery that runs through epicardial adipose tissue is often covered in part with myocardial tissue. This structure is known as a myocardial bridge (MB) 1 ; it exists almost exclusively in the left anterior descending coronary artery (LAD), 2 and it is regarded as a common anatomic variant rather than a congenital anomaly. 3 The frequency of an MB in the LAD is high, sometimes Ͼ50% by autopsy, 2 but it is Ͻ5% by angiography. 4 Because MBs have been identified angiographically indirectly through a "milking effect" phenomenon induced by systolic compression of the MB, a thin or short MB is often missed. 4 The use of other invasive imaging, such as intracoronary ultrasound and Doppler, has improved MB detection. 5,6 More recently, multidetector computed tomography (CT) has been used noninvasively to detect the MB itself directly, 7 and surprisingly, the use of multidetector CT for myocardial ischemia increases Editorial see p 357 Clinical Perspective on p 383The clinical outcome of patients with MBs has been considered benign 4 ; however, the significance of an MB to myocardial ischemia remains controversial. By multidetector CT imaging,...

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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...

Factors governing the folding pathways and the stability of apomyoglobin have been examined by replacing the distal histidine at position 64 with phenylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significantly more stable than wild-type apoMb. Kinetic refolding studies of this variant also show a significant difference from wild-type apoMb. The amplitude of the burst phase ellipticity in stopped-flow CD measurements is increased over that of wild-type, an indication that the secondary structure content of the earliest kinetic intermediate is greater in the mutant than in the wild-type protein. In addition, the overall rate of folding is markedly increased. Hydrogen exchange pulse labeling was used to establish the structure of the initial intermediate formed during the burst phase of the H64F mutant. NMR analysis of the samples obtained at different refolding times indicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic intermediate. Replacement of the polar distal histidine residue with a nonpolar residue of similar size and shape appears to stabilize the E helix in the early stages of folding due to improved hydrophobic packing. The presence of a hydrophilic histidine at position 64 thus exacts a price in the stability and folding efficiency of the apoprotein, but this residue is nevertheless highly conserved among myoglobins due to its importance in function.

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The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.