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To investigate protein folding dynamics in terms of compactness, we developed a continuous-flow mixing device to make smallangle x-ray scattering measurements with the time resolution of 160 s and characterized the radius of gyration (Rg) of two folding intermediates of cytochrome c (cyt c). The early intermediate possesses Ϸ20 Å of R g, which is smaller by Ϸ4 Å than that of the acid-unfolded state. The R g of the later intermediate is Ϸ18 Å, which is close to that of the molten globule state. Considering the ␣-helix content ( fH) of the intermediates, we clarified the folding pathway of cyt c on the conformational landscape defined by R g and fH. Cyt c folding proceeds with a collapse around a specific region of the protein followed by a cooperative acquisition of secondary structures and compactness. P roteins are unique heteropolymers that possess a remarkable property to fold quickly to compact and specific conformations. Interiors of proteins are densely packed with minimum void volumes (1), indicating the specific interresidue contacts that determine secondary and tertiary structures. The compactness is therefore an essential property of the folded conformations of proteins; however, the dynamics of compaction in the process of protein folding from the extended random-coil structures are still poorly understood. Two classical models of protein folding, the hydrophobic collapse (2) and the framework models (3), suppose that proteins acquire compactness in a distinct dynamical process separated from the secondary structure formations. In the hydrophobic collapse model (2), a nonspecific collapse of protein main chain is hypothesized to trigger the tertiary and secondary structure formations. In contrast, the framework model assumes that the initial formation of secondary structures urges the subsequent folding into compact conformations (3). Recent theoretical investigations, however, suggest that main-chain collapse and secondary structure formation are mostly concerted (4). Although the different equilibrium conformations of a certain protein indicate a linear correlation between the secondary structure content and compactness (5, 6), the relationship has not been confirmed directly for kinetic folding intermediates of proteins. Experimental investigations on the protein folding dynamics in terms of compactness are urgently needed to differentiate these models, that is, to understand how unfolded proteins kinetically explore for the native states on the conformational landscape defined by compactness and secondary structure content.Cytochrome c (cyt c) is a globular protein of 104 aa, whose folding dynamics has been the subject of extensive experimental investigations (7-21). A heme group is covalently connected to the main chain (22) and surrounded by the three major helices called N-terminal residues 6-14), C-terminal (residues 87-102), and 60's helices (residues 60-69). The time-resolved circular dichroism (CD) measurement on the folding process of cyt c clarified the stepwise formation of these helices...

An ultrarapid-mixing continuous-f low method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent f low created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 s to 3 ms. To eliminate the hemeligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The f luorescence decrease is biphasic. There is an unresolved process with < 50 s, followed by a slower, exponential process with ‫؍‬ 600 s at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.

The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f H) and radius of gyration (R g), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to Ϸ50% of the overall change in R g from the unfolded to native conformation, was observed within 300 s after the pH jump. The collapsed intermediate has a f H of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.

Cytochrome c folding was initiated using a new solution mixer that provides a time window which covers over 90% of the burst phase unresolved by conventional stop-flow measurements. Folding was followed by resonance Raman scattering. Kinetic analysis of the high frequency Raman data indicates that a nascent phase occurs within the mixing dead time of 100 microseconds. A significant fraction of the protein was found to be trapped in a misfolded bis-histidine form during the nascent phase at pH 4.5, thereby preventing the protein from folding rapidly and homogeneously. The nascent phase was followed by a haem-ligand exchange phase that populates the native histidine-methionine coordinated form through a thermodynamically controlled equilibrium.

Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser 20 -Lys 41 ) of ␤2-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly 13 C, 15 N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a ␤-strand-loop-␤-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of ␤2-microglobulin protofilament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR.2,2,2-trifluoroethanol ͉ amyloid fibril ͉ dialysis-related amyloidosis ͉ protein misfolding ͉ x-ray fiber diffraction

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Submillisecond folding of cytochrome c reveals that a nascent phase appears within the mixing dead time of 100 microseconds, followed by a ligand exchange reaction during which His 26/33, water and Met 80 are inter-exchanged as haem ligands through a thermodynamically controlled equilibrium. In the ligand exchange phase, the rate of formation of a misfolded histidine-histidine coordinated state (HH) decreases by two orders of magnitude as the pH is reduced from 5.9 to 4.5 due to the protonation of the misligated His 26/33. The activation energy barriers for the transitions from the histidine-water coordinated form (HW) to the histidine-methionine coordinated form and the HH form are 18 and 4 kcal mol-1 respectively, at pH 4.8. The activation energy barrier for protein to escape from the misligated HH to the HW form was measured to be 12 kcal mol-1, demonstrating the kinetic trapping effect of the misligated bis-histidine form. The development of the polypeptide tertiary structure near the haem is concomitant with the coordination of the native haem axial ligand.