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...

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.

Circadian clocks generate slow and ordered cellular dynamics but consist of fast-moving bio-macromolecules; consequently, the origins of the overall slowness remain unclear. We identified the adenosine triphosphate (ATP) catalytic region [adenosine triphosphatase (ATPase)] in the amino-terminal half of the clock protein KaiC as the minimal pacemaker that controls the in vivo frequency of the cyanobacterial clock. Crystal structures of the ATPase revealed that the slowness of this ATPase arises from sequestration of a lytic water molecule in an unfavorable position and coupling of ATP hydrolysis to a peptide isomerization with high activation energy. The slow ATPase is coupled with another ATPase catalyzing autodephosphorylation in the carboxyl-terminal half of KaiC, yielding the circadian response frequency of intermolecular interactions with other clock-related proteins that influences the transcription and translation cycle.