Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering

To investigate protein folding dynamics in terms of compactness, we developed a continuous-flow mixing device to make small-angle x-ray scattering measurements with the time resolution of 160 μs and characterized the radius of gyration (R(g)) of two folding intermediates of cytochrome c (cyt c). The early intermediate possesses ≈20 Å of R(g), which is smaller by ≈4 Å than that of the acid-unfolded state. The R(g) of the later intermediate is ≈18 Å, which is close to that of the molten globule state. Considering the α-helix content (f(H)) of the intermediates, we clarified the folding pathway of cyt c on the conformational landscape defined by R(g) and f(H). Cyt c folding proceeds with a collapse around a specific region of the protein followed by a cooperative acquisition of secondary structures and compactness.