Solvation and desolvation dynamics around helices during the kinetic folding process of apomyoglobin (apoMb) were investigated by using time-resolved infrared (IR) spectroscopy based on continuous-flow rapid mixing devices and an IR microscope. The folding of apoMb can be described by the collapse and search mechanism, in which the initial collapse occurring within several hundreds of microseconds is followed by the search for the correct secondary and tertiary structures. The time-resolved IR measurements showed a significant increase in solvated helix possessing a component of amide I' at 1633 cm-1 within 100 μs after initiating the folding by a pD jump from pD2.2 to 6.0. In contrast, there was a minor increase in buried helices having amide I' at 1652 cm-1 in this time domain. The observations demonstrate that the initially collapsed conformation of apoMb possesses a large amount of solvated helices, and suggest that much water is retained inside the collapsed domain. The contents of solvated and buried helices decrease and increase, respectively, in the time domain after the collapse, showing that the stepwise desolvation around helices is associated with the conformational search process. Interestingly, the largest changes in solvated and buried helices were observed at the final rate-limiting step of the apoMb folding. The persistence of the solvated helix until the final stage of apoMb folding suggests that the dissociation of hydrogen bonds between water and main-chain amides contributes to the energy barrier in the rate-determining step of the folding.
- protein folding
- rapid mixing
- time-resolved infrared spectroscopy