Time-resolved detection of association/dissociation reactions and conformation changes in photosensor proteins for application in optogenetics.

Photosensor proteins are important not only because of their biological functions but also because of their applications in optogenetics. To understand the molecular mechanism behind their biological functions and consequently seek possible applications to optogenetics, the dynamics of their intermolecular interaction (for example, association/dissociation reaction and conformational changes) upon photoexcitation need to be elucidated. Although it has been difficult to trace such reactions in the time domain using traditional spectroscopic techniques, the time-resolved diffusion method based on the transient grating technique has been demonstrated to possess a significant advantage in detecting such spectrally silent dynamics in a time-resolved manner. In this paper, the principle and studies on blue light sensor proteins, phototropins, are described. Reaction kinetics of dimerization, dissociation reactions, and conformational changes were measured, and reaction schemes were determined. This method can be employed to study protein reactions from the viewpoint of diffusion and to elucidate the reaction schemes and kinetics that cannot be detected by other spectroscopic methods.