Tetramer Formation Kinetics in the Signaling State of AppA Monitored by Time-Resolved Diffusion

The photoreaction kinetics of the BLUF domain of AppA5-125 was studied by monitoring time-dependence of an apparent diffusion coefficient (D) using the pulsed laser-induced transient grating technique. It was found that D of the photoproduct is time-dependent. From the concentration dependence of the reaction rate, it was concluded that the BLUF domain of AppA forms a dimer upon the photoexcitation. Since AppA exists as a dimeric form in the ground state, this dimerization reaction indicates the tetramer formation in the signaling state. From the slope of the plot of observed rate constants (kobs) against the AppA concentration, the second order rate constant is determined to be ∼2.5 × 105 M−1s−1, which is ∼4 orders in magnitude lower than the diffusion controlled reaction. It indicates that a relative orientation of the protein molecules during the dimerization process causes additional constraints, which slow down the reaction rate.