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

Partha Hazra ^*, Keiichi Inoue ^*, Wouter Laan , Klaas J. Hellingwerf  and Masahide Terazima ^*

^* Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan; and Laboratory for Microbiology, Swammerdam Institute for Life Science, Nieuwe Achtergracht, Amsterdam, The Netherlands

Correspondence: Address reprint requests to Masahide Terazima, E-mail: mterazima{at}kuchem.kyoto-u.ac.jp.

The photoreaction kinetics of the BLUF domain of AppA_5-125 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 (k_obs) against the AppA concentration, the second order rate constant is determined to be 2.5 x 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.

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