Tetramer Formation Kinetics in the Signaling State of AppA monitored by the time-resolved diffusion

¹ Kyoto University
² Swammerdam Institute for Life Science

^* To whom correspondence should be addressed. E-mail: mterazima{at}kuchem.kyoto-u.ac.jp.

Submitted on February 22, 2006
Revised on March 30, 2006
Accepted on 20 April 2006

	Abstract

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 photo-product 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 ~2.5x10⁵ M^-1s^-1, which is about 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.

Key Words: AppA, diffusion, photoreaction, tetramer

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