Kinetic Measurement of Transient Dimerization and Dissociation Reactions of Arabidopsis Phototropin 1 LOV2 Domain

摘要

Photochemical reaction of a plant blue-light photoreceptor, Arabidopsis phototropin 1-LOV (light-oxygen-voltage sensing) domain 2, was studied with a view to the diffusion coefficients (D) using the pulsed-laser-induced transient grating method. Although the reaction dynamics completes at a rate of several microseconds as long as it is monitored by the absorption change, the diffusion coefficient was found to be time-dependent in a time range of submilliseconds to seconds. The observed signal can be analyzed by the two-state model, which includes the D-value decrease from D of the reactant (9.8 ± 0.4) × 10⁻¹¹ m²/s to D of the product (8.0 ± 0.4) × 10⁻¹¹ m²/s. The D-value of the reactant implies that the dominant form in the ground state of phototropin 1 LOV2 is the monomeric form in a concentration range of 50–200 μM. According to the Stokes-Einstein relationship, the D-change can be explained by a volume increase of 1.8 times. Furthermore, the rate of the D-change was roughly proportional to the concentration of the sample. These two observations indicate that the LOV2 domain transiently forms a dimer upon photoexcitation. When the sample concentration is increased (>180 μM), a new signal component appears within a few milliseconds. This signal represents a D increase from 8.0 × 10⁻¹¹ m²/s to 9.8 × 10⁻¹¹ m²/s with a time constant of 300 μs. The completely opposite D-change from that observed in a lower concentration, as well as the concentration dependence, implies that a dimer is formed in the ground state in a higher concentration range, even though the fraction of the dimer is still minor in this range. This dimer is photodissociated, with a time constant of 300 μs. This research clearly shows that the time-resolved diffusion measurement is a very powerful tool for detecting spectrally silent association/dissociation processes during chemical reactions. The photoreaction of the LOV2 domain is discussed.