The LOV2 Domain of Phototropin:  A Reversible Photochromic Switch

Abstract

Light, oxygen, or voltage (LOV) domains constitute a new class of chromoprotein modules. 1 They form the blue-light-sensitive loci of the phototropins, a recently discovered class of plant photoreceptors that regulate a variety of responses. 2 LOV domains consist of approximately 100 amino acids and noncovalently bind a single flavin. 3,4 Blue-light absorption initiates a photochemical reaction which results in the formation of a covalent adduct between a conserved cysteine and the flavin. 5,6 It is believed that this species, referred to as S 390 given its absorption band in the near-UV, corresponds to the signaling state of the protein. The lifetime of the adduct in various LOV domains ranges from minutes to hours, 5,7-9 which implies that even under physiological illumination, there is a high probability for absorption of a second, near-UV photon. The resulting photochemistry in the LOV domain may have important consequences for its signaling function. For this reason, we have undertaken a time-resolved study of the molecular events that follow photolysis of S 390 in the LOV2 domain from the phy3 receptor of Adiantum.LOV2 was expressed and purified and transient absorption spectroscopy was carried out as previously described. 4,10 Continuous blue-light background illumination was applied to photoaccumulate S 390 , resulting in a steady-state S 390 population of about 85%. The remaining 15% can be assumed in the dark ground state D 447 , because the other photocycle intermediate, the FMN triplet, has a lifetime of only 2 µs 5 and will have a negligible concentration at steady state. The photoaccumulated sample was photolyzed with flashes of 100 fs duration at 400 nm, and the absorption changes were probed with a flash of white light at time delays ranging from -2 ps to 4.5 ns. To determine the dynamics of D 447 , we performed an experiment without background illumination but otherwise identical conditions. The resulting spectra were weighted and subtracted from the illuminated dataset. The resulting time-resolved spectra were subjected to a global analysis program 11 using a kinetic model consisting of sequentially interconverting species, that is, 1 f 2 f 3 f . . . , in which the arrows indicate successive monoexponential decays of increasing time constants. Associated with each species is a lifetime and a difference spectrum, denoted the species-associated difference spectrum (SADS). The results are shown in Figure 1. Four kinetic components are required to describe the data, with time constants of 500 fs, 9 ps, and 100 ps and a nondecaying component. The initially created excited species has a lifetime of 500 fs. The first SADS (thin solid line) representing this species shows a negative signal near 430 nm, which can be assigned to a combination of ground-state bleaching of the adduct and stimulated emission from the excited to the ground state. At wavelengths longer than 450 nm, it features an intense absorption with a maximum at 605 nm. We assign this SADS to the singlet excited state of S 390 . This spe...

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

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Contributions of green light to plant growth and development

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