About Phototropin

Although it was known for a long time that UV and blue light regulate a wide range of responses in plants, including phototropism (plant growth towards the light source) [15], chloroplast migration [16], and stomatal opening [17], the photoreceptors for these light responses have been identified only in recent years. Phototropins, recently renamed Phot proteins [2,3], represent a class of blue-light sensitive receptor kinases, which belong to the PAS domain superfamily originally characterized in PER, ARNT, SIM proteins. There are at least two kinds of types in this family, Phot1 and Phot2 These have formerly been named nph1 for nonphototropic hypocotyls-1 and npl1 for nph1-like, respectively [18]. Characteristic for phototropins is that each Phot contains two flavin mononucleotide (FMN)-binding LOV (light-, oxygen-, voltage-sensitive) domains at its N-terminus and a serine/threonine protein kinase domain at its C-terminus [19]. Generally, these two LOV domains are named LOV1 and LOV2. Each consists of approximately 100 amino acids, and differences in their sequences define them as either LOV1 or LOV2. They are separated by an intervening sequence of variable length. Upon blue light illumination of the protein the serine/threonine kinase is activated and multiple auto-phosphorylation is observed [20].

All LOV domains demonstrate qualitatively identical and fully reversible photocycles, governed by the same general reaction mechanism [21]. Thus they provide an excellent system for investigation of the photochemical properties of the blue light receptor phototropin. In recent years, phototropin has been successfully expressed in Escherichi. Coli, isolated, and purified. Subsequent spectroscopic experiments and biochemical analysis provided much insight into the photochemical properties and reaction mechanism underlying blue light perception by phototropin.

Introduction

When LOV domains are illuminated with blue light, they undergo a spectral change, characterized by a loss of absorption in the blue region of the spectrum combined with the appearance of a new absorption band with peak near 390 nm (see Figure 3.6). The absorbance changes are not those of the typical reduction reactions of FMN but resemble those observed for the formation of an adduct between a cysteine residue and the C(4a) carbon of the FMN chromophore previously observed for the mercuric ion reductase of the ACAA mutant [22]. In phototropin, the formation of this photoproduct occurs within a few microseconds following the absorption of the blue light photon [23]. When the illumination ceases, i.e. under dark condition, this adduct reverts to the dark form of the LOV domains. In addition, the mutation of a highly conserved cysteine in LOV domains to alanine or serine completely abolishes the photochemical reactivity. Therefore, the cysteinyl-adduct formation probably represents the primary photoreaction underlying light detection by the phototropins [24].

In recent years this hypothesis has been verified by NMR spectroscopy studies on LOV2 domain from Avena sativa [25], by FTIR spectroscopy studies on LOV2 domain from Oat [26], and by X-ray crystallography of a LOV2 domain from Adiantum capillus-veneris [27] and a LOV1 domain from Chlamydomonas reinhardtii [28]. The position of the reactive cystein is C450 in Avena sativa LOV2 domain, C966 in Adiantum capillus-veneris LOV2 domain, and C57 and C250 in Chlamydomonas reinhardtii LOV1 and LOV2 domains, respectively [29].

The photoproduct (LOV-390) produced by irradiation with blue light thermally reverts to the ground state (LOV-447) on the minute time scale in the dark, with a pH and salt concentration dependent time constant [30].

Many details of the mechanism of this photocycle of various LOV domains from different organisms are still unknown, though some progress has been made. At the moment the commonly accepted sequence of photophysical and photochemical reactions in the LOV

Introduction

domains involve ISC (intersystem crossing) from the excited singlet state of FMN to the triplet excited state (S1 → T1), characterized by a broad absorption band at about 660 nm and peak at 715 nm. Hence this species has been named LOV-715. The subsequent mechanistic steps of photoproduct formation are, however, still controversially discussed. It has been suggested that H⁺-transfer from the thiol group to the N(5)-position of the isoalloxazine ring is the rate-limiting step of the adduct formation [21]. But studies on a LOV1 mutant, in which the reactive cysteine was displaced by methionine, made it more likely that an electron transfer from the protein sulfur atom to FMN is the primary and rate-limiting step [31, 32]. This step would be followed by proton transfer to FMN resulting in a pair of neutral radicals which would eventually combine to form the adduct. This model is consistent with the results of time-resolved EPR measurements on several LOV domains, which lead to the conclusion that the radical pair mechanism is dominating the pathway from the triplet state to the adduct under most reaction conditions [33, 34]. Low-temperature studies with time-resolved EPR and UV/Vis spectroscopy on a LOV domain revealed that the light-induced flavin adduct can be formed even at temperature lower than 80K [34], which suggest that the process takes place without much structural rearrangement in the protein. This discovery is in remarkable contrast to other light sensor proteins, like rhodopsins, phytochromes, and PYP (photoactive yellow protein) [35]. In these photoreceptor systems photoisomerization occurs on a femtosecond or picosecond time scale, whereas the intermediates activating transducer appear in milliseconds or seconds, which might indicate that the phototropic response is the result of a complex signal transduction system.

So far the details of the interaction between the LOV domains and the kinase in phototropins is still not known. It was found by NMR spectroscopy that the short α-helix connecting LOV2 with the kinase associates with the LOV2 core under dark conditions but not in the light [36].

Introduction

Fluorescence measurements on LOV domains from several organisms displayed that the decay kinetics of LOV-390 in peptides containing both LOV domains is obviously slower than that of the isolated domains [37]. Through similar investigations on Phot2 from the fern Adiantum capillus-veneris, it was suggested that LOV1 reduces the lifetime of the domain pair LOV1+2 signal in order to limit the physiological response at strong light level [38]. Moreover, the data obtained from the tandem LOV domain fusion proteins closely resemble those from the full-length proteins. This certainly indicates a relevant interaction between the two LOV domains.

But how the photoreactions of LOV1 or LOV2 influence the interaction is not clear. In this dissertation, we also pay much attention to this problem and try to understand the interaction between two LOV domains through the studies on the individual and double domains.