1.A.4 (a) phototropin from Arabidopsis thaliana

(i) Phot1LOV1
　　The photochemical reaction of the LOV1 (light-oxygen-voltage 1) domain of phototropin 1 from Arabidopsis thaliana was investigated by the time-resolved transient grating method. As with other LOV domains, an absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 1.1 μs. After this reaction, a significant diffusion coefficient (D) change (D of reactant = 8.2 × 10^−11 m2/s, D of photoproduct = 6.4 × 10^−11 m2/s) was observed with a time constant of 14 ms at the protein concentration of 270 microM. From the D value of the ground state and the peak position in size exclusion chromatography, we have confirmed that the phot1LOV1 domain exists as a dimer in the dark. The D-value and the concentration dependence of the rate indicated that the phot1LOV1 domain associates to form a tetramer (dimerization of the dimer) upon photoexcitation. We also found that the chromophore is released from the binding pocket of the LOV domain when it absorbs two photons within a pulse duration, which occurs in addition to the normal photocycle reaction. On the basis of these results, we discuss the molecular mechanism of the light dependent role of the phot1LOV1 domain.

(ii) Phot1LOV2, Phot1LOV2-linker
　　We observed a time dependent D and it was interpreted in terms of the unfolding of alpha-helices in the linker region. The change of the a-helices was confirmed by observing the recovery of the circular dichroism intensity. The TrL signal showed that the molecular volume decreases with two time constants; 300 micros and 1.0 ms. The former time constant is close to the previously observed photo-dissociation reaction rate of the phot1LOV2 (without the linker) dimer, and the latter one agrees well with the rate of the D-change. Considering a similar time constant of the dissociation reaction of the LOV2 dimer, we interpreted this kinetics in terms of the dissociation step of the linker region from the LOV2 domain (T390pre state). After this step, the protein volume and D are decreased significantly with the lifetime of 1.0 ms. The D-decrease indicates the increase of the intermolecular interaction between the protein and water molecules. On the basis of these observations, a two step mechanism of the linker unfolding is proposed.
We also investigated the conformation change of A'a helix in the LOV domain. A mutant (T469I mutant) that renders the A'a helix unfolded in the dark state showed unfolding of the Ja helix with a time constant of 1 ms, which is very similar to the time constant reported for the wild-type LOV2-linker sample. Furthermore, a mutant (I608E mutant) that renders the Ja helix unfolded in the dark state exhibited an unfolding process of the A'a helix with a time constant of 12 ms. On the basis of these experimental results, it is suggested that the unfolding reactions of these helices occurs independently.



(iii) Phot2LOV1, Phot2LOV1-hinge
　　The TG signal of Phot2LOV1 showed a significant diffusion coefficient (D) change upon photoexcitation. This change was sensitive to the protein concentration and the observation time range. These observations were explained by assuming that there are reactive and non-reactive forms, and the fraction of these species is concentration dependent. From the concentration dependence of the dynamics, the monomer was found to form a dimer; however, the dimer does not exhibit an observable reaction. In the dark state, both species were in equilibrium and are not distinguishable spectroscopically. For the LOV1 domain with the hinge domain, the reaction scheme was the same as the LOV1 domain sample, but the D change was affected by the presence of the hinge region. This observation suggests that the hinge region undergoes a conformational change during the photoreaction.

(iv) Phot2LOV2, Phot2LOV2-linker
　　The diffusion coefficients of the adduct product after forming the chemical bond between the chromophore and Cys residue of Phot2LOV2 domain is found to be slightly smaller than that of the reactant, which fact implies that the core shrinks slightly on the adduct formation. After that change, no significant conformational change was observed. On the other hand, the signal of LOV2 with the linker part to the kinase domain clearly shows very different diffusion coefficients between the original and the adduct species. The large difference indicates significant global conformational change of the protein moiety upon the adduct formation. More interestingly, the diffusion coefficient is found to be time dependent in the observation time range. This dynamics representing the global conformational change is a clear indication of a spectral silent intermediate between the excited triplet state and the signaling product. From the temporal profile analysis of the signal, the rate of the conformational change is determined to be 2 ms.

(v) Phot2LOV2-kinase

(vi) Phot2LOV1-LOV2


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