1.A.5 (c) PixD

(i) SyPixD
　　SyPixD is a BLUF protein from Synechocystis sp. PCC6803 (Slr1694). According to the crystal structure, PixD consists of the BLUF domain and additional helices. The most striking characteristic of PixD is the unique formation of oligomers. PixD was found to crystallize to form a decamer in the asymmetric unit with two pentameric rings. This decameric structure may play an important role in the signal transduction of PixD proteins.
　　
　　A conformational change coupled with a volume contraction of 13 mL mol−1 was observed with a time constant of 45 ms following photoexcitation. At a weak excitation light intensity, there were no further changes in the volume and the diffusion coefficient (D). The determined D-value (3.7 × 10−11 m2 s−1) suggests that PixD exists as a decamer in solution, and this oligomeric state was confirmed by size exclusion chromatography (SEC) and blue native-polyacrylamide gel electrophoresis. Surprisingly, by increasing the excitation laser power, a large increase in D with a time constant of 350 ms was observed following the volume contraction reaction. The D-value of this photoproduct species (7.5 × 10−11 m2 s−1) is close to that of the PixD dimer. Combined with TG and SEC measurements under light-illuminated conditions, the light-induced increase in D was attributed to a transient dissociation reaction of the PixD decamer to a dimer. For the M93A-mutated PixD, no volume or D-change was observed. Furthermore, we showed that the M93A mutant did not form the decamer but only the dimer in the dark state. These results indicate that the formation of the decamer and the conformational change around the Met residue are important factors that control regulation of the downstream signal transduction by the PixD photoreceptor.
　　A protein-to-protein interaction between PixD and a response regulator PixE (Slr1693) is essential to achieve light signal transduction for phototaxis of the species. We studied interprotein reaction dynamics using time-resolved transient grating spectroscopy. The dissociation process was clearly observed as the light-induced diffusion coefficient change in the time domain and the kinetics was determined. More strikingly, disassembly was found to take place only after photoactivation of two PixD subunits in the complex. This result suggests that the biological response of PixD does not follow a linear correlation with the light intensity, but appears to be light-intensity-dependent.

(ii) TePixD
　　TePixD is a BLUF protein from Thermosynechococcus elongatus BP-1 (Tll0078). After formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as the expansion of the partial molar volume with a time constant of 40 microsec, whereas the other reaction phase represented a change in the diffusion coefficient (D) (i.e. the diffusion-sensitive conformational change (DSCC)). D decreased from 4.9 × 10−11 to 4.4 × 10−11 m2 s−1 upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 × 10−11 m2 s−1 upon formation of the second intermediate. From the global analysis of the signals at various grating wavenumbers, the time constant of the D-change was determined to be 4 ms. Although the magnitude and the rate constant of the faster volume change was independent of protein concentration, the amplitude of the signal which reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution; a reactive species exhibiting the DSCC and a second species which is non-reactive. The fraction of these species was found to be dependent on the concentration. The difference in the reactivity was attributed to the different oligomeric states of TePixD, i.e. pentamer and decamer. The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in signal transduction of this photosensory protein.
　　While the number of excited molecules increased monotonically as the laser power increased, the number of decamers exhibiting a global conformational change initially increased, and began to decrease with increasing the excitation intensity. This unusual power dependence was analyzed based on a Poisson distribution equation, demonstrating that decamers containing more than one excited monomer subunit do not undergo conformational change. Our results suggest that TePixD has a function of not only a photosensor but also sensing the light intensity.



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