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Laser-Induced Transient Grating Analysis of Dynamics of Interaction between Sensory Rhodopsin II D75N and the HtrII Transducer

Keiichi Inoue ^*, Jun Sasaki , John L. Spudich  and Masahide Terazima ^*

^* Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan; and Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas

Correspondence: Address reprint requests to M. Terazima, Tel.: 81-75-753-4026; E-mail: mterazima{at}kuchem.kyoto-u.ac.jp.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (HtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K₁–K₄) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the HtrII portion was found to cause a HtrII-dependent increase in volume rising in 8 µs in the K₄ state and a drastic decrease in the diffusion coefficient (D) of K₄ relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of HtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

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