1.A.3 (i) Sensory Rhodopsin from Natronobacterium pharaonis

　　Dynamics of protein conformational change of Natronobacterium pharaonis sensory rhodopsin II (NpSRII) and of NpSRII- transducer protein (delNpHtrII) complex are investigated in solution phase at room temperature by the laser flash photolysis and the transient grating methods in real time. The diffusion coefficients of both species indicate that the NpSRII-delNpHtrII complex exists in the dimeric form in 0.6 % dodecyl--maltopiranoside (DM) solution. Rate constants of the reaction processes determined from the transient absorption and grating methods agree quite well. Significant differences was found in the volume change and the molecular energy between NpSRII and NpSRII-delNpHtrII complex samples. The enthalpy of the second intermediate (L) of NpSRII-delNpHtrII is more stabilized compared with that of NpSRII. This stabilization indicates the influence of the transducer to the NpSRII structure in the early intermediate species by the complex formation. Relatively large molecular volume expansion and contraction were observed in the last two steps for NpSRII. Additional volume expansion and contraction were induced by the presence of delNpHtrII. This volume change, which should reflect the conformational change induced by the transducer protein, suggested that this is the signal transduction process of the NpSRII-delNpHtrII complex.
　　The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was also studied 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 showed only one spectrally distinct K-like intermediate in their photocycles, but the TG 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 micros 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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