Homotrimer formation and dissociation of pharaonis halorhodopsin in detergent system.
Halorhodopsin from NpHR is a light-driven Cl(-) pump that forms a trimeric NpHR-bacterioruberin complex in the native membrane. In the case of NpHR expressed in Escherichia coli cell, NpHR forms a robust homotrimer in a detergent DDM solution. To identify the important residue for the homotrimer formation, we carried out mutation experiments on the aromatic amino acids expected to be located at the molecular interface. The results revealed that Phe(150) was essential to form and stabilize the NpHR trimer in the DDM solution. Further analyses for examining the structural significance of Phe(150) showed the dissociation of the trimer in F150A (dimer) and F150W (monomer) mutants. Only the F150Y mutant exhibited dissociation into monomers in an ionic strength-dependent manner. These results indicated that spatial positions and interactions between F150-aromatic side chains were crucial to homotrimer stabilization. This finding was supported by QM calculations. In a functional respect, differences in the reaction property in the ground and photoexcited states were revealed. The analysis of photointermediates revealed a decrease in the accumulation of O, which is important for Cl(-) release, and the acceleration of the decay rate in L1 and L2, which are involved in Cl(-) transfer inside the molecule, in the trimer-dissociated mutants. Interestingly, the affinity of them to Cl(-) in the photoexcited state increased rather than the trimer, whereas that in the ground state was almost the same without relation to the oligomeric state. It was also observed that the efficient recovery of the photocycle to the ground state was inhibited in the mutants. In addition, a branched pathway that was not included in Cl(-) transportation was predicted. These results suggest that the trimer assembly may contribute to the regulation of the dynamics in the excited state of NpHR.
Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
Takashi
T
Tsukamoto
Takanori
T
Sasaki
Kazuhiro J
KJ
Fujimoto
Takashi
T
Kikukawa
Masakatsu
M
Kamiya
Tomoyasu
T
Aizawa
Keiichi
K
Kawano
Naoki
N
Kamo
Makoto
M
Demura
D000042:Absorption; D000595:Amino Acid Sequence; D002712:Chlorides / Q000378:metabolism; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology*; D006216:Halobacteriaceae; D025602:Halorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010782:Photolysis; D055503:Protein Multimerization / Q000187:drug effects*; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D012996:Solutions; D013329:Structure-Activity Relationship
D002712:Chlorides; D003902:Detergents; D025602:Halorhodopsins; D012996:Solutions