Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System

Abstract

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.

Salt bridge in the conserved His–Asp cluster in Gloeobacter rhodopsin contributes to trimer formation

“…4a in Ref. [24]), but the sign of the spectrum was opposite from BR [16] and HR [10,15,17,18]. One of possibility is that the molecular arrangement of the trimer structure of GR is different from that of BR and HR, but the further studies are awaited and in progress in our laboratory.…”

“…Thus, panel b reveals the correct assignment of each fraction. It is noteworthy that the sign of the bipolar CD spectrum is opposite to that of BR [16] and HR [10,15,17,18]. The CD spectrum of GR exhibited a pair of negative and positive bands near 500 and 570 nm, respectively, and a zero crossover point near the absorption maximum of 540 nm.…”

“…2a shows the chromatogram of the NpHR trimer, suggesting that peak B is attributable to the trimer. The F150W of NpHR forms a monomer in DDM solution [15], whose chromatogram is shown by the broken line in Fig. 2a.…”

Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation

“…It has been shown that this mutant protein forms a monomer in a solution containing the detergent DDM. 22 As shown in Figure 2A, peak B overlapped well with the peak of NpHR-F150W, indicating that peak B represents the monomeric form of TR. The visible CD spectrum was also changed from a biphasic curve to a monophasic one after thermal stimulation (81°C; Figure 2B, red line).…”

The light‐driven sodium ion pump: A new player in rhodopsin research

“…3A and B). While it has been reported that oligomerization affects the protein stability and kinetics of the photocycle [41,42], a number of biochemical and biophysical studies have proved that the functional unit of ion-pumping rhodopsins is the monomer [43][44][45], and the physiological role of oligomerizations is still unclear. Further studies are needed to determine why ion pumping rhodopsins form oligomers under physiological conditions.…”