Irreversible Trimer to Monomer Transition of Thermophilic
Rhodopsin upon Thermal Stimulation
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Abstract
Assembly is one of the keys to understand
biological molecules,
and it takes place in spatial and temporal domains upon stimulation.
Microbial rhodopsin (also called retinal protein) is a membrane-embedded
protein that has a retinal chromophore within seven-transmembrane
α-helices and shows homo-, di-, tri-, penta-, and hexameric
assemblies. Those assemblies are closely related to critical physiological
properties such as stabilizing the protein structure and regulating
their photoreaction dynamics. Here we investigated the assembly and
disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping
rhodopsin derived from a thermophile living at 75 °C. TR was
characterized using size-exclusion chromatography and circular dichroism
spectroscopy, and formed a trimer at 25 °C, but irreversibly
dissociated into monomers upon thermal stimulation. The transition
temperature was estimated to be 68 °C. The irreversible nature
made it possible to investigate the photochemical properties of both
the trimer and the monomer independently. Compared with the trimer,
the absorption maximum of the monomer is blue-shifted by 6 nm without
any changes in the retinal composition, p<i>K</i><sub>a</sub> value for the counterion or the sequence of the proton movement.
The photocycling rate of the monomeric TR was similar to that of the
trimeric TR. A similar trimer-monomer transition upon thermal stimulation
was observed for another eubacterial rhodopsin GR but not for the
archaeal rhodopsins AR3 and HwBR, suggesting that the transition is
conserved in bacterial rhodopsins. Thus, the thermal stimulation of
TR induces the irreversible disassembly of the trimer