Abstract
Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.
Highlights
Since the light-driven chloride ion pump halorhodopsin from Natromonas pharaonis was discovered to have the ability to silence the electrical activity of neurons [1], a number of ionpumping rhodopsins have proved useful for optical control of the neuron activity [2,3]
From the two absorption spectra of the lightadapted crystal that were measured with polarized light, it was shown that the absorption dipole moment of retinal is tilted largely from the c axis of the crystal, whereas the absorbance of bacterioruberin is significant only when the polarization plane of the measuring light is in parallel to the c axis
The trimeric assemblies of microbial rhodopsins The present result shows that cR3 forms a trimeric structure with bacterioruberin bound to the crevice between neighboring subunits
Summary
Since the light-driven chloride ion pump halorhodopsin from Natromonas pharaonis (pHR) was discovered to have the ability to silence the electrical activity of neurons [1], a number of ionpumping rhodopsins have proved useful for optical control of the neuron activity [2,3]. The light-driven proton pump archaerhodopsin-3 (Arch) was reported to be most powerful in optically silencing neurons [4]. Several proton-pumping rhodopsins [5,6,7,8,9,10,11,12,13,14] and two anionpumping rhodopsins [15,16] have been crystallized. It is still difficult to elucidate which structural factors affect the protein stability of ion-pumping rhodopsins in neuron cells. Against this background, it is important to accumulate more structural data of microbial rhodopsins
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