Abstract
Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.
Highlights
Microbial rhodopsins constitute a large group of photoactive membrane proteins with seven-transmembrane α-helices, which are found in archaea, bacteria, and lower eukaryotes (Fig. 1)[1]
The oligomeric states of microbial rhodopsins have been mainly studied by X-ray crystallography, gel filtration chromatography, cryo-electron microscopy, and circular dichroism (CD) spectroscopy
We comprehensively analyzed the oligomeric structures of various microbial rhodopsins, including Krokinobacter eikastus rhodopsin 2 (KR2: sodium pump), Gloeobacter rhodopsin (GR: proton pump), Fulvimarina rhodopsin (FR: chloride pump), Kineococcus radiotolerans actinorhodopsin (KrActR: proton pump), Quadrisphaera actinorhodopsin (QsActR: proton pump), green proteorhodopsin (GPR), Natronomonas pharaonis halorhodopsin (NpHR: chloride pump), Natronomonas pharaonis sensory rhodopsin II (NpSRII: phototaxis sensor), Parvularcula oceani xenorhodopsin (PoXeR: inward proton pump), and Anabaena sensory rhodopsin (ASR: photochromic sensor)
Summary
Oligomeric structure of sodium-pumping Krokinobacter rhodopsin, KR2. First, we analyzed a light-driven sodium-pumping rhodopsin KR2. This negative/positive couplet is similar to those of other eubacterial rhodopsins (KR2, FR, KrActR and QsActR), and consistent with the pentamer dominant oligomeric state of GPR observed by HS-AFM. The CD spectrum of NpHR shows a positive peak and negative peak on the blue (541 nm) and red sides (608 nm) of λmax = 576 nm, respectively (right panel in Fig. 5a)[38] This is similar to the CD spectra of trimeric BR and HsHR28,29, and the CD couplet is inverted compared to those observed in the pentameric rhodopsins (KR2, FR, GR, KrActR, QsActR and GPR).
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