X-ray structure analysis of bacteriorhodopsin at 1.3 Å resolution

Abstract

Bacteriorhodopsin (bR) of Halobacterium salinarum is a membrane protein that acts as a light-driven proton pump. bR and its homologues have recently been utilized in optogenetics and other applications. Although the structures of those have been reported so far, the resolutions are not sufficient for elucidation of the intrinsic structural features critical to the color tuning and ion pumping properties. Here we report the accurate crystallographic analysis of bR in the ground state. The influence of X-rays was suppressed by collecting the data under a low irradiation dose at 15 K. Consequently, individual atoms could be separately observed in the electron density map at better than 1.3 Å resolution. Residues from Thr5 to Ala233 were continuously constructed in the model. The twist of the retinal polyene was determined to be different from those in the previous models. Two conformations were observed for the proton release region. We discuss the meaning of these fine structural features.

NeoR, a near-infrared absorbing rhodopsin

“…The model was generated using the MODELLER (version 9.20) program ( https://salilab.org ) 54 . Multiple retinal proteins have served as a basis for the model generation: Haloquadratum walsbyi bacteriorhodopsin ( Hw BR, PDB ID: 4QI1) 55 , Halobacterium salinarum bacteriorhodopsin ( Hs BR, PDB ID: 5ZIM) 56 , Archærhodopsin-3 (AR3, PDB ID: 6GUX), Sensory Rhodopsin II (SRII, PDB ID: 5JJE) 57 , Acetabularia Rhodopsin I (ARI, PDB ID: 5AX0) 58 , Archærhodopsin-2 (AR2, PDB ID: 3WQJ) 59 , Blue-light-absorbing proteorhodopsin (BPR, PDB ID: 4JQ6) 60 , Exiguobacterium sibiricum rhodopsin (ESR, PDB ID: 4HYJ) 60 , Channelrhodopsin Chrimson, PDB ID: 5ZIH) 31 , Chlamydomonas reinhardtii channelrhodopsin-2 ( Cr ChR2, PDB ID: 6EID) 61 , Channelrhodopsin-1/−2 chimæra (C1C2, PDB ID: 3UG9) 62 , Krokinobacter eikastus rhodopsin-2 (KR2, PDB ID: 6RF6) 63 , Gloeobacter rhodopsin (GR, PDB ID: 6NWD) 64 and Coccomyxa subellipsoidea rhodopsin (CsR, PDB ID: 6GYH) 65 . Their sequences were aligned with ClustalO 66 .…”

Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs

“…bR has been studied extensively during the last decades using various biophysical techniques (Birge 1990a, b;Haupts et al 1999;Ernst et al 2014;Brown and Ernst 2017), as well as by solution- (Patzelt et al 2002;Schubert et al 2002) and solid-state NMR (Harbison et al 1984a, b;Smith et al 1984). Moreover, several high-resolution structures are available (Luecke et al 1999;Schobert et al 2002;Hasegawa et al 2018). Schubert et al (2002) determined assignments by solution-state NMR for bR in dodecylmaltoside (DM) micelles, and could assign approximately one-third of the amide signals.…”

Usefulness of oils for cleaning the host matrix and for cryoprotection of lipidic cubic phase crystals

“…The method assessed herein was actually used in our recent studies for bR and heme A synthase (HAS) (Hasegawa et al, 2018;Niwa et al, 2018). In the case of bR, squalane was used for cleaning and cryoprotection (Hasegawa et al, 2018). The size of the crystals used by Hasegawa et al was 300-400 mm, which was larger than the size of the crystals used in this work.…”

“…This type of bR crystal has typical type I packing in which 2D layers of membrane proteins stack into a 3D crystal. bR was purified and crystalized as described previously (Hasegawa et al, 2018). Briefly, the purple membrane was purified from Halobacterium salinarum strain R1 (JMC9409) by a standard procedure (Oesterhelt & Stoeckenius, 1971).…”