doi:10.1016/j.jmb.2007.11.030 J. Mol. Biol. (2008) 376,35–41 Available online at www.sciencedirect.com Folding and Assembly of Proteorhodopsin Adriana L. Klyszejko1†, Sarika Shastri2†, Stefania A. Mari3, Helmut Grubmüller4, Daniel J. Muller1⁎ and Clemens Glaubitz2 1Biotechnology Center, Proteorhodopsins (PRs), the recently discovered light-driven proton University of Technology pumps, play a major role in supplying energy for microbial organisms of Dresden, Germany oceans. In contrast to PR, rhodopsins found in Archaea and Eukarya are structurally well characterized. Using single-molecule microscopy and 2Institute of Biophysical spectroscopy, we observed the oligomeric assembly of native PR molecules Chemistry and Center for and detected their folding in the membrane. PR showed unfolding patterns Biomolecular Magnetic identical with those of bacteriorhodopsin and halorhodopsin, indicating Resonance, Goethe University that PR folds similarly to archaeal rhodopsins. Surprisingly, PR predomi- Frankfurt, Germany nantly assembles into hexameric oligomers, with a smaller fraction 3Institute of General assembling into pentamers. Within these oligomers, PR arranged into Physiology and Biological radial assemblies. We suggest that this structural assembly of PR may have Chemistry, University functional implications. of Milan, Italy © 2007 Elsevier Ltd. All rights reserved. 4Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany Received 5 October 2007; accepted 9 November 2007 Available online 19 November 2007 Keywords: membrane protein folding; oligomeric state; hexamer; high- Edited by W. Baumeister resolution atomic force microscopy; single-molecule force spectroscopy Introduction brane α-helices surrounding the light-sensitive retinal. However, the oligomeric assembly of rho- Rhodopsins occur in Archaea, Bacteria, and dopsins in Archaea and Eukarya differs and ranges Eukarya. Using a chromophore, rhodopsins sense from monomeric, to dimeric, to trimeric forms, which supposedly have structural and functional light of certain wavelengths. In Archaea, this 1,3,4 captured light is employed to pump protons or origins. chloride ions across the membrane and to create an The recent discovery of rhodopsins in bacteria electrochemical gradient, which powers cellular [proteorhodopsin (PR)] came after the sequence analysis of a cloned genome region from a marine machineries. Other archaeal rhodopsins sense light 5 for phototaxis.1 In vertebrates, rhodopsins function bacterium of the uncultivated SAR86 clade. Sub- primarily as sensory proteins and account for sequent DNA screening of microorganisms from vision.2 Archaeal and eukaryal rhodopsins show different oceans revealed a very large diversity of PR common structural motifs; they are embedded in belonging to divergent clades of the Alphaproteo- 6–11 the membrane bilayer and have seven transmem- bacteria and Gammaproteobacteria classes. Two related PR families that absorb light with different absorption maxima, ≈525 nm (green) and ≈490 nm *Corresponding author. E-mail address: (blue), were found, and their distribution was [email protected]. shown to be stratified with water depth. A single † A.K. and S.S. contributed equally to this work. amino acid at position 105 functions as a spectral Abbreviations used: AFM, atomic force microscopy; PR, tuning switch and seems to account for most of the proteorhodopsin; SMFS, single-molecule force spectral difference between green-absorbing PRs spectroscopy; F–D, force–distance; DOPC, and blue-absorbing PRs.10 The phototrophy con- 1,2-dioleolyl-sn-glycero-3-phosphocholine. ferred by PR can provide critical amounts of energy 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. 36 Proteorhodopsin Assembly not only for respiration and maintenance but also Results and Discussions for active growth of marine bacterioplankton in their natural environment.12 This potential to complement their chemotropic lifestyle by photo- Two-dimensional PR crystals show larger unit trophy has evolutionarily favored these oceanophi- cell size than bacteriorhodopsin lic microbial organisms and the wide oceanographic – distribution of PR genes.6,13 15 PRs overproduced in AFM imaging of reconstituted PR membranes in Escherichia coli have further established that PR buffer solution (Fig. 1a) showed crystalline (*) and functions as a light-driven proton pump with the densely packed (**) regions. At high resolution, the potential to generate energy for cell growth and surface of the two-dimensional PR crystals revealed maintenance.5,9,16 donut-like structures arranged in a hexagonal lattice PR is a light-driven proton pump like bacterior- of 8.8±0.7 nm (mean±SD; n=30) side length (Fig. hodopsin from archaeabacteria.1,17 In contrast to 1b), which agrees well with the low-resolution PR, the structure–function relationship of bacter- electron microscopy analysis of the same crystals.22 iorhodopsin and its homologues, the light-driven These donut-like structures featured pronounced chloride pump halorhodopsin18 and sensory protrusions extending 0.9±0.3 nm (n=50) above the rhodopsin,3 has been studied at molecular resolu- lipid surface. In comparison, the hexagonal lattice of tion. So far, the only structural insights available for bacteriorhodopsin trimers in native purple mem- PR have been revealed from spectroscopic studies brane patches shows a side length of ≈6.2 nm24 with and modeling.19,20 Recent low-resolution structural single bacteriorhodopsin molecules protruding data revealed from two-dimensionally crystallized between ≈0.5 and 0.8 nm from the lipid bilayer.25 PR suggested a hexagonal lattice constant of The hexagonal unit cell of the purple membrane ≈8.7 nm,21 showing a symmetric ringlike arrange- lattice covers an area of 16.64 nm2, hosting three ment.22 Initial solid-state NMR studies focused on bacteriorhodopsin molecules. In contrast, the area the characterization of retinal and Schiff base.23 covered by the unit cell of the hexagonal PR lattice is Here, we show high-resolution atomic force micro- 33.53 nm2. It was suggested that the secondary scopy (AFM) topographs of green PR reconstituted structures of PR and bacteriorhodopsin are very into the lipid bilayer. These topographs show similar.19 Thus, the unit cell of the PR lattice would oligomers of PRs assembled in a two-dimensional provide sufficient space to accommodate up to six lattice and densely packed in the membrane. PR molecules instead of three as observed for Single-molecule force spectroscopy (SMFS) shows bacteriorhodopsin. Recent calculations suggested PRs to unfold via similar structural intermediates ≈24,000 PR molecules per SAR86 cell.26 Packed as observed for bacteriorhodopsin and halorhodop- into a densely packed crystalline lattice, such as sin. This suggests that all three rhodopsins have observed for purple membrane, this would make a almost identical folding and three-dimensional 600-nm-diameter flat circular patch covering a structures. significant portion of a cell surface. Therefore, the Fig. 1. AFM of two-dimensionally crystallized and densely packed PR membranes. (a) Survey showing crystalline (*) and densely packed (**) patches of PRs embedded in the lipid (DOPC) bilayer. The lipid bilayer protruded 4.5±0.5 nm (n=10) from the support, the crystals protruded 7.1±0.5 nm (n=10), and the densely packed PRs protruded 6.3±0.5 nm (n=10). (b) High-resolution topography showing the hexagonal lattice (a=8.8±0.7 nm) of donut-shaped oligomers formed by PRs. (c) Calculated diffraction pattern of (b) documents spots (red circles), suggesting a lateral resolution of ≈1.5 nm. (d) Correlation average of (b). The symmetrized average showed a 9% deviation from 6-fold symmetry. AFM topographs recorded in buffer solution exhibit a full gray level corresponding to a vertical scale of 20 nm (a) and 2 nm (b and d). Proteorhodopsin Assembly 37 half packing density of PR would cover twice the The oligomeric assembly of PR area of the cell membrane. Hence, the enhanced size of the PR donuts leads to the question whether they High-resolution AFM of the noncrystalline areas reflect the oligomeric state of trimers, tetramers, of the reconstituted membrane patches showed pentamers, or hexamers. densely packed PR oligomers (Fig. 2a). The highest Most of the donut-shaped molecules observed in protrusions of these oligomers extended 1.3±0.2 nm the high-resolution AFM images (N90%) of the (n=50) from the surface of the lipid bilayer. The crystalline PR patches showed the same diameter majority of oligomers (N90%) observed were circu- (Fig. 1b). The donuts showed an average diameter of lar, with their protrusions being equally distributed 4.3±0.3 nm (n=83) and, in most cases, six protru- at the outer circle diameter. Surprisingly, indivi- sions, which became clearly visible after correlation dual oligomers exhibited either six (Fig. 2b) or five averaging (Fig. 1d). In contrast, some donut-like (Fig. 2c) protrusions, thus showing either 6- or 5-fold shapes exhibited smaller diameters (Fig. 1b, com- symmetries. In rare cases, incomplete oligomers pare circles) and less than six protrusions. However, missed a single subunit or more (Fig. 2, dotted from these topographs, it could not be unambigu- ellipse). The surface area occupied by a PR molecule ously answered how many PR molecules have ≈2×3 nm2 matches that observed for bacterio- established the protrusions. rhodopsin25 and halorhodopsin.27 This suggested Fig. 2. High-resolution AFM of densely packed PR oligomers. (a) PR molecules