About ten years ago the characterization of an uncultivated marine bacterium revealed a new type of retinal-binding, integral membrane protein called proteorhodopsin (PR).[1] Many variants of PR exist that are spectrally tuned to the light condition in their environment and can be classified into two major groups, the blue-and green-absorbing forms.[2] Functional assays confirmed their ability to pump protons in a light-dependent manner similar to other microbial rhodopsins.[3] The high abundance of bacteria living in oceanic surface waters makes PR highly interesting because of its potential role in non-chlorophyll-based phototrophy in oceanic carbon cycling and energy flux.[4] The green-absorbing variant of PR and in particular its retinal-binding pocket has been intensively investigated by mutational and spectroscopic analysis,[3, 5] solid-state NMR spectroscopy,[6, 7] and homology modeling.[8] In addition to the retinal-binding site K231, other functionally important residues include the primary proton acceptor D97, the Schiff base counterions R94 and D227 and the primary proton donor E108. Remarkably, D97 possesses an unusually high pKa value of about 7.5 which is stabilized by H75 near the photoactive center.[6, 9] A similar Asp–His cluster has also been observed in xanthorhodopsin.[10] Influenced by the protonation state of D97 the absorption maximum of the retinal cofactor in PR is highly sensitive to changes in pH, ranging from 520 to 540 nm between pH 10 and 4.[3] Furthermore, the direction of proton pumping switches in response to the pH value between an outward directed transport at alkaline pH and an inward directed transport at acidic pH.[11] In contrast to the functional analysis, structural data on PR are still sparse [8, 12, 13] mostly because of the lack of well-diffracting three-dimensional crystals. Recently, the potential of solution NMR spectroscopy to solve structures of helical membrane proteins has been reported [14, 15] and we show herein the de novo structure of the green variant of proteorhodopsin solved by solution NMR spectroscopy.

The structure of PR (Figure 1) was solved in the shortchain lipid diC7PC (diheptanoyl-phosphocholine) combining long-range NOEs with restraints derived from paramagnetic relaxation enhancement (PRE) and residual dipolar couplings (RDCs). The seven transmembrane helices are connected by short loops. Instead of the anti-parallel β-sheet that is observed between helices B and C in other microbial rhodopsins, torsion angles derived from the protein backbone dihedral angle prediction program TALOS+ suggest that PR residues G87–P90 form a short β-turn. The loop between helices D and E is longer than predicted by the secondary structure prediction program TMHMM.[16] In contrast, the loop region connecting helicesE and F is shorter than predicted as residues E170–N176 form a helical extension (Eо) of helix E. Helix Eо is connected to helix E through a slight helical distortion at G169. Without the extension, helix E has approximately the same length as its neighboring helix D and is thus significantly shorter than the other five helices. Transmembrane helixF is slightly kinked around P201 ending in the longest and most dynamic loop of PR connecting helices F and G. Helix G contains a kink at residue N230 similar to the π-bulge observed in other microbial retinal-binding proteins.[13, 17]