Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy^†

Abstract

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent-solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein-protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm(-1). In rapid-scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.

Studying the stoichiometries of membrane proteins by mass spectrometry: microbial rhodopsins and a potassium ion channel

“…Finally the photocycles of solubilized PR in Triton-X 100 and of 2D crystals were found to be almost identical. 25 Our results from the mass spectrum in Fig. 2(a) together with the visual data of the 2D crystals suggest that the photocycle seems to be independent of the oligomeric state.…”

Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR

“…Indeed, it was shown by CD spectroscopy that green PR in 2D crystals shows signals that indicate additional (aromatic) protein−protein contacts, resulting also in higher thermal stability. 58 3D crystal structures of two blue absorbing PRs have been reported. 10 The Med12BPR variant (57% identity to the green PR, PDB ID: 4JQ6) has been crystallized as the hexamer while two mutants of HOT75BPR variant (78% identity, PDB ID: 4KNF, 4JQ6) formed pentamers.…”

Crystallization and preliminary X-ray crystallographic analysis of a blue-light-absorbing proteorhodopsin

“…In order to understand the global ecological implications of PR and to solve the puzzle of its light harvesting and proton translocation, it is very important to resolve its three-dimensional structure. Various methods such as EM and NMR have been used to attempt to solve the structure of PR (Shastri et al, 2007;Liang et al, 2007;Schä fer et al, 2009;Pfleger et al, 2008;Shi et al, 2009), but no three-dimensional structure has been reported to date. To solve the crystal structure of a proteorhodopsin, we attempted to crystallize wild-type blue-lightabsorbing proteorhodopsin (BPR) and green-light-absorbing proteorhodopsin (GPR; Man et al, 2003) and various single and multiple mutants of these and other PRs.…”