The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.

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The function of membrane proteins is highly impacted by their membrane environment. One suitable approach to get insights into the interaction-induced dynamics of membrane proteins and lipid membranes is time-resolved infrared (IR) spectroscopy. Conclusions about environmental influences to the system can be drawn by correlating the observed kinetics to the well-characterized photocycles of light-driven transmembrane proton pumps like bacteriorhodopsin (BR). For the investigation of photoreceptor-membrane interactions, also minor changes in the absorption spectra must be resolved. Therefore, we applied IR laser spectroscopy using tunable quantum cascade lasers (QCLs) as IR light source. Several QCLs were implemented in a home-built spectrometer and provide a tunability in a broad spectral region covering protein, chromophore and lipid vibrational modes. Kinetics of the BR photocycle were monitored at single wavenumbers. This study demonstrates the high potential of QCL-based spectroscopy for the application to membrane protein studies.

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Membranes are crucial for the functionality of membrane proteins in several cellular processes. Time-resolved infrared (IR) spectroscopy enables the investigation of interaction-induced dynamics of the protein and the lipid membrane. The photoreceptor and proton pump bacteriorhodopsin (BR) was reconstituted into liposomes mimicking the native purple membrane. By utilization of deuterated lipid alkyl chains, corresponding vibrational modes are frequency-shifted into a spectrally silent window what allows to monitor lipid dynamics during the photoreaction of BR. Our home-built quantum cascade laser (QCL)-based IR spectrometer covers all relevant spectral regions to detect both lipid and protein vibrational modes. QCL-probed transients at single wavenumbers are compared with previously performed step-scan Fourier-transform infrared (FTIR) measurements. The absorbance changes of the lipids could be resolved by QCL-measurements with a much better signal to noise and with nanosecond time resolution. We found a correlation of the lipid dynamics with the protonation dynamics in the M intermediate. QCL spectroscopy extends the study of the protein's photocycle towards dynamics of the interacting membrane.