Effect of lipid bilayer properties on the photocycle of green proteorhodopsin

Abstract

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.

Characterization of fast‐tumbling isotropic bicelles by PFG diffusion NMR

“…With the latter, q = 0.3 bicelles were observed for amphiphile concentrations as low as 12 mM (Table F). Similarly, q = 0.5 CHAPS bicelles with 24 mM; amphiphile concentration have been reported . Yet, bicelles at low concentrations may have complex phase behaviors .…”

“…No systematic study of pH effects on isotropic bicelles has been conducted, but bicelles in the range from pH 3.2 to 7.8 have been used. Unnerståle et al studied the pH dependence of the interaction between the C‐peptide and phospholipid bicelles and reported a decrease in the diffusion coefficient of the bicelles upon pH increase (Table H).…”

“…Besides different head groups, also acyl chain length and degree of unsaturation can be varied, and studies using PFG NMR have been carried out on bicelles containing phosphatidylcholines (PC) with varying acyl chains: POPC (16 : 0–18 : 1 PC), 18 : 1 PC, and 20 : 1 PC bicelles as well as bicelles of increasing acyl chain length (12 : 0, 14 : 0, and 16 : 0)(Table B). The effects of such changes in lipid compositions have also been intensely studied by other methods, e.g.…”

“…A similar problem arises if different bicelle populations, e.g. with different sizes or morphology altogether, are present in a sample . Again, diffusion coefficients of the lipid populations differ while their spectra overlap.…”

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

“…The small contribution of PR to the energy metabolism in Synechocystis observed here will in part be due to its relatively slow turnover (i.e. 'pumping') rate, which may be close to only 10 protons per second (79)(80)(81). Taking the latter value as a starting point one can calculate that in Synechocystis, growing with a doubling time of about 8 h and carrying out predominantly linear electron flow, the retinal-based pump, when expressed at levels of up to 10 5 molecules per cell, contributes to proton motive force generation (and hence ATP production) less than 1 % (this is assuming that one CO 2 fixed requires the pumping of 10 protons).…”