Lipid−Rhodopsin Hydrophobic Mismatch Alters Rhodopsin Helical Content

The ability of photoactivated rhodopsin to achieve the enzymatically active metarhodopsin II conformation is exquisitely sensitive to bilayer hydrophobic thickness. The sensitivity of rhodopsin to the lipid matrix has been explained by the hydrophobic matching theory, which predicts that lipid bilayers adjust elastically to the hydrophobic length of transmembrane helices. Here, we examined if bilayer thickness adjusts to the length of the protein or if the protein alters its conformation to adapt to the bilayer. Purified bovine rhodopsin was reconstituted into a series of mono-unsaturated phosphatidylcholines with 14−20 carbons per hydrocarbon chain. Changes of hydrocarbon chain length were measured by 2H NMR, and protein helical content was quantified by synchrotron radiation circular dichroism and conventional circular dichroism. Experiments were conducted on dark-adapted rhodopsin, the photo-intermediates metarhodopsin I/II/III, and opsin. Changes of bilayer thickness upon rhodopsin incorporation and photoactivation were mostly absent. In contrast, the helical content of rhodopsin increased with membrane hydrophobic thickness. Helical content did not change measurably upon photoactivation. The increases of bilayer thickness and helicity of rhodopsin are accompanied by higher metarhodopsin II/metarhodopsin I ratios, faster rates of metarhodopsin II formation, an increase of tryptophan fluorescence, and higher temperatures of rhodopsin denaturation. The data suggest a surprising adaptability of this G protein-coupled membrane receptor to properties of the lipid matrix

The Bacteriorhodopsin Carboxyl-Terminus Contributes to Proton Recruitment and Protein Stability

We examined functional and structural roles for the bacteriorhodopsin (bR) carboxyl-terminus. The extramembranous and intracellular carboxyl-terminus was deleted by insertion of premature translation stop codons. Deletion of the carboxyl-terminus had no effect on purple membrane (PM) lattice dimensions, sheet size, or the electrogenic environment of the ground-state chromophore. Removal of the distal half of the carboxyl-terminus had no effect on light-activated proton pumping, however, truncation of the entire carboxyl-terminus accelerated the rates of M-state decay and proton uptake ∼3.7-fold and severely distorted the kinetics of proton uptake. Differential scanning calorimetry (DSC) and SDS denaturation demonstrated that removal of the carboxyl-terminus decreased protein stability. The DSC melting temperature was lowered by 6 °C and the calorimetric enthalpy reduced by 50% following removal of the carboxyl-terminus. Over the time range of milliseconds to hours at least 3 phases were required to describe the SDS denaturation kinetics for each bR construction. The fastest phases were indistinguishable for all bR’s, and reflected PM solubilization. At pH 7.4, 20 °C, and in 0.3% SDS (w/v) the half-times of bR denaturation were 19.2 min for the wild-type, 12.0 min for the half-truncation and 3.6 min for the full-truncation. Taken together the results of this study suggest that the bR ground state exhibits two “domains” of stability: (1) a core chromophore binding pocket domain that is insensitive to carboxyl-terminal interactions and (2) the surrounding helical bundle whose contributions to protein stability and proton pumping are influenced by long-range interactions with the extramembranous carboxyl-terminus