Biophysical studies of integral membrane proteins

Date of Completion

January 2005


Biology, Molecular|Chemistry, Biochemistry|Biophysics, General




Membrane proteins pose serious technical challenges to both X-ray crystallography and NMR. Recently, structural studies of peptides corresponding to fragments of rhodopsin and bacteriorhodopsin have demonstrated that these fragments retain their native secondary structure when isolated from the intact protein (Yeagle et al, Biochemistry, 2001:40(39);11932--7, Katraggadda et al, Biophys. J., 2001:81(2);1029--36). In this study, peptides corresponding to the transmembrane helices and helix-connecting loops of lactose permease were studied by solution NMR. The results reveal that the helices corresponding to the C-terminal domain of tend to form well-ordered helices in solution, while peptides corresponding to the N-terminal helices tended to be disordered in solution. These data suggest that the C-terminal domain of lactose permease is inherently more rigid than the N-terminal domain. ^ The role of chromophore structure in the kinetic stability of rhodopsin was studied using DSC. Regenerated rhodopsin and isorhodopsin were prepared by completely bleaching bovine disks, followed by overnight incubation with 11-cis retinal or 9-cis retinal. The activation energies of thermal denaturation for these pigments were according to the method of Sanchez-Ruiz et al (Biochemistry 1988 27(5); 1648--52) by collecting DSC thermograms at scanrates of 30, 60, and 90 °C/hr. The denaturation activation energies for rhodopsin and isorhodopsin were 170±20 and 140±10 kcal/mole, respectively. This suggests that the presence of a cis π-bond at carbon 9 of retinal causes a slight decrease in the kinetic stability of isorhodopsin relative to rhodopsin. ^ Recent studies have suggested that rhodopsin may form dimers in disk membranes, while many other studies have demonstrated its existence as a monomer under native conditions. In order to address this issue, DSC measurements of partially bleached disks were used to determine the influence of the opsin:rhodopsin ratio on the kinetic stability of rhodopsin. Disks containing opsin:rhodopsin mixtures of 1:3, 1:1, and 3:1 were prepared by partially bleaching disks with controlled light flashes. The denaturation activation energies were determined by collecting DSC thermograms at 15, 40, 65, and 90 °C/hr. The activation energy of rhodopsin was found to be unchanged by the opsin:rhodopsin ratio. Since the lower kinetic stability of opsin relative to rhodopsin would be expected to influence the denaturation of rhodopsin in partially bleached membranes, these data suggest that rhodopsin is monomeric in disk membranes. ^