The objective of the proposed research is to investigate by 2H-NMR the structure of the chromophore of bacteriorhodopsin, the key component of this light-driven proton pump. The rate of proton transfer reactions is critically dependent on the distances and bond angles of proton donors and acceptors. The protonated Schiff base is the source of protons in this pump and the structural information obtained will be essential in developing detailed models of its mechanism. The angles of each of the bonds connecting the five methyl groups of the chromophore with the polyene chain and beta-ionone ring will be determined with respect to the membrane normal. These angles will be obtained with high accuracy from the quadrupole splittings of the 2H-NMR spectra of oriented uniaxial samples of purple membranes regenerated with the corresponding selectively deuterated retinals. Experiments will be carried out in the dark initial state as well as in the key photocycle intermediates M, N and O, in which the chromophore is in a 13-cis configuration or has a deprotonated Schiff base. The changes in angle will thus provide structural information about curvature, twists and tilt of the chromophore in these functionally important intermediates. External parameters (pH, ionic strength and temperature) and mutants will allow trapping of the various intermediates. The M intermediate is the key intermediate in the proton translocation cycle, and particular attention will be paid to its potential role as a reprotonation switch. Complementary spin-lattice relaxation experiments will be performed to learn about the mobility of various parts of the chromophore in the different intermediates. This information will be valuable in understanding other medically important ion translocating membrane proteins, for which much less is known about the structure. Bacteriorhodopsin is moreover the prototype of the important family of receptor proteins with seven transmembrane alpha-helices, which includes the visual pigment rhodopsin. Activation of rhodopsin involves structural changes in its M-II intermediate which originates in the chromophore and propagate to the cytoplasmic loops. An investigation of the analogous steps in the M intermediate of bR is expected to contribute to an understanding of the mechanism of activation of rhodopsin in visual signal transduction.
| Heyn, M P; Borucki, B; Otto, H (2000) Chromophore reorientation during the photocycle of bacteriorhodopsin: experimental methods and functional significance. Biochim Biophys Acta 1460:60-74 |
| Moltke, S; Wallat, I; Sakai, N et al. (1999) The angles between the C(1)-, C(5)-, and C(9)-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: direct determination with solid-state (2)H NMR. Biochemistry 38:11762-72 |
| Moltke, S; Nevzorov, A A; Sakai, N et al. (1998) Chromophore orientation in bacteriorhodopsin determined from the angular dependence of deuterium nuclear magnetic resonance spectra of oriented purple membranes. Biochemistry 37:11821-35 |
