Location of the chromophore in bacteriorhodopsin.

Abstract

We present a location for the retinylidene chromophore in dark-adapted bacteriorhodopsin based on the differences in neutron scattering between purple membrane preparations reconstituted with retinal and with deuterated retinaL The Fourier difference density map contains more peaks than expected, and additional arguments are introduced to exclude artificial peaks caused by the reconstitution techniques or the limited resolution of the diffraction data. The membrane preparation used is necessarily dark-adapted and therefore contains 13-cis-and all-trans-retinal isomers in roughly equal amounts. However, we find only a single position for both isomers. Presumably, the difference in conformation caused by isomerization around the C13-C14 double bond is minimized by rotation around other bonds. The retinal is located between a-helical segments of the rotein and its nearest neighbor (intratrimer) distance is 26 A; the next-nearest neighbor (intertrimer) distance is 38 A.The purple membrane occurs as differentiated patches in the cell membrane of halobacteria. It contains only one protein, bacteriorhodopsin (bR), of molecular weight 26,500; the amino acid sequence is known (1, 2). It has a strong absorption band between 500 and 600 nm due to covalently bound retinal. bR converts light energy into an electrochemical proton gradient, which drives energy-requiring metabolic processes such as ATP synthesis and amino acid transport (3-5). The crystalline array of bR in the plane of the purple membrane has made it possible to determine the membrane structure at high resolution. Henderson and Unwin (6), using low-dose electron microscopy, obtained a three-dimensional scattering density map at 7-A resolution in the plane of the membrane and at 14-A resolution normal to it. The protein apparently consists of seven a-helical rods, about 40 A long and 10 A apart, all oriented roughly perpendicular to the membrane plane and arranged in two parallel rows. This model has been confirmed by Hayward et al. (7). The lipids occupy the interstices of the protein lattice in a bilayer orientation (8).The group of a-helical segments shown below in Fig. 2 Resonance Raman spectra of bR and its photocycle intermediates (17) strongly suggest that the retinylidene Schiff base is directly involved in proton transport across the membrane. This has led us to examine the location of retinal in the membrane. The retinylidene imine can be exchanged if the chromophore is bleached with hydroxylamine under intense illumination, followed by regeneration with added 13-cis-or alltrans-retinal (18,19). In this paper, we report the position of the retinylidene isomers in the membrane plane as determined by a neutron-scattering Fourier difference density map between samples regenerated with normal and with deuterated retinal, respectively. We have reported previously that the f3-ionone ring of the molecule is located near the center of the membrane profile (20). MATERIALS AND METHODSPreparation of Deuterated Membrane. Fully deuterated purple membrane...

A neutron diffraction study on the location of the polyene chain of retinal in bacteriorhodopsin.

“…All four studies concluded that the chromophore is located in the interior of bacteriorhodopsin. A clear discrepancy exists between the results of King et al (4) and Jubb et al. (7) concerning the location of the 0-ionone ring.…”

“…Thus far, the position of retinal could not yet be resolved by the latter method. In a pioneering study, King et al (4) determined this location, projected onto the plane of the membrane, by means of neutron diffraction with perdeuterated retinal containing 28 deuterons. Three main peaks were observed in the Fourier difference map and an unambiguous assignment was difficult.…”

“…Whereas in ref. 4 the Schiffbase position was assigned to one of several peaks, in ref. 8 two distinct density regions were identified corresponding to the hydrocarbon chain.…”

Rhodopsin and bacteriorhodopsin: structure—function relationships

“…The peptides (l-lo), (1 l-13) (14-24) and (25)(26)(27)(28)(29)(30) belonging to the N-terminal fragment were found in the supernatant. Papain action on the inside-out membranes cleaved another bond, 186-187.…”

“…Various physical methods, such as neutron diffraction [28] or fluorescence [29] provided some information about spatial arrangement of the /! !-ionone ring.…”