Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films

Abstract

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.

Effects of polyhydric alcohols on the conformational stability of the purple membrane

“…Furthermore, glycerol impregnation of an oriented PM film did not change the PM film far-UV CD spectrum. This indicates that glycerol does not affect the alignment of the helical polypeptide segments of the bR relative to the plane of the PM (Muccio & Cassim, 1979a;Papadopoulos & Cassim, 1981;Draheim & Cassim, 1985). Although there is no indication of a change in the net secondary structure of the bR, significant changes are possible in the tertiary structure of the bR without observably altering its far-UV CD spectrum (Becher & Cassim, 1977).…”

“…Similar results were obtained with dark-adapted PM (containing equimolar mixture of 13-cis and all-trans retinal). Oriented PM films were formed on optical quality flats of Suprasil-S quartz (Precision Cells, Inc., Hicksville, New York) according to the method of Muccio and Cassim (1979a). In order to impregnate a PM film with glycerol a very thin layer of 20% glycerol solution was first placed over the film.…”

“…568 nm with a predominant positive lobe. The 317-nm negative CD band has been attributed to a magnetic-dipole-allowed weak rr-Tr* retinyl transition (Muccio & Cassim, 1979a). The CD band system, centered at ca.…”

“…The CD band system, centered at ca. 568 nm is most likely due to the sum of at least two components (Heyn et al, 1975;Bauer et al, 1976;Becher & Cassim, 1976;Ebrey et al, 1977;Muccio & Cassim, 1979a). One component is a single positive Gaussian CD band centered at 568 nm resulting from asymmetric interactions between the retinyl chromophores and the apoproteins.…”

“…Impregnation of the PM film does not significantly change the light-scattering properties of the film in the optically transparent regions (such as 800 nm). In a PM film the optical activity due to excitonic coupling among the retinyl chromophores in the PM crystal lattice must vanish because of the p3 symmetry of the bR organization (Muccio & Cassim, 1979a). Thus only the positive monomeric CD band remains in the control spectrum together with a large negative CD band at 320 nm.…”