SPECTRAL RELATIONSHIP OF LIGHT-INDUCED REFRACTIVE-INDEX AND ABSORPTION CHANGES IN BACTERIORHODOPSIN FILMS CONTAINING WILDTYPE BRWT AND THE VARIANT BRD96N

Bacteriorhodopsin films containing the variant BR(D96N), Which differs from the wildtype BR(WT) by a single amino acid exchange, Asp 96 --> Asn, show significantly higher holographic diffraction efficiencies (eta) than BR(WT) films. The light-induced changes of both the absorption a(lambda,I) and the refractive index n(lambda,I), which influence the diffraction efficiency eta(lambda,I), have been investigated, and their spectral dependence has been measured in order to analyze both parameters separately. A maximal change of the refractive index of DELTA-n = 0.008 at 633 nm related to a modulation of the absorption of 0.55 OD, induced by actinic light of the wavelength 568 nm and an intensity of 20 MW/cm2, was observed for a BR film containing BR(D96N) at pH 9.5. From steady-state difference spectra of the absorption, the spectral refractive index change was calculated by the Kramers-Kronig relation, and a good correlation of the theoretically derived and experimentally measured values of the refractive index changes was found. This indicates that the chromophore system of bacteriorhodopsin, which is formed by the retinal molecule, its Schiff base linkage to the protein moiety, and an inner shell of amino acids, behaves like an almost undisturbed chromophore with respect to the photorefractive properties at low actinic light intensities, despite the fact that all components of the chromophoric system are covalently linked to the amino acid matrix. Further, it was demonstrated that it is possible to calculate the spectral dependence of the diffraction efficiency eta(lambda,I) from the easily accessible absorption changes DELTA-a(lambda,I) and the refractive index change DELTA-n(lambda,I) measured at a single wavelength. This method will be a useful tool for the characterization and optimization of bacteriorhodopsin films.