The purpose of this pilot study is to launch a two part high resolution electronic spectroscopy investigation of the purple membrane of Halobacterium halobium. One component deals with retinal, bacteriorhodopsin and its photocycle intermediates K610 and M412; the other component deals with the membrane and its blue and pink forms. We have already obtained interesting preliminary data on the purple and blue membrane species. The crystal-like membrane of Halobacterium halobrium is an important model for studying the structure and function of biological membranes and the visual process. It's primary component is the protein bacteriorhodopsin (bR) which acts as a light driven proton pump; the chromphore in bR is retinal. There is no high resolution data on the excited electronic states of retinal, bR or the purple membrane because of the extensive inhomogeneous broadening of their absorption bands. The approach here to obtaining detailed information on the isolated retinal molecule and its properties in the membrane will be to use a combination of optical hole- burning and Stark methods; the retinal chromophore will be placed in a series of low temperature (4.2K) environmental settings, ranging through an alkane crystal, bR monomer, purple membrane and photoproducts. Optical hole-burning involves burning a homogeneously broadened hole in an inhomogeneously broadened spectrum with an intense narrow band laser. (The hole results from the molecule's photochemistry or reorganization in the 4.2K lattice.) Measurement of the hole width gives the excited stat lifetime via the uncertainty principle. Excited state vibrational frequencies can also be determined; when a single hole is burned in the origin region of a broad absorption band, it will be followed by a pattern of the associated vibronic holes. Furthermore, when hole-burning is coupled with the Stark effect, they become a sensitive probe of excited state properties (e.g. dipole moment). In a brief preliminary investigation a sample of purple membrane was cooled in buffered glycerol/H2O glass to 4.2K. Upon irradiation with a pulsed dye laser (570 nm) the membrane bleached. As the sample was warmed to 298K the bleached area turned blue and then its original purple. This may be the first bleaching of purple membrane without a trapping agent (hydroxylamine) and we may have produced the blue membrane without the use of pH changes or ion complexing agents. In this pilot program we propose to make these observations under more controlled conditions of temperature and laser power to determine what species have been formed and how they are related photocycle dynamics.
