PICOSECOND RESONANCE COHERENT ANTI-STOKES-RAMAN SPECTROSCOPY OF BACTERIORHODOPSIN - QUANTITATIVE 3RD-ORDER SUSCEPTIBILITY ANALYSIS OF THE DARK-ADAPTED MIXTURE

Picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS) f rom a dark-adapted bacteriorhodopsin (DA/BR) sample containing compara ble amounts of two ground-state species having different retinal struc tures, BR-570 (13-trans, 15-anti) and BR-548 (13-cis, 15 syn) are repo rted. Two picosecond (6 ps FWHM) laser pulses (578 nm (omega1) and 604 -643 nm (omega(s))) are used to record PR/CARS data via electronically -resonant excitation in both BR-570 and BR-548. PR/CARS spectra from t he isomeric mixture in DA/BR requires analysis in terms of third-order susceptibility (chi(3)) relationships which permit a quantitative sep aration of the contributions from each species. PR/CARS spectra for BR -570 (light-adapted BR or LA/BR) in protonated and deuterated water sa mples together with their associated chi(3) analyses (published earlie r) are used in the chi(3) analysis of the DA/BR data to quantitatively separate the PR/CARS spectrum of BR-548. The chi(3) analysis of the P R/CARS data provides a detailed characterization of the vibrational mo des in BR-548 thought to contain a 13-cis, 15-syn retinal. As importan tly, the experimental and analytical methodologies developed for analy zing the DA/BR mixture are directly applicable to picosecond time-reso lved CARS experiments designed to examine BR mixtures containing inter mediates with distinct retinal structures formed during the BR photocy cle.