PICOSECOND RESONANCE COHERENT ANTI-STOKES-RAMAN SPECTROSCOPY OF LIGHT- AND DARK-ADAPTED BACTERIORHODOPSIN

Coherent anti-Stokes Raman scattering (CARS) from the C=C stretching b and region of retinal, recorded with low-power, 7-ps (fwhm) laser exci tation selected to be in electronic resonance with the retinal chromop hore, is presented for light- and dark-adapted bacteriorhodopsin (BR) samples. By minimizing photochemistry with low-power excitation, the p icosecond resonance CARS (PR/CARS) spectrum of BR-570 (13-trans, 15-an ti-retinal) is obtained directly from the light-adapted BR sample. The PR/CARS spectrum of BR-548 (13-cis, 15-syn retinal) is derived from a quantitative analysis of PR/CARS data from dark-adapted BR which cont ains comparable amounts of BR-570 and BR-548. Band origin positions, l ineshapes, relative intensities, and phase factors describing the elec tronic resonances are obtained from a quantitative fit of the PR/CARS spectra to third-order susceptibility (chi((3))) relationships. These PR/CARS data, the first reported for a molecular system as large as BR , demonstrate the experimental viability of CARS for recording the vib rational spectrum of a chromophore within a trans-membrane protein. In addition, the chi((3)), analysis shows that a CARS spectrum of a mixt ure of isomeric chromophores can be quantitatively separated into CARS data assignable to its individual components. The significance of the se results with respect to analyzing picosecond time-resolved CARS dat a from intermediates in the BR photocycle is discussed.