Picosecond time-resolved resonance Raman spectroscopy of bacteriorhodopsin's J, K, and KL intermediates

Abstract

Resonance Raman spectroscopy has been used to obtain structural and kinetic information on the primary photointermediates of bacteriorhodopsin with 3-ps time resolution. A synchronously pumped dye laser was amplified at 50 Hz to produce a probe pulse at 589 nm while a second, spectrally distinct, pump pulse at 550 nm was generated by amplification of a 10-nm portion of a continuum produced from the probe pulse. This apparatus was used to record Stokes Raman spectra of the photoproduct from 0 ps to 13 ns as well as anti-Stokes spectra from 0 to 10 ps. At 0 ps, the Stokes spectrum, assigned to J, has strong hydrogen out-of-plane (HOOP) intensity at 1000 and 956 cm-1, the fingerprint region consists of a broad band of lines from 1155 to 1200 cm-1, and the ethylenic line is found at 1518 cm-1. By 3 ps the relative HOOP intensity drops to its lowest value and the fingerprint collapses to a single strong mode at 1189 cm-1, while the ethylenic remains at 1518 cm-1. The lifetime of the initially strong anti-Stokes scattering is ∼2.5 ps, indicating that the J → K transition is due, in large part, to vibrational cooling of the chromophore. We conclude that the chromophore in J is highly twisted and thermally excited but that it cools and conformationally relaxes to a more planar 13-cis chromophore within 3 ps to form K. Between 3 and 40 ps there is a resurgence in Stokes HOOP intensity which remains large and nearly constant thereafter and the ethylenic frequency shifts from 1518 to 1521 cm-1 within 200 ps. These changes are assigned to the conversion of K to a more twisted and bluer-absorbing KL species between 20 and 100 ps which must be caused by an isomerization-induced protein conformational change. © 1991 American Chemical Society.