Picosecond resonance coherent anti-Stokes Raman spectroscopy of bacteriorhodopsin: spectra and quantitative third-order susceptibility analysis of the light-adapted BR-570

Abstract

Vibrational spectra from ground-state bacteriorhodopsin (BR) in light-adapted protonated and perdeuterated (D 2O) water samples (i.e. from BR-570) are recorded by picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Coherent excitation by pairs of 6 ps pulses at ω 1 (578 nm, Δω ⩽ 4 cm −1) and at ω s (604–643 nm, Δω ≈ 300 cm −1) simultaneously generates PR/CARS signals over ≈ 300 cm −1. The high S/N of the PR/CARS spectra obtained over the 760–1750 cm −1 region merit a quantitative χ (3) analysis to obtain band origin positions, band shapes, relative band intensities, and phase factors describing the electronic resonance. Excitation conditions are selected to minimize BR photochemistry and ensure the electronic resonance of ω 1 with the retinal chromophore and therefore, these PR/CARS data can be assigned to the all-trans, 15-anti retinal structure found in BR-570. Changes not only in the retinal structure itself, but also in the surrounding environment (protein and water molecules) which either alter the retinal structure or influence the electronic resonance in the retinal chromophore are reflected in these results. Thus, a more detailed view than previously available can be obtained of the vibrational degrees of freedom of all-trans, 15-anti retinal. Relative to resonance Raman data from BR-570, 8 new vibrational features in the PR/CARS spectra from BR-570 and changes in the positions of Raman bands directly attributable to the deuteration of the Schiff base nitrogen are found. Attention also is given to the influence of non-resonant and resonant contributions to the PR/CARS signal. These results are especially important in the analysis of PR/CARS data from samples in which mixtures of BR retinal chromophores, both static and transient, are found.