Biophysical Applications of Raman Spectroscopy

Because most of the molecules of interest in biophysics have very low symmetry, their vibrational modes cannot be described accurately as being either symmetric or antisymmetric, and the peaks in a Raman or resonance Raman spectrum cannot be assigned on the basis of simple selection rules. However, normal-mode analysis often can be used to identify the vibrational modes that are coupled most strongly to excitation of a chromophore. Isotopic labeling, chemical modifications of the chromophore, or site-directed mutagenesis can be used to shift a particular vibration to higher or lower frequency. 

Raman spectroscopy has been particularly useful in studies of rhodopsin and bacteriorhodopsin. As discussed in Chap. 4, excitatirai of rhodopsin or bacteriorho-dopsin by light causes isomerization of the retinyl chromophore. In rhodopsin, the chromophore changes from W-cis to all-trans in bacteriorhodopsin, it goes from dA -trans to 13-cA. Resonance Raman measurements showed that the isomerization is essentially complete in metastable intermediate states that form within a few ps [32-38]. The conformations of these states were ascertained by comparisons of the resonance Raman spectra with those of model compounds. 

Raman spectroscopy also has proved an effective way to study the ligation states and environments of hemes in proteins [39-49], and to examine the ligands and hydrogen bonding of the protein to the pigments in photosynthetic reaction centers 

12 Raman Scattering and Other Multi-photon Processes 

The experiments on GFP illustrated in Fig. 12.2 were aimed partly at the question of how excitation of the chromophore leads to dissociation of a proton from the phenolic -OH group (Fig. 5.9). Comparisons of resonance Raman spectra of GFP with spectra of the chromophore in ordinary and deuterated ethanol, together with normal-mode assignments of the Raman bands, indicated that stretching of the O-H bond is not strongly coupled to the initial excitation, and must develop later in the evolution of the excited state [59, 60]. 

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