Secondary structure determination in proteins from deep (192-223-nm) ultraviolet Raman spectroscopy

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Abstract
Raman intensities obtained with UV laser excitation at 223, 218, 204, 200, and 192 nm are reported for the amide I, II, III, and II'' bands of random-coil polylysine. The excitation profiles show enhancement via the .pi.-.pi.* electronic transition, at .apprx. 190 nm. Enhancement for amide I is weak, however, and most of the intensity can be accounted for by preresonance with a deeper UV transition at .apprx. 165 nm. The amide II'' band dominates the spectrum in D2O consistent with the suggestion that the main distortion coordinate in the .pi.-.pi.* excited state is the stretching of the C-N peptide bone. Amide II intensities with 200- and 192-nm excitation are reported for several proteins. The previously reported negative linear correlation with .alpha.-helix content (due to Raman hypochromism in the .alpha.-helices) is found not to apply to proteins with high .beta.-sheet content when the excitation wavelength is 200 nm. Much higher intensities are seen for these proteins and are attributed to a red shift of the .pi.-.pi.* absorption for the .beta.-structure. A linear correlation with .alpha.-helix content is found for excitation of 192 nm, which corresponds to an isosbestic point of the .beta.-sheet and random-coil absorption bands. Characteristic amide II Raman cross sections are derived for .alpha.-helical, .beta.-sheet, and random-coil elements and are used to determine secondary structure for .alpha.1- and .beta.-purothionin, by use of amide II intensities with 200- and 192-nm excitation. The results are in good agreement with a previous determination based on amide I band deconvolution in off-resonance Raman spectra.