Recent advances in laser technology will be used in the first study of proteins by resonance Raman spectroscopy with radiation in the 180 to 250 nm wavelength region. This new technique will be developed as a method for determining protein secondary structure. This alternative to UV CD is applicable to membrane and other particulate systems. Several amino acid side chains have absorption bands which should lead to resonance enhancement and permit the use of these groups as probes of their hydrogen bonding and specific conformational state. The refolding kinetics and isotope exchange behavior of globular proteins will be studied with this method. These experiments will also be used to probe the electronic structure of protein components, especialy the peptide bond. Recent experiments in our laboratory have demonstrated the feasibility of this laser technology and the ease with which it may be used to obtain resonance Raman spectra. The short wavelength of the laser radiation used in these studies permits the investigation of samples which are fluorescent but which emit at longer wavelengths, few, if any, biopolymer components appear to fluoresce in the region near 200 nm. Several interesting spectral enhancement, isotopic intensity and solvation bandwidth effects have been discovered in this preliminary work. For example, the amide II vibration of the peptide bond, which is very weak or absent in Raman spectra obtained with visible or near UV excitation, is quite strong when obtained with 213 nm laser radiation. Replacement of the amide proton by deuterium results in a large increase in the intensity of the amide II band with a corresponding decrease in the amide III intensity. At high dilution (1 mM) the amide I carbonyl stretch vibration appears to be very sensitive to aqueous solvation; this band is much broader in water than in acetonitrile. A wide range of solute concentrations can be used for these experiments down to a lower limit corresponding to roughly 0.1 OD at the excitation wavelength of 100 micromolar, whichever is lower. It has been found that the spectrum of biopolymer components is very sensitive to the excitation laser wavelength in the region near 200 nm. This can be used to sort out overlapping Raman bands and provides new information on electronic excitations.
| Hudson, B; Mayne, L (1986) Ultraviolet resonance Raman spectroscopy of biopolymers. Methods Enzymol 130:331-50 |
| Kubasek, W L; Hudson, B; Peticolas, W L (1985) Ultraviolet resonance Raman excitation profiles of nucleic acid bases with excitation from 200 to 300 nanometers. Proc Natl Acad Sci U S A 82:2369-73 |
