Deep-UV Raman Spectrometer Tunable Between 193 and 205 Nm for Structural Characterization of Proteins

A new deep-UV Raman spectrometer utilizing a laser source tunable between 193 and 205 nm has been designed, built, and characterized. Only selected wavelengths from this range have previously been accessible, by Raman shifting of the second, third, and fourth harmonics of the Nd:YAG fundamental in hydrogen. The apparatus was demonstrated to be a useful tool for characterizing hen egg white lysozyme structural rearrangements at various stages of fibril formation. High-quality deep-UV resonance Raman spectra were obtained for both a protein solution and a highly-scattering gelatinous phase formed by fibrillogenic species. In addition to amide bands, strong contribution of nu(12) and ring-C phenylalanine vibrational modes was observed at excitation wavelengths below 200 nm. Remarkably, the Raman cross-section of these modes revealed dramatic change of lysozyme in response to heat denaturation and fibril formation. These results indicate that phenylalanine could serve as a new deep-UV Raman probe of protein structure.