Ultraviolet Resonance Raman Spectroscopic Study of the Average Environment of Tyrosine in Native and Denatured Barnase

Ultraviolet resonance Raman spectroscopy was used to probe the change in the average environment of the seven tyrosine amino acids in barnase when the polypeptide chain folds from the denatured to the native state. The excitation radiation was continuous wave at a wavelength of 244 nm, and was generated from an intracavity-doubled argon ion laser, a BBO non-linear optical crystal being placed within the laser cavity. The change in the intensity ratio of the tyrosine Fermi-resonance doublet at 830/850 cm-1 was calibrated to provide a spectroscopic indicator of tyrosine hydrogen-bond strength. This was achieved by using p-cresol as a model compound, and measuring the resonance Raman spectra of p-cresol when dissolved in a range of solvents with known hydrogen-bond enthalpies of formation.