SIMULTANEOUS MONITORING OF THE ENVIRONMENT OF TRYPTOPHAN, TYROSINE, AND PHENYLALANINE RESIDUES IN PROTEINS BY NEAR-ULTRAVIOLET 2ND-DERIVATIVE SPECTROSCOPY

A method for deconvolution of the near-uv second-derivative spectra of proteins into their component tryptophan, tyrosine, and phenylalanine spectra is described. In this approach, the second-derivative spectra of tryptophan and tyrosine model compounds are numerically shifted to create a set of reference spectra corresponding to anticipated peak p ositions in protein environments of different polarity. The relative c ontributions of these individual standard spectra are varied until the best fit to the experimental protein spectrum is obtained. Separate a ddition of tryptophan and tyrosine standard spectra, weighted by their contributions as determined in the fitting procedure, yields an accur ate representation of the spectra of these residues in proteins. The p osition of the intersection of these spectra with the wavelength axis is used as a measure of spectral position in ethylene glycol perturbat ion experiments in which the average solvent accessibility is assessed by relating the observed shifts in the tryptophan and tyrosine spectr a to the shifts observed for corresponding model compounds. The phenyl alanine peak positions in the set of 16 proteins studied are determine d as described previously [H. Mach et al. (1991) Arch. Biochem. Biophy s. 287, 33-40]. For all three aromatic residues in proteins, no consis tent correlation between absolute spectral band positions and average solvent accessibility is observed, suggesting a significant influence of other local (e.g., electrostatic) effects on near-uv spectra of pro teins. The maximum spectral shift observed between solvent-exposed mod el compounds and side chains entirely buried in apolar protein core wa s found to be approximately 5 nm for tyrosine, 4 nm for tryptophan, an d 2 nm for phenylalanine residues. (C) 1994 Academic Press, Inc.