title: Characterization of individual tryptophan side chains in proteins using Raman spectroscopy and hydrogen-deuterium exchange kinetics.
abstract: Two Raman bands at 880 and 1360 cm-1 of tryptophan (Trp) side chains have been found useful in structural studies of the side chains in proteins. The frequency of the 880-cm-1 band reflects the strength of H bonding at the N1H site of the indole ring: the lower the frequency is, the stronger the H bonding is. The intensity of the 1360-cm-1 band, on the other hand, is a marker of the hydrophobicity of the environment of the indole ring: particularly strong in hydrophobic environments. It is also demonstrated that a combination of stepwise deuteration of the tryptophan side chains and difference spectrum techniques is useful to observe these marker bands due to each side chain separately. The states of six tryptophans in lysozyme revealed by this Raman spectroscopic method in solution are compared with those by X-ray diffraction in crystal. The Raman data on the outer four Trp's are consistent with the X-ray structure, whereas significant differences between solution and crystal are suggested for the strength of H bonding of the most and second most buried Trp's. Characterization of four Trp's in alpha-lactalbumin shows that the two outer Trp's are moderately H bonded to solvent water and closely surrounded by aliphatic side chains while the inner two are not H bonded nor closely surrounded by aliphatic side chains.
authors.affiliation: Pharmaceutical Institute, Tohoku University, Sendai, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Miura
authors.forename: H
authors.initials: H
authors.lastname: Takeuchi
authors.forename: I
authors.initials: I
authors.lastname: Harada
mesh_terms: D003903:Deuterium; D006859:Hydrogen; D006860:Hydrogen Bonding; D007700:Kinetics; D009113:Muramidase; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods; D014364:Tryptophan
chemical_list: D006859:Hydrogen; D014364:Tryptophan; D003903:Deuterium; D009113:Muramidase