N-terminal domain of the bacteriophage lambda repressor: investigation of secondary structure and tyrosine hydrogen bonding in wild-type and mutant sequences by Raman spectroscopy.

Laser Raman spectroscopy has been employed to investigate structures of the lambda repressor N-terminal fragment, which recognizes operator DNA. Examination of repressor fragments containing deuterated amide groups and specifically labeled deuteriotyrosines has enabled the assignment of many of the conformation-sensitive Raman bands. By use of Fourier deconvolution and signal averaging techniques, the spectra of both wild-type and mutant sequences have been obtained as a function of the total protein concentration in aqueous solution over the range 5-100 mg/mL. This analysis has permitted monitoring of the monomer-dimer association of the repressor fragment and determination of the effects of dimerization upon individual side-chain interactions and main-chain secondary structure. The spectra are interpreted to reveal the hydrogen-bonding environments of four tyrosines of the N-terminal fragment (Y22, Y60, Y85, and Y88). The fifth tyrosine (Y101) is known from NMR experiments to be exposed to solvent molecules. The results show that in the dimer Y22 and Y85 are each acceptors of a strong hydrogen bond from a positive donor group, while Y88 is the donor of a strong hydrogen bond to a negative acceptor and Y60, like Y101, is involved in both a donor role and an acceptor role. Y60, Y85, and Y88, which are all near the dimer interface, undergo a collective change in hydrogen-bonding environment with dissociation of the dimer. The net effect of this change is the conversion of one acceptor tyrosine, deduced to be Y88, to a combined donor and acceptor role. The Raman results also indicate a predominantly alpha-helical structure for the N-terminal fragment in aqueous solution, with 70 +/- 4% of the residues incorporated into helical domains. The amount of alpha-helix determined from the Raman spectrum is consistent with X-ray and prediction results and is altered neither by the mutations C85----Y85 and C88----Y88 nor by dissociation of the dimer.