CHARACTERIZATION OF THE 2 TRYPTOPHAN RESIDUES OF THE LACTOSE REPRESSOR FROM ESCHERICHIA-COLI BY PHOSPHORESCENCE AND OPTICAL-DETECTION OF MAGNETIC-RESONANCE

The native lactose repressor from Escherichia coli (Lac Rep) and two s ingle-point mutants, W220Y and W201Y, were investigated using low-temp erature phosphorescence and optical detection of magnetic resonance (O DMR) spectroscopy. Emission from two tryptophan residues was evident i n the phosphorescence spectrum of native Lac Rep at 77 K. Using the si ngle-point mutants, the triplet-state properties of tryptophans 201 an d 220 were obtained independently. Trp 220 was characterized as a part ially solvent-exposed residue (0,0 band centered at 409.5 nm), while t ryptophan 201 exhibited the properties of a buried residue (0,0 band c entered at 413.5 nm). Both single-point mutant proteins experienced ch anges in tryptophan triplet-state properties as a result of binding ei ther of two inducer sugars: isopropyl beta-D-thiogalactoside, a monosa ccharide, or melibiose, a disaccharide. Putative singlet-singlet energ y transfer from tryptophan 220 to tryptophan 201 was also investigated , but the quantitative results must be viewed with some caution.