title: Histidine-tryptophan interactions in T4 lysozyme: 'anomalous' pH dependence of fluorescence.
abstract: A variant of T4 lysozyme which contains only a single tryptophan residue (at position 138) has been prepared (W126Y/W158Y designated 'YWY'). Two additional mutations to YWY have been prepared involving replacement of glutamine 105, which hydrogen bonds to the indole N-H of trp 138 in wild type, with either a histidine (YWY/Q105H) or an alanine (YWY/Q105A). The fluorescence properties of these two species are investigated as a function of pH. YWY/Q105A exhibits essentially a single exponential fluorescence decay (5% tau = 0.35 ns 95% tau = 5 ns) and almost no pH dependence in steady state or time resolved fluorescence behavior. In contrast, YWY/Q105H exhibits complex fluorescence decay over the entire pH range used in these experiments. As the pH is lowered from 8 to 4, there is an increase in the quantum yield and a change in the average lifetime (from 2.0 to 3.1 ns). Using this data, the pKa of histidine 105 has been determined to be 5.9. These results are contrasted to those from other proteins which show a pH dependent tryptophan fluorescence associated with a neighboring histidine or other residue. Quenching behavior in terms of the stereochemistry of the tryptophan-histidine interaction and implications of these results for current models of complex fluorescence behavior of single tryptophan proteins are also discussed.
authors.affiliation: Department of Chemistry, University of Oregon, Eugene 97403, USA.
authors.forename: M
authors.initials: M
authors.lastname: Van Gilst
authors.forename: B S
authors.initials: BS
authors.lastname: Hudson
mesh_terms: D005453:Fluorescence; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009113:Muramidase / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics; D013604:T-Phages / Q000201:enzymology*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
chemical_list: D006639:Histidine; D014364:Tryptophan; D009113:Muramidase