title: Spectral properties of Trp182, Trp194, and Trp250 on the alpha subunit of bacterial luciferase.
abstract: The phosphorescence and fluorescence properties of bacterial luciferase (alphabeta) mutants from Xenorhabdus luminescens were investigated. All tryptophans in the alpha and beta subunits were replaced with tyrosines except for one or two tryptophans in the alpha subunit. Because one luciferase mutant (W250) retained only a single tryptophan in the alpha subunit while two other mutants (W182/250 and W194/250) each contained two tryptophans in the alpha subunit, it was possible to deduce the spectral properties of these specific tryptophans (Trp182, Trp194, Trp250). Analyses of the phosphorescence properties were particularly revealing as only a single phosphorescence emission peak at 411-414 nm was observed for the W250 and W194/250 mutants while peaks at 409 and 414 nm could be clearly observed for the W182/250 mutant. Coupled with intrinsic fluorescence quenching experiments, these results show that alphaTrp182 is in a distinctly polar environment while alphaTrp250 is in a hydrophobic region and illustrate the advantages of using phosphorescence to recognize different microenvironments for tryptophan residues.
authors.affiliation: Department of Biochemistry, McGill University, 3655 Drummond Street, Montreal, Quebec, H3G 1Y6, Canada.
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: N
authors.initials: N
authors.lastname: Valkova
authors.forename: E
authors.initials: E
authors.lastname: Meighen
mesh_terms: D019943:Amino Acid Substitution; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism*; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan; D020593:Xenorhabdus / Q000201:enzymology*
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014364:Tryptophan; D008156:Luciferases