title: Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation.
abstract: Light-activation of photoactive yellow protein (PYP) is followed by a series of dynamical transitions in the structure of the protein. Tryptophan fluorescence is well-suited as a tool to study selected aspects of these. Using site-directed mutagenesis eight 'single-tryptophan' mutants of PYP were made by replacement of either a tyrosine, phenylalanine or histidine residue by tryptophan, while simultaneously eliminating the endogenous W119. Surprisingly, only three of these eight mutants turn out to emit measurable tryptophan fluorescence: F6W/W119F, F96W/W119F and H108W/W119F. Significantly, all three show altered tryptophan fluorescence upon formation of the pB state. As F96 is located very close to the chromophore, the F96W/W119F mutant protein is particularly suitable for further studies on the dynamical changes of the polarity in the chromophore-binding pocket of PYP. Furthermore, WT PYP can be photo-activated by a UV photon via the highly conserved W119 and subsequent Förster resonance energy transfer. Placing a unique tryptophan residue elsewhere in the protein shows that position 119 is favoured for UV-activation of PYP.
authors.affiliation: Laboratory for Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1090 GE, Amsterdam, The Netherlands.
authors.forename: Marijke
authors.initials: M
authors.lastname: Hospes
authors.forename: Johnny
authors.initials: J
authors.lastname: Hendriks
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: Hellingwerf
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008163:Luminescent Measurements / Q000379:methods*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria; D014364:Tryptophan