title: Photokinetic, biochemical and structural features of chimeric photoactive yellow protein constructs.
abstract: Of the 10 photoactive yellow protein (PYPs) that have been characterized, the two from Rhodobacter species are the only ones that have an additional intermediate spectral form in the resting state (λmax  = 375 nm), compared to the prototypical Halorhodospira halophila PYP. We have constructed three chimeric PYP proteins by replacing the first 21 residues from the N-terminus (Hyb1PYP), 10 from the β4-β5 loop (Hyb2PYP) and both (Hyb3PYP) in Hhal PYP with those from Rb. capsulatus PYP. The N-terminal chimera behaves both spectrally and kinetically like Hhal PYP, indicating that the Rcaps N-terminus folds against the core of Hhal PYP. A small fraction shows dimerization and slower recovery, possibly due to interaction at the N-termini. The loop chimera has a small amount of the intermediate spectral form and a photocycle that is 20 000 times slower than Hhal PYP. The third chimera, with both regions exchanged, resembles Rcaps PYP with a significant amount of intermediate spectral form (λmax  = 380 nm), but has even slower kinetics. The effects are not strictly additive in the double chimera, suggesting that what perturbs one site, affects the other as well. These chimeras suggest that the intermediate spectral form has its origins in overall protein stability and solvent exposure.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA. jkyndt@email.arizona.edu
authors.forename: John A
authors.initials: JA
authors.lastname: Kyndt
authors.forename: Terry E
authors.initials: TE
authors.lastname: Meyer
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Olson
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cusanovich
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020607:Halorhodospira halophila / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010782:Photolysis; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D016354:Rhodobacter capsulatus / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria