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Photokinetic, Biochemical and Structural Features of Chimeric Photoactive Yellow Protein Constructs
Author(s) -
Kyndt John A.,
Meyer Terry E.,
Olson Kenneth T.,
Beeumen Jozef,
Cusanovich Michael A.
Publication year - 2012
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2012.01235.x
Subject(s) - chimera (genetics) , chemistry , kinetics , biophysics , crystallography , stereochemistry , biochemistry , biology , gene , physics , quantum mechanics
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 (λ max = 375 nm), compared to the prototypical H alorhodospira 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 R b. 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 (λ max = 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.