Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface.
Protein-chromophore interactions in photoreceptors often shift the chromophore absorbance maximum to a biologically relevant spectral region. A fundamental question regarding such spectral tuning effects is how the electronic ground state S(0) and excited state S(1) are modified by the protein. It is widely assumed that changes in energy gap between S(0) and S(1) are the main factor in biological spectral tuning. We report a generally applicable approach to determine if a specific residue modulates the energy gap, or if it alters the equilibrium nuclear geometry or width of the energy surfaces. This approach uses the effects that changes in these three parameters have on the absorbance and fluorescence emission spectra of mutants. We apply this strategy to a set of mutants of photoactive yellow protein (PYP) containing all 20 side chains at active site residue 46. While the mutants exhibit significant variation in both the position and width of their absorbance spectra, the fluorescence emission spectra are largely unchanged. This provides strong evidence against a major role for changes in energy gap in the spectral tuning of these mutants and reveals a change in the width of the S(1) energy surface. We determined the excited state lifetime of selected mutants and the observed correlation between the fluorescence quantum yield and lifetime shows that the fluorescence spectra are representative of the energy surfaces of the mutants. These results reveal that residue 46 tunes the absorbance spectrum of PYP largely by modulating the width of the S(1) energy surface.
Philip
Andrew F
AF
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Nome
Rene A
RA
Papadantonakis
George A
GA
Scherer
Norbert F
NF
Hoff
Wouter D
WD
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055592:Biophysical Phenomena; D020134:Catalytic Domain / Q000235:genetics; D020607:Halorhodospira halophila / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; C050162:photoactive yellow protein, Bacteria