Structure of the I1 early intermediate of photoactive yellow protein by FTIR spectroscopy.
To understand how proteins translate the energy of sunlight into defined conformational changes, we have measured the photocycle reactions of photoactive yellow protein (PYP) using time-resolved step scan Fourier transform infrared (FTIR) spectroscopy. Global fit analysis yielded the same apparent time constants for the reactions of the chromophore, the protonation changes of protein side chains and the protein backbone motions, indicating that the light cycle reactions are synchronized. Changes in absorbance indicate that there are at least four intermediates (I1, I1', I2, I2'). In the intermediate I1, the dark-state hydrogen bond from Glu 46 to the aromatic ring of the p-hydroxycinnamoyl chromophore is preserved, implying that the chromophore undergoes trans to cis isomerization by flipping, not the aromatic ring, but the thioester linkage with the protein. This excludes an I1 structural model proposed on the basis of time resolved Laue crystallography, but does agree with the cryotrapped structure of an I1 precursor.
Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA. rbrudler@scripps.edu
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D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism; D003624:Darkness; D018698:Glutamic Acid / Q000378:metabolism; D020607:Halorhodospira halophila / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D007259:Infrared Rays; D007536:Isomerism; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010777:Photochemistry; D020283:Photoreceptors, Microbial; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D014732:Vibration
D001426:Bacterial Proteins; D004396:Coloring Agents; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria; D018698:Glutamic Acid