Structure of the I-1 early intermediate of photoactive yellow protein by FTIR spectroscopy

To understand how proteins translate the energy of sunlight into defined co nformational changes, we have measured the photocycle reactions of photoact ive yellow protein (PYP) using time-resolved step scan Fourier transform in frared (FTIR) spectroscopy. Global fit analysis yielded the same apparent t ime constants for the reactions of the chromophore, the protonation changes of protein side chains and the protein backbone motions, indicating that t he light cycle reactions are synchronized. Changes in absorbance indicate t hat there are at least four intermediates (I-1, I-1', I-2, I-2') In the int ermediate I-1, the dark-state hydrogen bond from Glu 46 to the aromatic rin g of the p-hydroxycinnamoyl chromophore is preserved, implying that the chr omophore undergoes trans to cis isomerization by flipping, not the aromatic ring, but the thioester linkage with the protein. This excludes an I-1 str uctural model proposed on the basis of time resolved Laue crystallography, but does agree with the cryotrapped structure of an I-1 precursor.