SPECTRAL TUNING, FLUORESCENCE, AND PHOTOACTIVITY IN HYBRIDS OF PHOTOACTIVE YELLOW PROTEIN, RECONSTITUTED WITH NATIVE OR MODIFIED CHROMOPHORES

Photoactive yellow proteins (PYPs) constitute a new class of eubacteri al photoreceptors, containing a deprotonated thiol ester-linked 4-hydr oxycinnamic acid chromophore. Interactions with the protein dramatical ly change the (photo)chemical properties of this cofactor. Here we des cribe the reconstitution of apoPYP with anhydrides of various chromoph ore analogues. The resulting hybrid PYPs, their acid-denatured states, and corresponding model compounds were characterized with respect to their absorption spectrum, pK for chromophore deprotonation, fluoresce nce quantum yield, and Stokes shift. Three factors contributing to the tuning of the absorption of the hybrid PYPs were quantified: (i) thio l ester bond formation, (ii) chromophore deprotonation, and (iii) spec ific chromophore-protein interactions. Analogues lacking the 4-hydroxy substituent lack both contributions (chromophore deprotonation and sp ecific chromophore-protein interactions), confirming the importance of this substituent in optical tuning of PYP. Hydroxy and methoxy substi tuents in the 3- and/or 5-position do not disrupt strong interactions with the protein but increase their pK for protonation and the fluores cence quantum yield, Both deprotonation and binding to apoPYP strongly decrease the Stokes shift of chromophore fluorescence, Therefore, cou pling of the chromophore to the apoprotein not only reduces the energy gap between its ground and excited state but also the extent of reorg anization between these two states. Two of the PYP hybrids show photoa ctivity comparable with native PYP, although with retarded recovery of the initial state.