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Volume 271, Number 50, Issue of December 13, 1996 pp. 31949-31956
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Spectral Tuning, Fluorescence, and Photoactivity in Hybrids of Photoactive Yellow Protein, Reconstituted with Native or Modified Chromophores

(Received for publication, July 9, 1996, and in revised form, September 3, 1996)

From the ^¶ Laboratory for Microbiology, E. C. Slater Institute, BioCentrum Amsterdam, Nieuwe Achtergracht 127, 1018 WS, Amsterdam and the ^" Department of Organic Chemistry, University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS, Amsterdam, The Netherlands

Photoactive yellow proteins (PYPs) constitute a new class of eubacterial photoreceptors, containing a deprotonated thiol ester-linked 4-hydroxycinnamic acid chromophore. Interactions with the protein dramatically change the (photo)chemical properties of this cofactor. Here we describe the reconstitution of apoPYP with anhydrides of various chromophore 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, fluorescence quantum yield, and Stokes shift. Three factors contributing to the tuning of the absorption of the hybrid PYPs were quantified: (i) thiol ester bond formation, (ii) chromophore deprotonation, and (iii) specific chromophore-protein interactions. Analogues lacking the 4-hydroxy substituent lack both contributions (chromophore deprotonation and specific chromophore-protein interactions), confirming the importance of this substituent in optical tuning of PYP. Hydroxy and methoxy substituents in the 3- and/or 5-position do not disrupt strong interactions with the protein but increase their pK for protonation and the fluorescence quantum yield. Both deprotonation and binding to apoPYP strongly decrease the Stokes shift of chromophore fluorescence. Therefore, coupling of the chromophore to the apoprotein not only reduces the energy gap between its ground and excited state but also the extent of reorganization between these two states. Two of the PYP hybrids show photoactivity comparable with native PYP, although with retarded recovery of the initial state.

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