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

Abstract

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.

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

“…Also, hydrogen bonding by water to the proximal carbonyl oxygen, which is solventaccessible in Ppr-PYP (see above), could also contribute to a blue shift in absorption because of resonance stabilization between quinonic and phenolic forms of the chromophore (19). However, it is difficult to correlate the structural changes observed in this structure with the specific and nonspecific protein environmental effects that affect chromophore absorption in PYPs (45,46).…”

Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

“…The absorption maximum of PYP M is 355 nm (12), which is close to that of denatured PYP at neutral pH (11,23). Because the phenolic oxygen of PYP is protonated, the largely blue-shifted absorption spectrum of PYP M is due mainly to protonation of the chromophore.…”

Resonance Raman Evidence for Two Conformations Involved in the L Intermediate of Photoactive Yellow Protein

“…Photoexcitation of PYP dark triggers a photocycle that involves two major intermediate states denoted PYP L (also called I 1 or pR) and PYP M (also called I 2 or pB) (7,8). Although it is well established that the PYP L 3 PYP M process involves protonation of the phenolic O1 (9,10), it is still not clear what structural factors promote the protonation reaction. To address this issue, we have performed resonance Raman (RR) investigations of PYP L and report a novel finding for two conformations in the active site of this intermediate state.…”

“…The PYP L 3 PYP M process involves the protonation of the phenolic O1 of the chromophore (9,10,12,13,18). In this study, we suggest that the absence or weakening of the hydrogen bond between the phenolic O1 and Glu 46 in PYP L l .…”