Ultrafast light-induced response of photoactive yellow protein chromophore analogues

Abstract

The fluorescence decays of several analogues of the photoactive yellow protein (PYP) chromophore in aqueous solution have been measured by femtosecond fluorescence up-conversion and the corresponding time-resolved fluorescence spectra have been reconstructed. The native chromophore of PYP is a thioester derivative of p-coumaric acid in its trans deprotonated form. Fluorescence kinetics are reported for a thioester phenyl analogue and for two analogues where the thioester group has been changed to amide and carboxylate groups. The kinetics are compared to those we previously reported for the analogues bearing ketone and ester groups. The fluorescence decays of the full series are found to lie in the 1-10 ps range depending on the electron-acceptor character of the substituent, in good agreement with the excited-state relaxation kinetics extracted from transient absorption measurements. Steady-state photolysis is also examined and found to depend strongly on the nature of the substituent. While it has been shown that the ultrafast light-induced response of the chromophore in PYP is controlled by the properties of the protein nanospace, the present results demonstrate that, in solution, the relaxation dynamics and pathway of the chromophore is controlled by its electron donor-acceptor structure: structures of stronger electron donor-acceptor character lead to faster decays and less photoisomerisation.

Two Photon‐Induced Electron Injection From a Nanotrigger in Native Endothelial NO‐Synthase

“…[4][5][6] Additional specific interactions of the compounds with protein residues also tune isomerization. [7][8]24] The decay of bound NT* to eNOS was found to be similar with one-and two-photon excitation within experimental error ( Figure 3& Figure 4). The proposed reaction sequence that accounts for decay of NT* bound to eNOS in the presence of the cofactor Ca 2 + /calmodulin is described in Equations (1)- (5):…”

“…[7] For example, the shorter excited state lifetime of p-coumaric acid increases with the viscosity. [7,8] This excited state either undergoes a non-radiative transition back to the trans ground state via a ground-state intermediate or decay via the formation of the cis-photoisomer. [8,24] In pseudocyanine, trans-stilbene or p-coumaric acid, all carrying the shortest p-conjugated chain, the barrier height to isomerization is either negligible or very low.…”

“…[7,8] This excited state either undergoes a non-radiative transition back to the trans ground state via a ground-state intermediate or decay via the formation of the cis-photoisomer. [8,24] In pseudocyanine, trans-stilbene or p-coumaric acid, all carrying the shortest p-conjugated chain, the barrier height to isomerization is either negligible or very low. [6-8, 18, 24] In the nanotrigger carrying a more extended p-conjugated chain, we do not have evidence of isomerization in solvents like THF or DMSO or in NADPH sites of proteins eNOS, DHFR or glucose-6-phosphate dehydrogenase.…”

“…[3][4][5][6] In naturally organized systems as in protein interiors, isomerization may be enhanced by the protein environment compared to homogeneous solution as exemplified by the stilbene-like chromophore of the yellow protein. [7][8] Additionally, photochemical reactions are obviously tuned by the exciting wavelength and two-photon-induced processes depend on the two-photon absorption cross-sections (TPA). [1,9,10] The main and most advantageous use of two photon-induced fluorescence is imaging because it provides high resolution and improved depth penetration in biological samples as well as reduced photodamage.…”

Hydrogen Bonding Controls Excited-State Decay of the Photoactive Yellow Protein Chromophore

“…Time-resolved fluorescence decay measurements of chromophore analogues in water indicate that also in solution excited-state decay is very efficient. (5) In contrast, calculations on the isolated chromophore by Gromov et al demonstrate that in vacuum the intersection seam lies rather far away from the accessible S 1 minima. (6) These findings suggest that specific interactions that are present in the protein as well as in water control the ultrafast decay of the chromophore.…”