Photoisomerization and Photoionization of the Photoactive Yellow Protein Chromophore in Solution

Abstract

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.

Uncovering the hidden ground state of green fluorescent protein

“…GFPuv (including surface exposed mutations F99S, M153T, and V163A) was prepared as described (19) The sample was loaded in a f low system of 2-ml volume and pumped through a f low cuvette of 1-mm path length. The pump-probe setup has been described in detail (20) and has been modified to include an additional pulse (15,17). The basis of the system is a 1-kHz amplified Ti:sapphire laser system (Coherent Radiation, Mountain View, CA; BMI, Evry, France) delivering 450-J 60-fs 800-nm pulses.…”

“…Multipulse spectroscopy can be used to control and influence the course of reactions as they evolve, and its power lies in the ability to selectively remove or transfer the population of transient species with carefully timed laser pulses (11)(12)(13). We have recently expanded this technique by combining it with broad-band spectral probing and elaborate global analysis techniques, enabling us to disentangle complex branched reaction schemes (14)(15)(16)(17)(18). In this work, we describe the direct observation of the hidden anionic ground state of GFP as an integral part of the photocycle, revealed through the use of multipulse spectroscopy in combination with global analysis techniques.…”

Initial Photoinduced Dynamics of the Photoactive Yellow Protein

“…[48,50] The PDP data favor the inhomogeneous over the homogeneous model, since each ESI does not have the same yield in initiating the PYP photocycle. This observation, in combination with the global analysis of the kinetic trace PDP signals, supports the observation that ESI1 has the highest yield ( % 40 %) followed by ESI2 (20 %), whereas ESI3 has a near-negligible yield ( % 1 %) in initiating the photocycle.…”

Ultrafast Photoisomerization of Photoactive Yellow Protein Chromophore Analogues in Solution: Influence of the Protonation State

“…[35] A transient ground-state of a different nature had also been proposed previously from pump-dump-probe experiments. [34] It was thus inferred that, within the natural PYP photoreceptor, photoisomerization of the deprotonated thioester chromophore does occur because the expected large photoinduced charge shift is counteracted by the presence of a positive charge on the nearby Arg52 residue. [35,80] Interestingly, a study of the R52Q mutant in which positively charged arginine is replaced by the neutral glutamine, gave evidence that the isomerization reaction in the protein nanospace is slowed down by the loss of the Coulombic interaction between the chromophore and Arg52.…”