Sandrine Charier

Excited-state relaxation dynamics of a PYP chromophore model in solution: influence of the thioester group

Abstract Cis–trans photoisomerization of a photoactive yellow protein chromophore model, the deprotonated trans S -phenyl thio- p -hydroxycinnamate, is studied in aqueous solution by subpicosecond transient absorption and gain spectroscopy. The excited-state deactivation is found to involve the formation, in 1.7 ps, of an intermediate state which decays in 2.8 ps. A persistent bleaching signal is observed at longer times indicating that the excited state not only relaxes to the ground state but also partly forms a stable photoproduct, possibly the cis isomer. This behavior is analogous to that of the native photoactive yellow protein.

Molecular probes for nonlinear optical imaging of biological membranes

Second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) are nonlinear optical (NLO) phenomena that scale with excitation intensity squared, and hence give rise to an intrinsic 3-dimensional resolution when used in microscopic imaging. TPEF microscopy has gained widespread popularity in the biology community whereas SHG microscopy promises to be a powerful tool because of its sensitivity to local asymmetry. We have implemented an approach toward the design of NLO-probes specifically adapted for SHG and/or TPEF imaging of biological membranes. Our strategy is based on the design of nanoscale amphiphilic NLO-phores. We have prepared symmetrical bolaamphiphilic fluorophores combining very high two-photon absorption (TPA) cross-sections in the visible red region and affinity for cellular membranes. Their incorporation and orientation in lipid membranes can be monitored via TPEF anisotropy. We have also prepared amphiphilic push-pull chromophores exhibiting both large TPA cross-sections and very large first hyperpolarizabilities in the near-IR region. These NLO-probes have proved to be particularly useful for imaging of biological membranes by simultaneous SHG and TPEF microscopy and offer attractive prospects for real-time imaging of fundamental biological processes such as adhesion, fusion or reporting of membrane potentials.

Chapter 82 - Chemical structure effect on the excited-state relaxation dynamics of the PYP chromophore

In order to better understand the early photophysics of PYP, this chapter compares three model chromophores, the deprotonated trans-p-coumaxic acid (pCA 2- ) and its amide (pCM - ) and phenyl thioester (pCT - ) analogues, in aqueous solution. The excited-state relaxation dynamics is followed by subpicosecond transient absorption and gain spectroscopy. The excited-state deactivation pathway of PYP model chromophores is found to depend strongly on the substituent adjacent to the carbonyl group. The photoisomerization reaction of the deprotonated p-coumaric acid (pCA 2- ) and of its amide analogue (pCM - ) in solution does not show any spectroscopically detectable intermediate, which is quite different from PYP. On the contrary, the phenyl thioester derivative pCT - exhibits a photo-physical behavior in solution surprisingly close to that of the protein during its initial deactivation step. This chapter highlights the determining role of the thioester bond in the primary molecular events in PYP.