Fluorescence anisotropy of photoactive yellow protein (PYP) and related systems: Is the low-lying excited state of the PYP quasi-degenerate?

Abstract

Photoactive yellow protein (PYP) is a blue-light photoreceptor responsible for the negative phototaxis of the purple sulfur bacterium Ectothiorhodospira halophila. Photoexcitation of PYP with a blue light triggers a photocycle that involves several intermediate states. High-resolution gas-phase fluorescence excitation experiments performed on the neutral form of PYP's pCA chromophore involved excitation wavelength-dependent photochemistry. This is explained by the existence of a low-lying second electronic state (n → π* origin). Based on steady state and transient absorption measurements, it is concluded that upon excitation, simultaneous population of those states may occur and the complex photophysics of PYP arise due to competing relaxation pathways from those states. This chapter uses femtosecond time-resolved fluorescence up-conversion spectroscopy to elucidate the quasi-degenerate nature of the PYP's excited state. Detailed fluorescence anisotropy measurements are carried out on native PYP, 3,4-dihydroxy cinnamic acid analogue, and several mutant PYP systems.