Ultrafast Carbonyl Motion of the Photoactive Yellow Protein Chromophore Probed by Femtosecond Circular Dichroism

Abstract

Motions of the trans-p-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of photoactive yellow protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near-ultraviolet, over a time scale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (≪0.8 ps) and unidirectional flipping motion in the excited state with an angle of ca. 17-53°. For the subset of proteins that do not enter the signaling photocycle, TRCD provides strong evidence that the carbonyl group moves back to its initial position, leading to the formation of a nonreactive ground-state intermediate of trans conformation. The initial ground state is then restored within ca. 3 ps. Comparative study of R52Q and wild-type PYP provides direct evidence that the absence of Arg52 has no effect on the conformational changes of the chromophore during those steps.

Enzyme transient state kinetics in crystal and solution from the perspective of a time-resolved crystallographer

“…2) together with a plausible chemical, kinetic mechanism. In solution time-resolutions as good as 8 fs are reached 61,62 . However, with spectroscopy, structural information is sparse 63 .…”

Communication: Broadband and ultrasensitive femtosecond time-resolved circular dichroism spectroscopy

“…The first one is better sensitivity. In the recently reported single wavelength TRCD measurements, 11 the measurement error was about 0.3 mOD, which equals to 10 mdeg. Also in broadband TRCD spectroscopy, the sensitivity was estimated to be the order of 0.1 mOD.…”

“…Nevertheless, this type of TRCD measurements using a BS compensator is only practicable at a single wavelength due to the strong wavelength-dependent behavior of the phase retarder. 3,9,11 Recently, Eom et al have developed new methods for ellipticity measurement employing a heterodyne-detection technique. 12,13 They successfully obtained the steady-state CD and optical rotatory dispersion (ORD) spectra of Ni-(tartrate) 2 by measuring the relative phase and amplitude of the transmitted electric field perpendicular to the incident polarization.…”

Four resonance structures elucidate double-bond isomerisation of a biological chromophore

“…Photoactive yellow protein (PYP) 1 is a remarkable model system for studying double-bond isomerisation in photoreceptor proteins, as this photoreceptor has amply been characterised using a wide variety of advanced methods such as time-resolved X-ray crystallography, [2][3][4][5] neutron crystallography, 6 NMR, 7 and ultrafast spectroscopy. [8][9][10][11][12] These studies provided important insights into photoinduced double-bond isomerisation of the PYP chromophore that triggers the PYP photoresponse. The chromophore is derived from the anionic phenolate form of the p-coumaric thioester (pCTM À ), which is in the E(trans)-configuration and stabilised by hydrogen bonds (H-bonds) with the protein (Fig.…”