Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila

Abstract

The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.

Evidence for trans‐cis isomerization of the p‐coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein

“…3A,B), which was subsequently confirmed by ~H NMR spectra of intact PYP [I] and X-ray crystallography [4]. In order to be able to extract the chromophore from the blue-shifted photocycle intermediate, pB [6,7], a solution of PYP was illuminated at acidic pH (i.e. pH 4, see section 2), because under the latter conditions the last step of the photocycle of PYP (the recovery of the ground state pG) is decelerated and consequently, the intermediate pB accumulates to a larger extent than at neutral pH [16].…”

“…PYP from E. halophila was isolated as previously described [15] with mine-modifications [7]. A sample of I ml, containing 17 I~M purified PYP, was exposed for 15 s to a Schott KL1500 150 W halogen lamp containing a long.wavelength band-pass filter (50% cut-off at 430 nm), to accumulate pB.…”

“…After absorption of a blue photon, the ground state of PYP (pG, ~,,,,,x = 446 nm) enters a photocycle in which a red.shifted intermediate, pR (~,,,,,x =~65 nm), and a blue-shifted intermediate, pB (~,,,= =355 nm), are formed sequentially, followed by the reformation of the ground state [6,7]. This photocycle strongly resembles the photochemistry of the archaebacterial sensory rhodopsins.…”

Rhodopsin(s) in eubacteria

“…lA,[4,6,13,14]). The absorbance spectra of these intermediates have been estimated using a global analysis approach ( Fig.…”

“…The absorbance spectra of these intermediates have been estimated using a global analysis approach ( Fig. 1B; [14]). At 77 K a bathointermediate of PYP (which is similar to pR) is trapped, indicating that the photocycle is blocked after its first step at this temperature.…”