Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin

Abstract

A water-soluble yellow protein, previously discovered in the purple photosynthetic bacterium Ectothiorhodospira halophila, contains a chromophore which has an absorbance maximum at 446 nm. The protein is now shown to be photoactive. A pulse of 445-nm laser light caused the 446-nm peak to be partially bleached and red-shifted in a time less than 1 microsecond. The intermediate thus formed was subsequently further bleached in the dark in a biphasic process occurring in approximately 20 ms. Finally, the absorbance of native protein was restored in a first-order process occurring over several seconds. These kinetic processes are remarkably similar to those of sensory rhodopsin from Halobacterium, and to a lesser extent bacteriorhodopsin and halorhodopsin; although these proteins are membrane-bound, they have absorbance maxima at about 570 nm, and they cycle more rapidly. In attempts to remove the chromophore for identification, it was found that a variety of methods of denaturation of the protein caused transient or permanent conversion to a form which has an absorbance maximum near 340 nm. Thus, by analogy to the rhodopsins, the absorption at 446 nm in the native protein appears to result from a 106-nm red shift of the chromophore induced by the protein. Acid denaturation followed by extraction with organic solvents established that the chromophore could be removed from the protein. It is not identical with all-trans-retinal and remains to be identified, although it could still be a related pigment. The E. halophila yellow protein has a circular dichroism spectrum which indicates little alpha-helical secondary structure (19%).(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Reactivity and Spectroscopy of the Thiol Ester-Linked p-Coumaric Acid Chromophore in the Photoactive Yellow Protein from Ectothiorhodospira halophila

“…However, it has recently been reported that this crystal structure is erroneous (Baca et al, 1994); it has been redetermined and refined to 1.4 Å, showing that PYP has an R/ fold (Borgstahl et al, 1995). This is in line with early circular dichroism spectra of PYP indicating 19% R-helix (Meyer et al, 1987;W. D. Hoff et al, unpublished results).…”

Thiol ester-linked p-coumaric acid as a new photoactive prosthetic group in a protein with rhodopsin-like photochemistry

“…On a submillisecond time scale this photocycle intermediate is converted to a state absorbing at 355 nm, which recovers to the ground state on a subsecond time scale (Meyer et al, 1987;Hoff et al, 1994b). We propose that the photochemical basis of this process is the cis-trans isomerization of the vinyl trans double bond in the chromophore.…”

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].…”

“…This spectral tuning of the chromophore can be explained by contributions of (i) the thiol-ester linkage, causing a shift to 335 nm, (ii) the deprotonation of the phydroxy group of the chromophore, shifting the absorbance maximum to 410 nm, and (iii) unidentified aspects of tile protein environment, resulting in the observed absorption maximum at 446 nm [2,4,27]. After excitation with blue light, pG is converted into the short-lived intermediate pR [6,7], in which the chromophore is probably in the ci~ configuration (low 8re,x) and still deprotonated (long ~.,,,x). In the dark, pR is subsequently converted to the long-lived intermediate pB (low 8,,,,=x, short ~,,,,~x), in which the chromophore is in the cis configuration, as shown here, and protonated.…”

“…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.…”