Reconstitution photoactive yellow protein from apoprotein and p‐coumaric acid derivatives

Abstract

We report reconstitution of photoactive yellow protein (PYP) from apoPYP and p-coumaric acid derivatives. The addition ofp-coumaric acid to the apoPYP sample did not result in the recovery of PYP. In contrast, yellow products were obtained by the addition of p-coumaryl thiophenyl ester or pcoumaric anhydride to the apoPYP sample, the absorption spectra of which were indistinguishable from the spectrum of intact PYP. Our findings provide strong evidence that PYP has the p-coumaryl chromophore. This reconstitution technique opens the way for further biophysical studies of PYP using artificial chromophore analogs.Key words'." Photoactive yellow protein; Photoreceptor protein; p-Coumaric acid; Chromophore; Reconstitution; Ectothiorhodospira halophila 1, IntroductionPhotoactive yellow protein (PYP) is present in Ectothiorhodospira halophila as a soluble small chromoprotein [1]. It has a broad absorption band, the absorption maximum of which is at 446 nm [1]. It is considered a photoreceptor protein for the negative phototaxis of E. halophila [2]. PYP has a photoreaction cycle comprised of several intermediates, the spectral and kinetic properties of which are very similar to those of the retinal proteins in halobacteria [3 5]. Formation of the bathochromic photoproduct occurs upon light absorption, the photoproduct being converted to a near-UV intermediate in a microsecond time scale and reverting to the ground state in a subsecond time scale. These intermediates correspond to the K (batho) or L (lumi) and M (meta) intermediates of the retinal proteins. The relation between PYP and the retinal proteins, therefore, is a matter of interest, but the nature of the PYP protein moiety is very different from the moieties of the retinal proteins [6,7]. Retinal proteins are membrane proteins composed of seven transmembrane helices [8,9] and have molecular weights of 24,000-40,000. PYP is a soluble small protein of 14 kDa which crystal structure analysis to 1.4-A resolution has shown to have the ot/fl-fold structure [7].The PYP chromophore first was thought to be retinal because its photochemical properties are similar to those of retinal proteins [10]. The chromophore-binding site was tentatively assigned to the lysine residue at the 111 position spectroscopy has shown that the molecular weight of the chromophore is 147, smaller than that of retinal [6]. Moreover, the chromophore binding site is a unique cysteine residue at position 69 [6]. These findings suggest that the chromophore is not retinal. Recent studies have shown that the PYP chromophore is p-coumaric acid (4-hydroxycinnamic acid) which binds to the cysteine residue via a thioester bond [11,12] (Fig. la,b).Although studies based on NMR analysis of the chromophore released from digested PYP [11] or high-resolution crystallography of PYP [12] clearly have shown that PYP has the p-coumaryl chromophore, direct confirmation would depend on the reconstitution of PYP from its apoprotein and pcoumaric acid. Moreover, an effective reconstitution technique would pr...

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

“…After growth for a further 4 h, the cells were spun down and lysed. The chromophore was then reconstituted with the apoprotein as described (29). After centrifugation, the supernatant containing Ppr-PYP was applied to a chelating fast flow Sepharose resin charged with Ni 2ϩ (Amersham Pharmacia), followed by dialysis against 10 mM Tris (pH 8.0) and anion exchange chromatography using high performance Q Sepharose (Amersham Pharmacia).…”

Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

“…p-Coumaric-8-13 C-acid was prepared by p-hydroxybenzaldehyde and triethylphosphonoacetate-2-13 C followed by alkaline hydrolysis of the ester. 13 C-Labeled PYP was prepared by reconstitution of PYP with 13 C-labeled p-coumaric anhydride and apoPYP (9). They were then desalted by dialysis and applied to a small DEAE-Sepharose column (Pharmacia Biotech Inc.).…”

“…Namely, the protein moiety of PYP has an ␣/␤ fold structure (5) composed of 125 amino acids (6,7). The chromophore is a p-coumaric acid (7)(8)(9) bound to a cysteine residue via a thioester bond.…”

Protonation/Deprotonation Reactions Triggered by Photoactivation of Photoactive Yellow Protein from Ectothiorhodospira halophila

“…ApoPYP can be produced heterologously in Escherichia coli (15) and can subsequently be converted to functional holoprotein through reconstitution with activated derivatives of its chromophore (16,17). This capability has made PYP available in sufficient amounts to allow detailed analysis of its photocycle using biophysical techniques like x-ray diffraction, Fourier transform infrared, Raman, NMR, transient absorption, and fluorescence spectroscopy (see e.g.…”