1.4 .ANG. Structure of Photoactive Yellow Protein, a Cytosolic Photoreceptor: Unusual Fold, Active Site, and Chromophore

Abstract

A photosensing protein directs light energy captured by its chromophore into a photocycle. The protein's structure must accommodate the photocycle and promote the resulting chemical or conformational changes that lead to signal transduction. The 1.4 A crystallographic structure of photoactive yellow protein, determined by multiple isomorphous replacement methods, provides the first view at atomic resolution of a protein with a photocycle. The alpha/beta fold, which differs from the original chain tracing, shows striking similarity to distinct parts of the signal transduction proteins profilin and the SH2 domain. In the dark state structure of photoactive yellow protein, the novel 4-hydroxycinnamyl chromophore, covalently attached to Cys69, is buried within the major hydrophobic core of the protein and is tethered at both ends by hydrogen bonds. In the active site, the yellow anionic form of the chromophore is stabilized by hydrogen bonds from the side chains of Tyr42 and buried Glu46 to the phenolic oxygen atom and by electrostatic complementarity with the positively charged guanidinium group of Arg52. Thr50 further interlocks Tyr42, Glu46, and Arg52 through a network of active site hydrogen bonds. Arg52, located in a concavity of the protein surface adjacent to the dominant patch of negative electrostatic potential, shields the chromophore from solvent and is positioned to form a gateway for the phototactic signal. Overall, the high-resolution structure of photoactive yellow protein supports a mechanism whereby electrostatic interactions create an active site poised for photon-induced rearrangements and efficient protein-mediated signal transduction.

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

“…Deprotonation of p-coumaric acid is also crucial in determining the position of the absorbance maximum of intact PYP. In the ground state of PYP the chromophore occurs in its deprotonated form, as proposed by Baca et al (1994), confirmed by crystallographic data (Borgstahl et al, 1995), and proven by resonance Raman measurements (Kim et al, 1995). The final red shift from 410 to 446 nm remains unexplained.…”

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.…”

“…In pG the maximal absorbance of the chromophore is strongly shifted to the red (from 284 nm to 446 nm). 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).…”

“…The crystal st; ucture of PYP has recently been re-determined down to 1.4 resolution and shows that the protein has an a/B fold similar *Corresponding author. Fax: (31) (20) to th,,t of eukaryotic proteins involved in signal transduction [4]. Evide,~ee has been obtained indicating that PYP functions as the blue, light photoreceptor for a new type of negative phototactic ~esponse [5].…”

Unraveling the similarity of the photoabsorption of deprotonated p-coumaric acid in the gas phase and within the photoactive yellow protein

“…Thereby, all Asp and Glu residues, except Glu64, were assumed to be negatively charged. Glu64 was assumed protonated and thus neutral 32 . From a detailed study 33 of the 95 hydrogen-bond structure and the solvent exposure, it was decided that the two His residues in the protein are protonated on the Nε2 atom.…”