Dux, P.; Hard, K.; Devreese, B.; Nugteren-Roodzand, I.M.; Crielaard, W.; Boelens, R.; Beeumen, J.; Kaptein, R.; Hellingwerf, K.J. Published in: Biochemistry DOI:10.1021/bi00251a001Link to publication Citation for published version (APA):Hoff, W. D., Dux, P., Hard, K., Devreese, B., Nugteren-Roodzand, I. M., Crielaard, W., ... Hellingwerf, K. J. (1994). p-Coumaric acid, a new photoactive chromophore of a yellow photoreceptor protein with rhodopsin-like characteristics. Biochemistry, 33, 13959-13963. DOI: 10.1021/bi00251a001 General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. would make it the first eubacterial rhodopsin. Here we report the chemical structure of this chromophoric group to be p-coumaric acid, which is covalently bound to a unique cysteine in the apoprotein via a thiol ester bond, and thus not retinal. This makes PYP the first example of a protein containing p-coumaric acid, a metabolite previously found only in plants, as a prosthetic group and establishes the photoactive yellow proteins as a new type of photochemically active receptor molecule.The photoactive yellow proteins (PYP) constitute a responsible for the yellow color of the protein have been advanced (Meyer, 1985;McRee at al, 1989;Van Beeumen et al., 1993), but the true nature of this chromophore et al, 1993) and crystal structure ( M~R~~ et al., 1989) of pyp at 2.4-ij resolution have been and show that the protein is composed of two perpendicular plates of P-sheet, forming a p-clam structure very similar to the fold homologous group of proteins found in many Eubacteria (Meyer, 1985; M e w et al., 1990;Hoff et al., 1994a). The isolated from Ecfothiorhodospira halophila have been studied in some detail. Since PYP was isolated in 1985, a number of proposals concerning the chemical structure of the cofactor structural and photochemical characteristics Of the PYP remained unclear. The amino acid sequence (Van Beeumen

Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.

The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.

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