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

Green Fluorescent Protein (GFP) is a bioluminescence protein from the jelly fish Aequorea victoria. It can exist in at least two spectroscopically distinct states: GFP395 and GFP480, with peak absorption at 395 and 480 nm, respectively, presumably resulting from a change in the protonation state of the phenolic ring of its chromophore. When GFP is formed upon heterologous expression in Escherichia coli, its chromophore is mainly present as the neutral species. UV and visible light convert (the chromophore of) GFP quantitatively from this neutral- into the anionic form. On the basis of X-ray diffraction, it was recently proposed (Brejc, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311; Palm, G. J. et al. (1997) Nat. Struct. Biol. 4, 361-365) that the carboxylic group of Glu222 functions as the proton acceptor of the chromophore of GFP, during the transition from the neutral form (i.e., GFP395) to the ionized form (GFP480). However, X-ray crystallography cannot detect protons directly. The results of FTIR difference spectroscopy, in contrast, are highly sensitive to changes in the protonation state between two conformations of a protein. Here we report the first characterization of GFP, and its photoconversion, with FTIR spectroscopy. Our results clearly show the change in protonation state of the chromophore upon photoconversion. However, they do not provide indications for a change of the protonation state of a glutamate side chain between the states GFP395 and GFP480, nor for an isomerization of the double bond that forms part of the link between the two rings of the chromophore.

The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signal sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disulphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.

Chemical reactivity and spectroscopy of the thiol ester-linked p-coumaric acid chromophore in the photoactive yellow protein from Ectothiorhodospira halophila Hoff, W.D.; Devreese, B.; Fokkens, R.H.; Nugteren-Roodzant, J.M.; Beeumen, J.; Nibbering, N.M.M.; Hellingwerf, K.J. Published in: Biochemistry DOI:10.1021/bi951755zLink to publication Citation for published version (APA):Hoff, W. D., Devreese, B., Fokkens, R. H., Nugteren-Roodzant, J. M., Beeumen, J., Nibbering, N. M. M., & Hellingwerf, K. J. (1996). Chemical reactivity and spectroscopy of the thiol ester-linked p-coumaric acid chromophore in the photoactive yellow protein from Ectothiorhodospira halophila. Biochemistry, 35, 1274-1281/1284. DOI: 10.1021/bi951755z 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. Here we report on the chemistry of the linkage of this new photoactive cofactor to apoPYP: (i) Analysis of chromophore-peptide conjugates of PYP by high-resolution mass spectrometry unambiguously shows that the p-coumaric acid molecule is bound to Cys 69 via a thiol ester bond. The PYP chromophore is the first cofactor known to be stably thiol ester-linked to its apoprotein.(ii) The chemical reactivity of this thiol ester bond with respect to dithiothreitol, performic acid, and high pH is similar to that of disulfide bridges. These treatments result in the cleavage of the thiol ester bond, concomitant with strong shifts in the UV/vis absorbance band of the chromophore. (iii) The spectral properties of the PYP chromophore under different conditions are related to the structural integrity of the protein, the presence of the thiol ester bond, and the ionization state of the phenolic proton of the chromophore. These results are important for the general problem of spectral tuning in photoreceptor proteins.