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

Mersacidin belongs to the type B lantibiotics (lanthi-onine

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to R-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an R-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.Eukaryotic gene transcription by RNA polymerase II requires the assembly of many proteins on the promoter region (1-3). The rate of transcription is enhanced by transcriptional activators, through recruitment of chromatin remodeling enzymes and through facilitating the assembly of the preinitiation complex (PIC) 1 (4, 5). One of the best characterized activators is the herpes simplex virion protein 16 (VP16), also known as Vmw65, ICP25, or R-TIF (6, 7). The carboxy-terminal region (residues 410-490) of this protein contains two potent transcription activation domains (TADs) (8, 9) that can target many proteins of the RNA polymerase II transcription machinery, such as TBP (10), TFIIA (11,12), TFIIB (13), the RAP74 subunit of TFIIF (14), the p62 component of TFIIH (15), the TBP-associatedfactors hTAF II 31 (16), dTAF II 40 (17), hTAF II 32 (18), the human cofactor PC4 (19,20), CBP (21,22), and p300 (23,24). The two functional regions in this acidic VP16 activation domain (VP16ad) are independently able to activate transcription in vivo via distinct pathways (8,17,22,(25)(26)(27). Both subdomains, VP16ad/n (412-453) and VP16ad/c (454-490), have been extensively studied by mutational analysis, indicating key roles for specific hydrophobic and acidic residues (26-29). The TADs of transcription factors often lack a folded structure under physiological conditions †

The yeast Paf1 complex consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and regulates histone H2B ubiquitination, histone H3 methylation, RNA polymerase II carboxy-terminal domain (CTD) Ser2 phosphorylation, and RNA 3' end processing. We provide structural insight into the Paf1 complex with the NMR structure of the conserved and functionally important Plus3 domain of human Rtf1. A predominantly beta-stranded subdomain displays structural similarity to Dicer/Argonaute PAZ domains and to Tudor domains. We further demonstrate that the highly basic Rtf1 Plus3 domain can interact in vitro with single-stranded DNA via residues on the rim of the beta sheet, reminiscent of siRNA binding by PAZ domains, but did not detect binding to double-stranded DNA or RNA. We discuss the potential role of Rtf1 Plus3 ssDNA binding during transcription elongation.

Structural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant protein production with the E. coli expression systems. To circumvent this problem we decided to focus on separate protein domains. We describe here a fast microtiterplate based, expression and solubility screening procedure, using a combination of in vitro and in vivo expression, and purification with nickel-NTA magnetic beads. All steps are optimized for automatic HTP processing using a liquid handling station. Furthermore, large-scale expression and protein purification conditions are optimized, permitting the purification of 24 protein samples per week. We further show that results obtained from the expression screening can be extrapolated to the production of protein samples for NMR. Starting with 81 cloned human protein domains, in vivo expression was detected in 54 cases, and from 28 of those milligrams of protein were purified. An informative HSQC spectrum was recorded for 18 proteins (22%), half of which were indicative of a folded protein. The success rate and quality of the HSQC spectra suggest that the domain approach holds promise for human proteins.