Ž . Biochimica et Biophysica Acta 1385 1998 1–6 Short sequence-paper Sequence, chromophore extraction and 3-D model of the photoactive yellow protein from Rhodobacter sphaeroides 1 Remco Kort a , Mary K. Phillips-Jones b , Daan M.F. van Aalten c , Andrea Haker a , Sally M. Hoffer a , Klaas J. Hellingwerf a , Wim Crielaard a, ) a Laboratory for Microbiology, EC Slater Institute, UniÕersity of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, Netherlands b Department of Microbiology, UniÕersity of Leeds, Leeds LS2 9JT, UK c Keck Structural Biology, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA Received 2 March 1998; accepted 19 March 1998 Abstract Ž . The photoactive yellow protein pyp gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product. A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs. Downstream from pyp, a number of adjacent open reading frames were identified, including a gene Ž . encoding a CoA-ligase homologue pCL . This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein. We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R. sphaeroides cells by capillary zone electrophoresis. The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R. sphaeroides PYP, constructed by homology-based molecular modelling. In addition, this model shows that R. sphaeroides PYP contains a characteristic, positively charged patch. q 1998 Elsevier Science B.V. All rights reserved. Ž . Keywords: Photoactive yellow protein; Chromophore extraction; Rhodobacter sphaeroides Ž Photoactive yellow protein is a small 125 amino . acids , water-soluble protein found in the three halophilic purple bacteria Ectothiorhodospira halophila, Rhodospirillum salexigens and Chro- w x matium salexigens 1–3 . The encoding gene has w x been cloned from two of these species 4,5 . The protein is proposed to play a role as a photoreceptor ) Corresponding author. 1 The nucleotide sequences reported in this paper have been submitted to the EMBL nucleotide sequence database under accession numbers AJ002398 and X98889. wx for negative phototaxis 6 . Upon blue light absorp- tion, it enters a rhodopsin-like photocycle, starting with the fast formation of a red-shifted intermediate, followed by the formation of a blue-shifted interme- diate and a relatively slow recovery of the ground w x state 7,8 . The crystal structure of PYP has been ˚ wx elucidated to 1.4 A resolution 9 . Recently, structural information about the long-lived photocycle interme- diate has also become available, showing conforma- tional changes, including the ejection of the chro- w x mophore from the binding pocket 10 . In addition to these studies, it was shown that the chromophore of 0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 98 00050-8