PRIMARY STRUCTURE OF A PHOTOACTIVE YELLOW PROTEIN FROM THE PHOTOTROPHIC BACTERIUM ECTOTHIORHODOSPIRA-HALOPHILA, WITH EVIDENCE FOR THE MASS AND THE BINDING-SITE OF THE CHROMOPHORE

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined t o be DDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP GKFKEGVASGNLNT MFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homo logy to any other protein, although the crystal structure, known at 2. 4 angstrom resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sc i. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eu karyotic proteins that bind hydrophobic molecules. The amino acid sequ ence exhibits no greater similarity between PYP and FABP than for prot eins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers withi n a second. Here we demonstrate that the chromophore is bound covalent ly to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, pl us Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not unif ormly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chem ical nature of the chromophore remains undetermined, but we here propo se a possible structure based on precise mass analysis of a chromophor e-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon r eduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/-0.5 Da); the cysteine residue t o which it is bound is at sequence position 69.