An objective of this proposal is to identify the amino acid residue in E. coli outer membrane protein OmpA that post-translationally forms a covalent bond to polyhydroxy-butyrate (PHB). Our preliminary studies indicate that the binding site for PHB is in the region of the eighth ?-strand across the outer membrane, i.e., residues 162-174. PHB can form covalent bonds to amino acid side-chains only at its carboxy or hydroxy termini. Covalent bonds may be formed to basic, hydroxy, or sulfhydryl amino acids. The PHB fragment 162-174 contain 4 amino acids that are possible covalent binding sites--2 serines, 1 tyrosine, and 1 arginine. The site of covalent attachment of PHB will be determined by a peptide mass mapping approach. The 162-174 peptide, or other PHB-peptides, will be analyzed by MALDI-MS. Peptides modified by PHB will differ in mass from the predicted sequence and can be identified by direct sequencing by post-source decay (MALDI-PSD-MS) or by complete proteolysis followed by MALDI-MS identification of the modified residue. To aid in the identification of covalently-linked amino acids(s), model derivatives of short-chain PHBs with homopolymers of amino acids containing functionalized side chains (amino and hydroxyl) have been prepared by the laboratory of Professor Dieter Seebach, Professor of Organic Chemistry, ETH, Switzerland. The mass spectrometric data from PHB-modified peptides will be used to aid in identifying the PHB covalent binding sites.
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