title: Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies.
abstract: Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.
authors.affiliation: Department of Microbiology, Montana State University, Bozeman 59717-3520, USA. umbaj@gemini.oscs.montana.edu
authors.forename: A J
authors.initials: AJ
authors.lastname: Jesaitis
authors.forename: D
authors.initials: D
authors.lastname: Gizachew
authors.forename: E A
authors.initials: EA
authors.lastname: Dratz
authors.forename: D W
authors.initials: DW
authors.lastname: Siemsen
authors.forename: K C
authors.initials: KC
authors.lastname: Stone
authors.forename: J B
authors.initials: JB
authors.lastname: Burritt
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000276:immunology; D000906:Antibodies / Q000032:analysis; D015153:Blotting, Western; D003198:Computer Simulation; D018604:Epitope Mapping / Q000379:methods*; D008958:Models, Molecular; D010047:Ovalbumin / Q000378:metabolism; D019151:Peptide Library; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D000199:Actins; D000906:Antibodies; D019151:Peptide Library; D010047:Ovalbumin