Actin surface structure revealed by antibody imprints: Evaluation of phage-display analysis of anti-actin antibodies

Phage-display peptide Library analysis of an anti-F actin polyclonal antibo dy identified 12 amino acid residues of actin that appear, in its X-ray cry stal structure, to be grouped together in a surface accessible conformation al epitope. Phage epitope mapping was carried out by isolating immune compl exes containing members of the J404 nonapeptide phage-display Library forme d in diluted antiserum and isolated on a protein A affinity matrix. Immunor eactive clones were grown as plaques, replica plated onto nitrocellulose, a nd labeled with anti-actin immune serum. One hundred and forty-four positiv ely staining clones identified in this way were sequenced. Of these, 54 dis played peptides with sequence similarities. When the most abundantly select ed sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on West ern 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 ov albumin, was not recognized by the antiserum and minimally inhibited bindin g of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four resi dues that corresponded, in frame, to a highly conserved six residue region of the chicken p-actin sequence (351)TFQQMW(356) (identical residues are sh own in bold). Examination of the rabbit skeletal muscle X-ray crystal struc ture suggested that within a 15 Angstrom radius of W-356 nine additional re sidues 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 d eviation fits of 2.1-2.5 Angstrom. We conclude that phage-display analysis can provide information about the relative location of amino acids on the s urfaces of proteins using antibody imprints of the protein surface structur e.