Structure and folding of bacteriophage T4 gene product 9 triggering infection. II. Study of conformational changes of gene product 9 mutants using monoclonal antibodies

Gene product 9 (gp9) of bacteriophage T4, whose spatial structure we have r ecently solved to 2.3 Angstrom resolution, is a convenient model for studyi ng the folding and oligomerization mechanisms of complex proteins. The gp9 polypeptide chain consists of 288 amino acids forming three domains. Three monomers, packed in parallel, assemble to a functionally active protein. Th e main aim of this work was to study conformational changes and trimerizati on of gp9 deletion mutants using monoclonal antibodies (mAbs). We selected a set of mAbs interacting with the amino, middle, and carboxyl regions of t he protein, respectively. Eighteen mAbs bind to native as well as to denatu red protein, and two mAbs bind to denatured protein only Using mAbs, we fou nd that deletions of the gp9 N-terminal region result in conformational cha nges in the middle and C-terminal domains. The study of mAb binding to the C Delta 7 truncated mutant by competitive ELISA and immunoblotting shows th at the C-terminus of the gp9 sequence is essential for protein trimerizatio n and stability. a single point substitution of the Gln282 residue causes f ormation of a labile trimer that has significant conformational changes in the protein domains. The results of our study show that folding and trimeri zation of gp9 is a cooperative process that involves all domains of the pro tein.