Properties of monoclonal antibodies selected for probing the conformation of wild type and mutant forms of the P22 tailspike endorhamnosidase.

Eleven species of monoclonal antibodies directed against the trimeric P22 tailspike endorhamnosidase have been selected and characterized. Seven of these antibodies recognize the native tailspike, both isolated and assembled onto the virion, and prevent phage infection. Four antibodies react with denatured forms of the tailspike as well as with the plastic absorbed tailspike. Three of these latter prevent the tailspike from assembling onto the phage head. The antibodies have been tested against tailspike proteins carrying single amino acid substitutions at 15 different sites on the protein. Two of these mutations interfere with binding by a set of the monoclonals, indicating that they disrupt the epitopes for these antibodies. Since amino acid replacements corresponding to the temperature-sensitive folding mutations do not change the conformation of the native protein, these mutant proteins may be particularly useful for mapping epitopes. Amber fragments of the tailspike chain are recognized predominantly by the anti-denatured antibodies suggesting either that they are conformationally closer to folding intermediates than to the native tailspike or that the epitopes recognized by anti-native antibodies are carried by the C-terminal end of the native protein. Immunochemical detection by an anti-denatured antibody, after sucrose gradient sedimentation of a large 55-kDa amber fragment, indicates a monomeric rather than a trimeric state. This suggests that the missing C-terminal region is important for the trimerization reaction. Such N-terminal amber fragments may be useful models for studying with the monoclonal antibodies the nascent chain emerging from the ribosome.