STRUCTURAL STUDIES OF THE CONTRACTILE TAIL SHEATH PROTEIN OF BACTERIOPHAGE-T4 .2. STRUCTURAL-ANALYSES OF THE TAIL SHEATH PROTEIN, GP18, BY LIMITED PROTEOLYSIS, IMMUNOBLOTTING, AND IMMUNOELECTRON MICROSCOPY

The molecular structure of the T4 phage tail sheath protein, gpl8, was studied by limited proteolysis, immunoblotting, and immunoelectron microscopy. Gpl8 is extremely resistant to proteolysis in the assembled form of either extended or contracted sheaths, but it is readily cleaved by proteases in the monomeric form, giving rise to stable protease-resistant fragments. Limited proteolysis with trypsin gave rise to a trypsin-resistant fragment, Ala82-Lys316, with a molecular weight of 27K. Chymotrypsinand thermolysin-resistant fragments were also mapped close to the trypsin-resistant region. The time course of trypsin digestion of the monomeric gpl8 as monitored by SDS-polyacrylamide gel electrophoresis and immunoblotting of the gel revealed that the polypeptide chain consisting of 658 amino acid residues is sequentially cleaved at several positions from the C terminus. The N-terminal portion, Thrl-Arg81, was then removed to form the trypsin-resistant fragment. Immunoelectron microscopy revealed that the polyclonal antibodies against the trypsin-resistant fragment bound to the tail sheath. This supported the idea that at least part of the protease-resistant region of gpl8 constitutes the protruding part of the sheath protein as previously revealed with three-dimensional image reconstruction from electron micrographs by Amos and Klug [Amos, L. A., & Klug, A. (1975) J. Mol. Biol. 99, 51–73]. © 1990 American Chemical Society. All rights reserved.