Assembly of the bacteriophage P22 requires a 303 amino acid residue scaffolding protein. Two scaffolding protein deletion mutants, consisting of residues 141 to 303 and 141 to 292, have been described. We report here that the 141-303 fragment, but not the 141-292 fragment, promoted procapsid assembly in vitro, bound to preformed shells of coat protein, and bound to a coat protein affinity column. These findings suggest that the carboxyl-terminal half of the scaffolding protein is sufficient for promoting assembly, and that the 11 amino acid residues at the extreme carboxyl terminus are required for binding to the coat protein. Analysis of the products of in vitro assembly reactions suggests that the maximum amount of scaffolding protein that can pack into a procapsid is dictated by the internal volume of the procapsid rather than by a finite number of binding sites. However, when the amount of scaffolding protein was reduced to limiting values, both the wild-type protein and the 141-303 fragment assembled procapsids with the same number, rather than the same mass, of scaffolding protein molecules. When the 141-292 fragment was added to a mixture of coat and scaffolding proteins, the initial phase of procapsid assembly was inhibited, but the final yield and composition of the procapsids were not affected. Assembly by a covalent dimeric mutant scaffolding protein (R74C/L177I) was not inhibited by the 141-292 fragment, which suggests that the inhibition is due to the formation of inactive heterodimers between the 141-292 fragment and the monomeric scaffolding protein. The 141-303 fragment, which has less tendency to self-associate than the wild-type protein, formed aberrant species as well as normal procapsid-like particles when the rate of assembly was high, suggesting that scaffolding protein dimerization may play a role in ensuring fidelity of assembly. Alternatively, residues 1 to 140 may play a direct structural role in preventing inappropriate scaffolding/coat protein interactions.
Copyright 1998 Academic Press.