FUNCTIONAL DOMAINS OF BACTERIOPHAGE-P22 SCAFFOLDING PROTEIN

Assembly of the bacteriophage P22 requires a 303 amino acid residue sc affolding protein. Two scaffolding protein deletion mutants, consistin g of residues 141 to 303 and 141 to 292, have been described. We repor t here that the 141-303 fragment, but not the 141-292 fragment, promot ed procapsid assembly in vitro, bound to preformed shells of coat prot ein, and bound to a coat protein affinity column. These findings sugge st that the carboxyl-terminal half of the scaffolding protein is suffi cient for promoting assembly, and that the 11 amino acid residues at t he extreme carboxyl terminus are required for binding to the coat prot ein. Analysis of the products of in vitro assembly reactions suggests that the maximum amount of scaffolding protein that can pack into a pr ocapsid is dictated by the internal volume of the procapsid rather tha n by a finite number of binding sites. However, when the amount of sca ffolding protein was reduced to limiting values, both the wild-type pr otein and the 141-303 fragment assembled procapsids with the same numb er, rather than the same mass, of scaffolding protein molecules. When the 141-292 fragment was added to a mixture of coat and scaffolding pr oteins, the initial phase of procapsid assembly was inhibited, but the final yield and composition of the procapsids were not affected. Asse mbly by a covalent dimeric mutant scaffolding protein (R74C/L177I) was not inhibited by the 141-292 fragment, which suggests that the inhibi tion is due to the formation of inactive heterodimers between the 141- 292 fragment and the monomeric scaffolding protein. The 141-303 fragme nt, which has less tendency to self-associate than the wild-type prote in, formed aberrant species as well as normal procapsid-like particles when the rate of assembly was high, suggesting that scaffolding prote in dimerization may play a role in ensuring fidelity of assembly. Alte rnatively, residues 1 to 140 may play a direct structural role in prev enting inappropriate scaffolding/coat protein interactions. (C) 1998 A cademic Press