BACTERIOPHAGE P22 SCAFFOLDING PROTEIN FORMS OLIGOMERS IN SOLUTION

The scaffolding protein of Salmonella typhimurium bacteriophage P22 is a 33.6 kDa protein required both in vivo and in vitro for the polymer ization of the viral coat protein into closed T = 7 icosahedral procap sids. In vitro assembly reaction kinetics have previously been found t o vary between second and third order with respect to scaffolding prot ein concentration, suggesting that dimers and/or higher-order oligomer s may be the active species in assembly. Analytical ultracentrifugatio n experiments suggest that scaffolding protein undergoes a rapidly-rev ersible monomer/dimer/tetramer equilibrium, with higher association co nstants at 4 degrees C than at 20 degrees C. Under conditions in which in vitro assembly reactions are carried out (30 to 1000 mu g/ml scaff olding protein, 20 degrees C), monomers are the predominant species, b ut the concentration of dimers is significant. A mutant scaffolding pr otein, R74C/L177I, which forms disulfide-linked dimers, catalyzed proc apsid assembly at a higher rate than did the wildtype scaffolding prot ein; preincubation in dithiothreitol had little effect on the wild-typ e protein, but greatly reduced the activity of the mutant. These findi ngs suggest that dimers and/or higher-order oligomers of scaffolding p rotein are active species in the assembly of P22. (C) 1997 Academic Pr ess Limited.