Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro.
Coat and scaffolding subunits derived from P22 procapsids have been purified in forms that co-assemble rapidly and efficiently into icosahedral shells in vitro under native conditions. The half-time for this reaction is approximately five minutes at 21 degrees C. The in vitro reaction exhibits the regulated features observed in vivo. Neither coat nor scaffolding subunits alone self-assemble into large structures. Upon mixing the subunits together they polymerize into procapsid-like shells with the in vivo coat and scaffolding protein composition. The subunits in the purified coat protein preparations are monomeric. The scaffolding subunits appear to be monomeric or dimeric. These results confirm that P22 procapsid formation does not proceed through the assembly of a core of scaffolding, which then organizes the coat, but requires copolymerization of coat and scaffolding. To explore the mechanisms of the control of polymerization, shell assembly was examined as a function of the input ratio of scaffolding to coat subunits. The results indicated that scaffolding protein was required for both initiation of shell assembly and continued polymerization. Though procapsids produced in vivo contain about 300 molecules of scaffolding, shells with fewer subunits could be assembled down to a lower limit of about 140 scaffolding subunits per shell. The overall results of these experiments indicate that coat and scaffolding subunits must interact in both the initiation and the growth phases of shell assembly. However, it remains unclear whether during growth the coat and scaffolding subunits form a mixed oligomer prior to adding to the shell or whether this occurs at the growing edge.
Prevelige
P E
PE
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Thomas
D
D
King
J
J
D002213:Capsid; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D012483:Salmonella Phages / Q000648:ultrastructure*; D014764:Viral Proteins
D046911:Macromolecular Substances; D014764:Viral Proteins