Kinetic and calorimetric evidence for two distinct scaffolding protein binding populations within the bacteriophage P22 procapsid

A wide variety of viruses require the transient presence of scaffolding pro teins to direct capsid assembly. In the case of bacteriophage P22, a model in which the scaffolding protein selectively stabilizes on-pathway growing intermediates has been proposed. The stoichiometry and thermodynamics of bi nding of the bacteriophage P22 scaffolding protein within the procapsid wer e analyzed by light scattering and isothermal titration calorimetry. Calori metric experiments carried out between 10 and 37 degreesC were consistent w ith the presence of at least two distinct populations of binding sites, in agreement with kinetic evidence obtained by a light scattering assay. Bindi ng to the high-affinity sites occurred at 20 degreesC with a stoichiometry of approximately 60 scaffolding molecules per procapsid and an apparent K-d of approximately 100-300 nM and was almost completely enthalpy-driven. For the second binding population, precise fitting of the data was impossible due to small heats of binding, but the thermodynamics of binding were clear ly distinct from the high-affinity phase. The heat capacity change (DeltaC( p)) of binding was large for the high-affinity sites and negative for both sets of sites. Addition of sodium chloride (1 M) greatly reduced the magnit ude of the apparent DeltaH, in agreement with previous evidence that electr ostatic interactions play a major role in binding. A mutant scaffolding pro tein that forms covalent dimers (R74C/L177I) bound only to the high-affinit y sites. These data comprise the first quantitative measurements of the ene rgetics of the coat protein/scaffolding protein interaction.