The folding and assembly of bacteriophage P22 coat protein

Date of Completion

January 2007


Chemistry, Biochemistry




Single amino acid substitutions in a protein can cause misfolding and aggregation to occur. Protein misfolding can be rescued by second site amino acid substitutions called suppressor substitutions (su), commonly through stabilizing the native state of the protein or by increasing the rate of folding. In addition, molecular chaperones can aid in the folding process to alleviate aggregation caused by misfolding. I have characterized the folding of coat protein containing two types of su substitutions for the bacteriophage P22, both in vivo and in vitro, and have learned that the global mechanism of suppression differs for the two suppressors. One suppressor, T166I, functions through a concerted chaperone network by inducing and recruiting the major bacterial co-chaperone complex, GroEL/S, as well as more effectively binding an assembly chaperone, the scaffolding protein of phage P22. The other global suppressor, F170L, functions through native state stabilization. Our characterization of the folding mechanisms of these global suppressors has shed light on the substrate recognition mechanism of GroEL/S. ^ Icosahedral capsid assembly is an example of a reaction controlled solely by the protein:protein interactions of the viral subunits. Bacteriophage P22 procapsid assembly proceeds through a nucleation-limited reaction, where scaffolding protein directs the proper assembly of coat protein. Here I have investigated the nucleation and elongation reactions of procapsids assembled in vitro. Nucleation in low salt concentrations readily occurred but led to bowl-like partial procapsids, which are kinetically trapped, metastable intermediates. I further characterized phage P22 assembly by showing that procapsid assembly is an equilibrium process, with a demonstrated pseudo-critical concentration of left-over reactants. Lastly, a rigorous thermodynamics approach was used to characterize the scaffolding:coat protein interactions that occur at equilibrium. Since bacteriophage P22 assembly closely resembles the assembly pathway of more complicated eukaryotic viruses, such as herpesvirus and adenovirus, the characterization employed here can ultimately be applied to other virus assembly pathways. ^