|Native PAGE shows that bacterioferritin mutants R30A, R61A, Y114A and E128A exhibit no detectable 24-mers.
Many proteins self-assemble into larger, multisubunit complexes held together by protein-protein interactions. Although the interfaces between subunits are often quite large, the binding energies usually are localized to a few “hot-spot” residues, thus providing key pharmaceutical targets. In this study, the researchers used Escherichia coli bacterioferritin (BFR), a cage protein composed of 24 subunits, as a model to identify key amino acid residues that control self-assembly and protein stability. They first identified nine potential hot-spot residues by inspecting the BFR crystal structure and then designed, expressed and purified alanine mutants at these sites for a shaving mutagenesis study. Four residues— Arg-30, Arg-61, Tyr-114 and Glu-128— shut down formation of the 24-mer complex when mutated and led to the formation of a cooperatively folded dimer. This suggests that these residues are crucial “switch residues” that promote higher-order assembly. These findings provide an excellent starting point for future work analyzing how structure relates to function in supramolecular proteins as well as for the design of drugs to disrupt protein self-assembly and novel protein nanostructures.
Alanine Shaving Mutagenesis to Determine Key Interfacial Residues Governing the Assembly of a Nanocage Maxiferritin
Yu Zhang, Siti Raudah Mohamed Lazim, Huihian Teo, Gwenda W. S. Teo, Rongli Fan, Xiaoming Sun and Brendan P. Orner
J. Biol. Chem., published online Feb. 5, 2010