Method Overview
Our project had two phases: a computational phase in which modifications to protein structure were modeled and the most energetically favorable protein structures were selected. And, a wet lab phase in which the gene of the proposed computational designs were constructed, expressed, and their protein products were characterized using analytical methods.
First, the protein design suite Rosetta was used to model the fusion proteins and determine if mutations will promote the formation of fractal assemblies. Since the fusion proteins had 0 residue linkers, computational protein design via Rosetta was used to prevent clashing between the enzyme and the binding domain/peptide. Several designs were selected for experimental characterization.
Second, the fusion proteins were made using molecular cloning techniques. The triple-mutant superbinder SH2 domain was cloned into the C-terminus of atzC, and the superbinder SH2 binding peptide was cloned into the N-terminus of atzA. AtzC and atzA were cloned in the his-tagged expression vectors pET29b and pET15b, respectively. Site-directed mutagenesis was performed to make mutations in the resulting genes.
Third, the proteins were expressed in E. coli BL21(DE3) and purified using Ni-NTA affinity chromatography. The proteins were then buffer exchanged out of elution buffer and into 50mM HEPES, 100mM NaCl, 5% glycerol, pH 7.6. Proteins were then assayed for phosphorylation by ELISA, binding by BLI, assembly formation by DLS, disassembly by DLS, and structure by helium-ion microscopy.
Fourth, a fractal assembly generator and a enzyme kinetics simulator were prepared to model the resulting fractal assemblies and metabolic pathway kinetics.
Below are detailed protocols for all methods used.