Alanine-shaving Mutagenesis to Determine Key Interfacial Residues Governing the Assembly of a Nano-cage Maxi-ferritin

Abstract

The fundamental process of protein self-assembly is governed by protein-protein interactions between subunits, which combine to form structures that are often on the nano-scale. The nano-cage protein, bacterioferritin from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for an alanine-shaving mutagenesis study to discover key amino acid residues at symmetry-related protein-protein interfaces that control protein stability and self-assembly. By inspection of these interfaces and "virtual alanine scanning," nine mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, dynamic light scattering, native PAGE, and temperaturedependent CD. Many of the selected amino acids act as hot spot residues. Four of these (Arg-30, which is located at the two-fold axis, and Arg-61, Tyr-114, and Glu-128, which are located at the three-fold axis), when individually mutated to alanine, completely shut down detectable solution formation of 24-mer, favoring a cooperatively folded dimer, suggesting that they may be oligomerization "switch residues." Furthermore, two residues, Arg-30 and Arg-61, when changed to alanine form mutants that are more thermodynamically stable than the native protein. This investigation into the structure and energetics of this self-assembling nano-cage protein not only can act as a jumping off point for the eventual design of novel protein nanostructures but can also help to understand the role that structure plays on the function of this important class of proteins.

Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

“…In analogy to our work with E. coli bacterioferritin (BFR), DPS was subjected to a strategy where amino acids at protein-protein interfaces were mutated to identify residues that control assembly and stability. 19 As opposed to traditional ''alanine shaving'' studies where the energetic role of each residue is determined relative to the wild-type through the measurement of the equilibrium between the oligomerized state and disassociated state, our strategy, due to the complexities associated with highly oligomeric structures and mathematical challenges in measuring their equilibrium, focused on measuring the energetic effect of mutating a residue to alanine on the equilibrium between the folded, oligomeric state and the unfolded, disassociated state through denaturation. 12,27 All nine of the mutants formed cooperatively folded a-helical structures (Supporting Information Fig.…”

“…In that previous research, we discovered four residues that completely obliterate cage formation when mutated to alanine. 19 Surprisingly, one of these is located at the twofold axis of symmetry suggesting that there either is a global conformational change or that an oligomerization nonproductive dimer is possible. In addition, we also discovered two residues that increase the overall stability of the protein when mutated.…”

“…It should be mentioned that in similar systems we have observed that conditions for obtaining TEM micrographs can encourage oligomerization that is not observed in solution. 19 …”

“…Although no clear trends are evident, thermal stability does not seem to follow that of reversibility which is consistent with other work we have done in these systems. 18,19 Interestingly, the double mutant R83A/R133A, which was the least stable, is among the most reversible which is possibly due to the fact that it only forms dimer in solution. As we have reported that BFR alanine-shaved mutants were more reversible than the DPS series, the fact that the thermal stability of BFR was more sensitive to mutation than was DPS, suggests that that competing thermodynamic versus kinetic control exists in the unfolding of the two proteins, or simply that an aggregation pathway is more accessible to DPS in general.…”

“…14 We have recently performed alanine shaving of the proteinprotein interfaces in the maxi-ferritin BFR from Escherichia coli to identify residues that control its stability and oligomerization. 18,19 As an extension of this latter work, we describe here the application of this strategy to E. coli DPS (EcDPS). Because the structural energetics of the two proteins are quite unique, this is a distinct application of the strategy.…”