Stabilization of a Protein Nanocage through the Plugging of a Protein–Protein Interfacial Water Pocket

Abstract

The unique structural properties of the ferritin protein cages have provided impetus to focus on the methodical study of these self-assembling nanosystems. Among these proteins, Escherichia coli bacterioferritin (EcBfr), although architecturally very similar to other members of the family, shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states. Through computational analysis and comparison to its homologues, we have found that this protein has a smaller than average dimeric interface on its 2-fold symmetry axis mainly because of the existence of an interfacial water pocket centered around two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, we have used a semiempirical computational method to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to that of wild-type EcBfr. This computational study also converged on the water-bridged asparagines. Replacing these two asparagines with hydrophobic amino acids resulted in proteins that folded into α-helical monomers and assembled into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirmed that, in all cases, these proteins, in agreement with the calculations, possessed increased stability. One of the three mutations shifts the population in favor of the higher-order oligomerization state in solution as evidenced by both size exclusion chromatography and native gel electrophoresis. These results taken together suggest that our low-level design was successful and that it may be possible to apply the strategy of targeting water pockets at protein--protein interfaces to other protein cage and self-assembling systems. More generally, this study further demonstrates the power of jointly employing in silico and in vitro techniques to understand and enhance biostructural energetics.

Thermophilic Ferritin 24mer Assembly and Nanoparticle Encapsulation Modulated by Interdimer Electrostatic Repulsion

“…21 However, in E. coli bacterioferritin, mutations designed to plug an interdimer water pocket with hydrophobic residues led to significantly enhanced thermal stability (Δ T m >20 °C) but greater dimer population compared to the wt, as the geometry of the more stable dimers prevented cage formation. 13 Although A127R appears to favor dimer at low-salt conditions, its subunit thermal stability is identical to wt. For D138K, its enhanced 24mer stability at low-salt conditions also does not appear to be linked to thermal stability, as it too exhibits the wt T m .…”

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

“…We referred to this as the ''Arch and Keystone'' hypothesis, the possibility of which we are currently exploring. 38 These complexities contrasted with the relatively very rational and straightforward DPS, further emphasizing the structural energetic distinctness of these mini-and maxi-ferritins. Furthermore, fundamental studies of how proteinprotein interactions control the stability and oligomerization of cage proteins could aid in the development of future applications, provide controls for further studies, and direct investigations into using these proteins as building blocks in the design and discovery of novel oligomerized architectures.…”

Concerted Motions Networking Pores and Distant Ferroxidase Centers Enable Bacterioferritin Function and Iron Traffic

“…Twenty four subunits and 12 hemes assemble into a spherical and hollow structure (Figure 1B) with an outer diameter of ∼120 Å and an inner diameter of ∼80 Å, where up to ∼3,500 iron atoms can be stored in the form of a Fe 3+ mineral(10). Details pertaining the self-assembly and stability of the 24-mer bacterioferritin shells are beginning to emerge (11, 12). The formation of an iron core (iron uptake) requires binding of ferrous iron (Fe 2+ ) to a ferroxidase catalytic site, where it is oxidized to the ferric (Fe 3+ ) state (10, 13, 14), and then translocated to the interior cavity.…”