Publication Details (including relevant citation information):
Biochemistry, Just Accepted Manuscript
Publication Date (Web): April 13, 2011
Copyright © 2011 American Chemical Society
The unique structural properties of the ferritin protein cages have provided impetus to focus on the methodical study of these self-assembling nano-systems. Among these proteins, E. 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 homologs, we have found that this protein has a smaller than average dimeric interface on its two-fold symmetry axis mainly due to 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 semi-empirical computational method to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to the 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 bio-structural energetics.
Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi200207w