Maziar Soleymani Ardejani - Stabilization of a Protein Nano-cage through the Plugging of a Protein-Protein Interfacial Water Pocket

Version 1

      Publication Details (including relevant citation   information):

      Biochemistry, Just Accepted Manuscript

      DOI: 10.1021/bi200207w

      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):