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Biochemistry. 2011 May 17;50(19):4029-37. doi: 10.1021/bi200207w. Epub 2011 Apr 26.

Stabilization of a protein nanocage through the plugging of a protein-protein interfacial water pocket.

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Division of Chemistry and Biological Chemistry, Nanyang Technological University, 21 Nanyang Link, Singapore 637371.


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.

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