Strategies for protein-based nanofabrication: Ni-NTA as a chemical mask to control biologically imposed symmetry

Background: Technologies that improve control of protein orientation on surfaces or in solution, through designed molecular recognition, will expand the range of proteins that are useful for biosensors, molecular devices and biomaterials. A limitation of some proteins is their biologically imposed symmetry, which results in indistinguishable recognition surfaces. Here, we have explored methods for modifying the symmetry of an oligomeric protein that exhibits useful self-assembly properties.Results:Escherichia coli glutamine synthetase (GS) contains 24 solvent-exposed histidines on two symmetry-related surfaces. These histidines drive a metal-dependent self-assembly of GS tubes. Immobilization of GS on the affinity resin Ni²⁺-NTA followed by on-column modification with diethyl pyrocarbonate affords asymmetrically modified GS that self-assembles only to the extent of ‘short’ dimeric GS tubes, as demonstrated by electron microscopy, dynamic light scattering and atomic force microscopy. The utility of Ni²⁺-NTA as a chemical mask was also demonstrated for asymmetric modification of engineered cysteines adjacent to the natural histidines.Conclusions: Current genetic methods do not provide distinguishable recognition elements on symmetry-related surfaces of biologically assembled proteins. Ni²⁺-NTA serves as a mask to control chemical modification in vitro of residues within symmetry-related pairs, on proteins containing functional Histags. This strategy may be extended to modification of a wide range of amino acids with a myriad of reagents.