Strategies for protein-based nanofabrication: Ni2+-NTA as a chemical mask to control biologically imposed symmetry

Background: Technologies that improve control of protein orientation on sur faces or in solution, through designed molecular recognition, will expand t he range of proteins that are useful for biosensors, molecular devices and biomaterials. A limitation of some proteins is their biologically imposed s ymmetry, which results in indistinguishable recognition surfaces. Here, we have explored methods for modifying the symmetry of an oligomeric protein t hat exhibits useful self-assembly properties. Results: Escherichia coil glutamine synthetase (GS) contains 24 solvent-exp osed histidines on two symmetry-related surfaces. These histidines drive a metal-dependent self-assembly of GS tubes. Immobilization of GS on the affi nity resin Ni2+-NTA followed by on-column modification with diethyl pyrocar bonate affords asymmetrically modified GS that self-assembles only to the e xtent of 'short' dimeric GS tubes, as demonstrated by electron microscopy, dynamic light scattering and atomic force microscopy. The utility of Ni2+-N TA 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 recogni tion elements on symmetry-related surfaces of biologically assembled protei ns. Ni2+-NTA serves as a mask to control chemical modification in vitro of residues within symmetry-related pairs, on proteins containing functional H is-tags. This strategy may be extended to modification of a wide range of a mino acids with a myriad of reagents.