Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags

We report oriented immobilization of proteins using the standard hexahistidine (His(6))-Ni2+ :NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His(6)-tag on the TagRFP proteins. All TagRFP variants with His(6)-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His(6)-tags on opposite sides of the so-called beta-barrel. Time resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His(6)-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in end-on and side-on orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His(6)-tag forms a full coordination bond with the Ni2 :NTA SAMs, which varied with the position of the His(6)-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+ :NTA moieties and by additional electrostatic interactions between different fractions of the beta-barrel structure and charged NTA. moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect.