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

Abstract

We report oriented immobilization of proteins using the standard hexahistidine (His₆)-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 His₆-tag on the TagRFP proteins. All TagRFP variants with His₆-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His₆-tags on opposite sides of the so-called β-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 His₆-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 His₆-tag forms a full coordination bond with the Ni²⁺:NTA SAMs, which varied with the position of the His₆-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 β-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.