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

ACS Nano. 2017 Sep 26;11(9):9068-9083. doi: 10.1021/acsnano.7b03717. Epub 2017 Sep 13.


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.

Keywords: molecular dynamics simulations; monolayers; multivalency; protein immobilization; self-assembly.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Histidine / chemistry*
  • Immobilized Proteins / chemistry*
  • Luminescent Proteins / chemistry*
  • Molecular Dynamics Simulation
  • Nickel / chemistry*
  • Nitrilotriacetic Acid / chemistry*
  • Oligopeptides / chemistry*
  • Red Fluorescent Protein
  • Sea Anemones / chemistry*
  • Surface Properties


  • His-His-His-His-His-His
  • Immobilized Proteins
  • Luminescent Proteins
  • Oligopeptides
  • Histidine
  • Nickel
  • Nitrilotriacetic Acid