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

Dorothee Wasserberg, Jordi Cabanas-Danés, Jord Prangsma, Shane O'Mahony, Pierre Andre Cazade, Eldrich Tromp, Christian Blum, Damien Thompson, Jurriaan Huskens, Vinod Subramaniam, Pascal Jonkheijm*

*Corresponding author for this work

    Research output: Contribution to JournalArticleAcademicpeer-review


    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.

    Original languageEnglish
    Pages (from-to)9068-9083
    Number of pages16
    JournalACS Nano
    Issue number9
    Publication statusPublished - 26 Sept 2017


    D.W. thanks the German Academic Exchange Service for financial support under postdoctoral research grant D/08/ 46093. Work by J.C.-D., E.T., and P.J. was funded by Project P2.02 OAcontrol of the research program of the BioMedical Materials Institute, cofunded by the Dutch Ministry of Economic Affairs. J.P. and V.S. acknowledge support from Stichting Technische Wetenschappen (STW) under the nanoscopy program (project no. 12149). The work by D.W. and P.J. was cofunded by Starting ERC Grant (259183 Sumoman). D.T. acknowledges financial support from the European Union’s Seventh Framework Programme (FP7/ 2007−2013) under grant number 604530-2 (CellulosomePlus) and thanks Science Foundation Ireland (SFI) for financial support under Grant Number 15/CDA/3491 and for provision of computing resources at the SFI/Higher Education Authority Irish Centre for High-End Computing (ICHEC). The authors thank Dr. M. Koay from the University of Twente for discussions and comments.

    FundersFunder number
    SFI/Higher Education Authority Irish Centre for High-End Computing
    Seventh Framework Programme259183, 604530
    Science Foundation Ireland15/CDA/3491
    Deutscher Akademischer AustauschdienstD/08/ 46093, P2.02 OAcontrol
    Stichting voor de Technische Wetenschappen12149
    Ministry of Economic Affairs
    Seventh Framework Programme


      • molecular dynamics simulations
      • monolayers
      • multivalency
      • protein immobilization
      • self-assembly


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