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

    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.

    Original languageEnglish
    Pages (from-to)9068-9083
    Number of pages16
    JournalACS Nano
    Volume11
    Issue number9
    DOIs
    Publication statusPublished - 26 Sep 2017

    Keywords

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

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