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Controlling protein surface orientation by strategic placement of oligo-histidine tags

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journal contribution
posted on 2017-10-31, 11:59 authored by Dorothee Wasserberg, Jordi Cabanas-Danés, Jord Prangsma, Shane O'Mahony, Pierre-André Cazade, Eldrich Tromp, Christian Blum, DAMIEN THOMPSONDAMIEN THOMPSON, Jurriaan Huskens, Vinod Subramaniam, Pascal Jonkheijm
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. Timeresolved 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.


BGP Doctoral 2010 Grant (Royal Holloway, University of London)

Arts and Humanities Research Council

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Japan Society for the Promotion of Science

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ACS Nano;11, pp. 9068-9083


American Chemical Society



Other Funding information

German Academic Exchange Service, Stichting Technische Wetenschappen, ERC, SFI



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