University of Limerick Institutional Repository

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

DSpace Repository

Show simple item record

dc.contributor.author Wasserberg, Dorothee
dc.contributor.author Cabanas-Danés, Jordi
dc.contributor.author Prangsma, Jord
dc.contributor.author O'Mahony, Shane
dc.contributor.author Cazade, Pierre-André
dc.contributor.author Tromp, Eldrich
dc.contributor.author Blum, Christian
dc.contributor.author Thompson, Damien
dc.contributor.author Huskens, Jurriaan
dc.contributor.author Subramaniam, Vinod
dc.contributor.author Jonkheijm, Pascal
dc.date.accessioned 2017-10-31T11:59:55Z
dc.date.available 2017-10-31T11:59:55Z
dc.date.issued 2017
dc.identifier.uri http://hdl.handle.net/10344/6208
dc.description peer-reviewed en_US
dc.description.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. 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. en_US
dc.language.iso eng en_US
dc.publisher American Chemical Society en_US
dc.relation info:eu-repo/grantAgreement/EC/FP7/259183 en_US
dc.relation.ispartofseries ACS Nano;11, pp. 9068-9083
dc.relation.uri http://dx.doi.org/10.1021/acsnano.7b03717
dc.subject protein immobilization en_US
dc.subject multivalency en_US
dc.subject monolayers en_US
dc.subject molecular dynamics simulations en_US
dc.subject self-assembly en_US
dc.title Controlling protein surface orientation by strategic placement of oligo-histidine tags en_US
dc.type info:eu-repo/semantics/article en_US
dc.type.supercollection all_ul_research en_US
dc.type.supercollection ul_published_reviewed en_US
dc.identifier.doi 10.1021/acsnano.7b03717
dc.contributor.sponsor German Academic Exchange Service en_US
dc.contributor.sponsor Stichting Technische Wetenschappen en_US
dc.contributor.sponsor ERC en_US
dc.contributor.sponsor SFI en_US
dc.relation.projectid D/108/46093 en_US
dc.relation.projectid 12149 en_US
dc.relation.projectid 259183 en_US
dc.relation.projectid 604530-2 en_US
dc.relation.projectid 15/CDA/3491 en_US
dc.rights.accessrights info:eu-repo/semantics/openAccess en_US


Files in this item

This item appears in the following Collection(s)

Show simple item record

Search ULIR


Browse

My Account

Statistics