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

• Published in: ACS nano Vol. 11; no. 9; pp. 9068 - 9083
• Main Authors: Wasserberg, Dorothee, Cabanas-Danés, Jordi, Prangsma, Jord, O’Mahony, Shane, Cazade, Pierre-Andre, Tromp, Eldrich, Blum, Christian, Thompson, Damien, Huskens, Jurriaan, Subramaniam, Vinod, Jonkheijm, Pascal
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.09.2017
• Subjects: 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; molecular dynamics simulations; self-assembly; protein immobilization; monolayers; multivalency
• DOI: 10.1021/acsnano.7b03717
• ISSN: 1936-0851

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.