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

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 in

SUBMITTER: Wasserberg D 

PROVIDER: S-EPMC5618149 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature