A coiled-coil strategy for the directional display of multiple proteins on the surface of iron oxide nanoparticles

Abstract

Current methods for attachment of proteins to inorganic nanoparticle surfaces utilize strategies that can destroy the tertiary structure of large proteins and/or do not allow for directional attachment, thereby diminishing functionality. We report a novel approach for the conjugation of proteins to iron oxide nanoparticles (IONPs) through coiled-coil interactions. With this strategy, coiled-coiled interactions drive attachment of the desired protein, fused to a K-coil peptide, and E-coil peptides linked to the IONP surface via a nanoparticle attachment peptide (NAP). Importantly, this method allows for directional attachment of proteins without the use of either chemical conjugation or extreme pH solutions and, thus, allows maintenance of correct protein structure with functional orientation relative to the IONP surface. In our studies, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (EGFP) were used as model proteins to demonstrate the coiled-coil approach can be utilized to attach large proteins to IONP surfaces while maintaining complex tertiary structures. These proof-of-concept studies showed EGFP- and mRFP-K-coil fusion proteins interacted specifically with E-coil-coated nanoparticles and multiple different proteins could be displayed on individual IONPs. We did not observe fluorescence after conjugation of fluorescent proteins to either E-coil-coated IONPs or dopamine-coated IONPs, which was likely due to quenching or FRET activity induced by the iron oxide core. However, substituting an E-coil-bound chromatography resin for E-coil-coated IONPs allowed the preservation of protein fluorescence following coiled-coil attachment of K-coil-fused mRFP, demonstrating the coiled-coil attachment strategy provided maintenance of the mRFP tertiary structure required for fluorescence. The coiled-coil nanoparticle attachment strategy developed herein should have broad applications in nanomedicine.