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Site-specific Labeling of Active Proteins with Gold Nanoparticles

  • Marie-Eve Aubin-Tam (a1) and Kimberly Hamad-Schifferli (a2)

Abstract

Covalent conjugation of nanoparticles to proteins is challenging as proteins have numerous residues to which the nanoparticle can non-specifically adsorb. This is problematic as non-specific adsorption is known to denature the protein, altering its structure and thus compromising protein activity. We study site specific gold nanoparticle labeling of two enzymes, Ribonuclease S and Cytochrome c, with the goal of understanding conditions that minimize non-specific adsorption and optimize protein structure and activity. Ribonuclease S is a two-piece protein made of S-peptide and S-protein. 3nm gold nanoparticle is attached to a mutated cysteine residue on the S-peptide. The altered enzymatic activity of gold labeled Ribonuclease S is determined using RNA substrate with a fluorophore-quencher couple. Cytochrome c is linked to 1.5nm nanoparticles with ligands having neutral, negatively, or positively charged endgroups, through covalent attachment of gold with a specific surface cysteine residue. The labeled protein is characterized by circular dichroism spectroscopy and UV-visible absorption. For both proteins, agarose gel electrophoresis was used to determine optimal reaction stoichiometry and also probe non-specific adsorption between the nanoparticle and protein.

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Keywords

Site-specific Labeling of Active Proteins with Gold Nanoparticles

  • Marie-Eve Aubin-Tam (a1) and Kimberly Hamad-Schifferli (a2)

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