Site-specific Labeling of Active Proteins with Gold Nanoparticles
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Site-Specific Labeling of Active Proteins with Gold Nanoparticles Marie-Eve Aubin-Tam1 and Kimberly Hamad-Schifferli2,1 1 Biological Engineering; 2Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 U.S.A. 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 nonspecific 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. INTRODUCTION Conjugation of nanoparticles (NP) to biomolecules is of particular importance because of numerous applications in sensing, imaging, assembly, and control[1-4]. While labeling techniques have been extensively developed for DNA, nanoparticle attachment to active proteins is more intricate as they are inherently much more difficult to handle. Proteins often distort when linked to a nanoparticle, which is evidently problematic as their complex three-dimensional structure must be preserved for proper function. For most applications of NP-protein conjugates, it is crucial that labeling is targeted so that it does not hamper interactions of the protein with its partners or obstruct its active site. Furthermore, a labeling approach that does not depend on pH, salt concentration, temperature or other physical characteristics of the milieu broadens the range of potential applications. Therefore, site-specific covalent labeling in combination with knowledge of the protein three dimensional structure is desirable. Structural studies have been conducted previously for NP-enzymes conjugates[5-8], but often non-specific electrostatic interactions have been utilized for the linkage. Multiple amino acids are known to form weak or strong covalent interaction with gold. As a result, conjugation of a protein at a specific amino acid with NP presents major challenges. Clearly, it is of greatest significance to study how the NP affects the activity and the structure of the protein when it is forced to link at a s
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