title: Site-directed nanoparticle labeling of cytochrome c.
abstract: Although nanoparticle-protein conjugates have been synthesized for numerous applications, bioconjugation remains a challenge, often resulting in denaturation or loss of protein function. This is partly because the protein-nanoparticle interface is poorly understood, which impedes the use of nanoparticles in nanomedicine. Although the effects of nanoparticle ligand and material on protein structure have been explored, the choice of the labeling site on the protein has not yet been systematically studied. To address this issue, we label cytochrome c site-specifically with a negatively charged Au nanoparticle via a covalent thiol-Au bond. The attachment site is controlled by cysteine mutations of surface residues. The effect of labeling on protein structure is probed by circular dichroism. Protein unfolding is the most severe when the nanoparticle is attached to the N- and C-terminal foldon, the core motif of cytochrome c. Also, when the nanoparticle is attached in the vicinity of charged residues, the amount of structural damage is greater because of salt-dependent electrostatic interactions with charged ligand bis(p-sulfonatophenyl) phenylphosphine on the nanoparticle. Molecular dynamics simulations also elucidate local to global structural perturbation depending on labeling site. These results suggest that the labeling site must be considered as one of the main design criteria for nanoparticle-protein conjugates.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Aubin-Tam
authors.forename: Wonmuk
authors.initials: W
authors.lastname: Hwang
authors.forename: Kimberly
authors.initials: K
authors.lastname: Hamad-Schifferli
mesh_terms: D001665:Binding Sites; D045304:Cytochromes c / Q000737:chemistry*; D006046:Gold; D053768:Metal Nanoparticles / Q000737:chemistry*; D015336:Molecular Probe Techniques; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D013438:Sulfhydryl Compounds; D006046:Gold; D045304:Cytochromes c