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.bioconjugation ͉ protein folding ͉ circular dichroism ͉ molecular dynamics simulation ͉ nanoscale interface N anomaterials hold much promise for biomedical applications such as multiscale imaging (1-3), novel therapeutic approaches (4), and biomolecular sensing (5). The emerging field of nanomedicine is based on exploiting the unique size-and material-dependent properties of nanomaterials. However, the biggest barrier in effectively using them is their biological interface. Historically, biological-inorganic interfaces suffer complications such as surface fouling in medical devices and nonspecific adsorption of proteins on sensors, inhibiting practical use. The problem significantly worsens for nanoscale systems because of their high surface-to-volume ratio. Nanoscale surfaces are physically and chemically different from the bulk, requiring nontraditional tools to effectively probe them. These factors suggest a pressing need for deeper understanding of the ''nano-bio'' interface.One particular issue related to the interface that limits the potential of nanoparticles (NPs) is conjugation to a biomolecule. Covalently linking a NP to a protein can affect both its structure and function. Even if protein structure is retained, the NP can sterically hinder substrate binding. NPs are large compared with dye molecules, and their interface with proteins is highly complex. NPs are not hard spheres but crystals with facets, edges, and vertices, and their passivating ligands can change conformation or come on and off the NP. Consequently, there are numerous nonspecific interactions between the NP and protein, where a...

Monolayer coated gold nanoparticles for delivery applications

“…A number of different approaches have been adopted for direct conjugation of proteins to AuNPs through thiols or amines on the proteins [112], but retaining the native structure of the proteins remains a challenge [113]. Antibody conjugated AuNPs provide a useful tool for cellular application including imaging and photothermal therapy.…”

On the difference of equilibrium constants of DNA hybridization in bulk solution and at the solid‐solution interface

“…Furthermore it is of primary importance for the effective development of several (other) biological applications of nanotechnology, including nanoparticle-protein conjugates (Aubin-Tam et al, 2008), and solid-state nanopores (Dekker, 2007).…”

Protein thin film machines

“…These hydration forces oscillate with distance, varying between attraction and repulsion with a periodicity equal to the water diameter. Since the inter-protein separation in the YCC-MC and YCC-BPS-MC films differ due to BPS intercalation 23 ( Figure 3) the intermolecular forces that are responsible for the mechanical work may be these hydration forces.This result is similar to recent studies of self-assembled monolayers (SAMs) of single strand DNA 36 which points at the key role played by the structural forces in water in the mechanisms Before concluding, it is worth noticing that the presented experiments as well as their thermodynamic analysis are of primary interest also for interface biological systems other than thin film switches. Consider Eq.…”