Electrostatic Interactions and Protein Competition Reveal a Dynamic Surface in Gold Nanoparticle–Protein Adsorption

Abstract

Gold nanoparticle– (AuNP–) protein conjugates are potentially useful in a broad array of diagnostic and therapeutic applications, but the physical basis of the simultaneous adsorption of multiple proteins onto AuNP surfaces remains poorly understood. Here, we investigate the contribution of electrostatic interactions to protein–AuNP binding by studying the pH-dependent binding behavior of two proteins, GB3 and ubiquitin. For both proteins, binding to 15-nm citrate-coated AuNPs closely tracks with the predicted net charge using standard pKa values, and a dramatic reduction in binding is observed when lysine residues are chemically methylated. This suggests that clusters of basic residues are involved in binding, and using this hypothesis, we model the pKa shifts induced by AuNP binding. Then, we employ a novel NMR-based approach to monitor the binding competition between GB3 and ubiquitin in situ at different pH values. In light of our model, the NMR measurements reveal that the net charge, binding association constant, and size of each protein play distinct roles at different stages of protein adsorption. When citrate-coated AuNPs and proteins first interact, net charge appears to dominate. However, as citrate molecules are displaced by protein, the surface chemistry changes, and the energetics of binding becomes far more complex. In this case, we observed that GB3 is able to displace ubiquitin at intermediate time scales, even though it has a lower net charge. The thermodynamic model for binding developed here could be the first step toward predicting the binding behavior in biological fluids, such as blood plasma.

Surface Plasmon Resonance, Formation Mechanism, and Surface Enhanced Raman Spectroscopy of Ag+-Stained Gold Nanoparticles

“…Indeed, AuNP surface chemistries, including ligand interfacial interactions of plasmonic AuNPs, have evolved as one of the most active research areas due to their importance in essentially every aspect of AuNP applications (Perera et al, 2016a,b; Athukorale et al, 2018). The most common ligands used for AuNP surface functionalization are proteins (Vangala et al, 2012; Siriwardana et al, 2013; Wang et al, 2016; Woods et al, 2016), organothiols (Ansar et al, 2011, 2013b, 2014), and thiolated chemicals such as poly(ethylene glycol) (Siriwardana et al, 2014).…”

Protein Interactions with Nanoparticle Surfaces: Highlighting Solution NMR Techniques

“…Characterization by both 1D and 2D NMR spectroscopy suggests that Ubq and GB3 have a site‐specific orientation on 15 nm citrate AuNPs . In both of these cases, binding is in very slow exchange, allowing the quantification of adsorbed protein .…”

“…Relative concentrations are calculated with respect to the standard, which can be compared to a sample with no NPs in order to obtain absolute concentrations. This method was used to measure the competition between the wild‐type (WT) GB3 protein and all of its lysine to alanine variants, allowing an investigation of which lysine residues matter most in NP binding . The advantage of the 1D half‐filter experiments is its ability to detect two protein‐NP mixtures in situ in the same sample.…”

“…Different analytical tools have been employed to characterize biocorona formation on NP surfaces. These techniques include surface plasmon resonance (SPR), transmission electron microscopy (TEM), mass spectrometry (MS) based proteomics, chromatography, fluorescence spectroscopy, CD spectroscopy, electrophoresis (gel, capillary, and 2D electrophoresis), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR) . These techniques have been used to identify the structural and conformational changes of various proteins onto the NP surface.…”

Interaction between cyanine dye IR‐783 and polystyrene nanoparticles in solution

“…Nuclear Magnetic Resonance (NMR) is the most versatile tool for determining molecular structure. NMR is being increasingly used in recent years to examine molecules that interact noncovalently with nanoparticle surfaces . Previously, we have used Saturation‐Transfer Difference (STD) NMR to examine the binding between small molecules and the surface of polystyrene (PS) nanoparticles.…”