Mechanistic Insights into Ultrasmall Gold Nanoparticle–Protein Interactions through Measurement of Binding Kinetics

Gold nanoparticles (NPs) in the ultrasmall size regime provide a new paradigm in the way that nanomaterials can be used to regulate protein structure and function. However, the rational design of ultrasmall NPs as viable synthetic effectors of protein function requires detailed quantitative understanding of their biomolecular interactions. Herein, we focused on the kinetics of NP–protein complexation—an often neglected factor in studies at the bio-nanointerface—to gain molecular-level insights into mechanisms of interactions. The protein α-thrombin and ultrasmall gold NPs coated with p-mercaptobenzoic acid (AuMBA) and glutathione (AuGSH) were used as model systems in our studies. Binding kinetics was quantified by surface plasmon resonance (SPR) biosensing and stopped-flow spectroscopy. The results revealed strong and weak interactions of AuMBA and AuGSH toward thrombin (KD ∼30 nM and 20 μM, respectively), as well as “fast” and “slow” association kinetics (kₒₙ ∼10⁶–10⁷ and 10⁴ M–¹ s–¹, respectively). The significantly smaller kₒₙ for AuGSH implied the presence of a larger energy barrier along the association pathway, presumably related to the penalty required to remove interfacial ions. Analysis of dissociation reactions revealed that thrombin interactions with both NPs formed transient, weakly adhesive complexes characterized by short residence times (tᵣ = 1/kₒff ∼0.1–16 s). Interestingly, the reverse reactions were best described by multiple dissociation processes suggesting a heterogeneous population of complexes stabilized to different extent.