Ultrasmall gold nanoparticles (NPs; &lt; 3 nm) decorated with suitable surface moieties can resemble protein receptors in both size and surface chemistry. In this context, a detailed characterization of NP-protein interactions is required to understand how NP structure can impact protein function and, ultimately, biological activity. Previous studies on NP-protein interactions have focused on the thermodynamics of binding (e.g., KD), while the importance of kinetics has been largely overlooked. However, dissecting the individual contributions of the reaction rate constants to KD can provide unique insights into the interaction mechanisms. Here, the binding kinetics of two types of ultrasmall NPs (AuMBA and AuGSH) with three distinct proteins (CrataBL, thrombin and ubiquitin) were investigated by means of two different techniques (surface plasmon resonance, SPR, and fluorescence stopped-flow spectroscopy, SF). The SPR measurements revealed much stronger interactions of CrataBL and thrombin towards AuMBA relative to AuGSH (KD ~ 50 nM vs. ~ 10-20 uM). The higher affinity of the proteins towards AuMBA compared to AuGSH was mainly the result of a ~ 100-fold larger kon rate constant (~ 106 M-1s-1 vs. ~ 104 M-1s-1), therefore suggesting that the AuMBA- and AuGSH-protein association reactions followed diffusion- and reaction-controlled processes, respectively. For AuGSH, the reaction-limited mechanism was presumed to originate from an energy barrier caused by the removal of water and ions from the binding interface. SF measurements of AuMBA binding with FITC-labeled ubiquitin, in turn, revealed interesting salt-dependent changes in the binding constants: the apparent kon decreased 10-fold (from ~ 107 to 106 M-1s-1) as the NaCl concentration increased from 5 to 300 mM, whereas the koff remained unchanged. The reason for this behavior is currently being investigated in more detail. Overall, these findings contribute to advance our understanding of the molecular mechanisms of NP-protein interactions.

Molecular insights into ultrasmall nanoparticle-protein interactions through measurement of binding kinetics

2019 Fall National Meeting

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