Binding kinetics of ultrasmall gold nanoparticles with proteins

Abstract

Synthetic ultrasmall nanoparticles (NPs) can be designed to interact with biologically active proteins in a controlled manner. However, the rational design of NPs requires a clear understanding of their interactions with proteins and the precise molecular mechanisms that lead to association/dissociation in biological media. Although much effort has been devoted to the study of the kinetics mechanism of protein corona formation on large NPs, the nature of NP-protein interactions in the ultrasmall regime is radically different and poorly understood. Using a combination of experimental and computational approaches, we studied the interactions of a model protein, CrataBL, with ultrasmall gold NPs passivated with p-mercaptobenzoic acid (AuMBA) and glutathione (AuGSH). We have identified this system as an ideal in vitro platform to understand the dependence of binding affinity and kinetics on NP surface chemistry. We found that the structural and chemical complexity of the passivating NP layer leads to quite different association kinetics, from slow and reaction-limited (AuGSH) to fast and diffusion-limited (AuMBA). We also found that the otherwise weak and slow AuGSH-protein interactions measured in buffer solution are enhanced in macromolecular crowded solutions. These findings advance our mechanistic understanding of biomimetic NP-protein interactions in the ultrasmall regime and have implications for the design and use of NPs in the crowded conditions common to all biological media.

Allosteric inhibition of α-thrombin enzymatic activity with ultrasmall gold nanoparticles

“…AuMBA–thrombin binding affinity was therefore measured by surface plasmon resonance (SPR) by flowing AuMBA over a streptavidin sensor surface containing immobilized thrombin. Importantly, the spatial constraints of the surface immobilization in SPR hinder aggregation and thereby allow the measurement of binding affinity 49. Immobilization was accomplished through a biotin–PPACK label attached to thrombin's active site; therefore, the protein was uniformly oriented on the sensor surface with both exosites presumably exposed to allow interactions with AuMBA.…”

“…Immobilization was accomplished through a biotin–PPACK label attached to thrombin's active site; therefore, the protein was uniformly oriented on the sensor surface with both exosites presumably exposed to allow interactions with AuMBA. The AuMBA–thrombin interactions were analyzed with a continuous surface-site distribution model,40,41,49,50 which can allow the distinction between mono- and multi-valently attached AuMBA populations based on their different dissociation kinetics. It can also naturally accommodate multiple binding sites on the immobilized thrombin.…”

High Surface Reactivity and Biocompatibility of Y2O3 NPs in Human MCF-7 Epithelial and HT-1080 Fibro-Blast Cells

“…Protein adsorption to NPs is a dynamic process in fluids be it culture media, blood or other bio fluids independent of nature of the NPs either polymeric [28] or metallic [30]. One of the most notable alterations when NP is under biological fluids is the formation of NP-protein corona [28].…”

Identification of the binding site between bovine serum albumin and ultrasmall SiC fluorescent biomarkers

“…This picture is in agreement with the binding kinetics results for negatively charged ultrasmall gold nanoparticles. 84 Indeed, nanoparticles between 1 and 2 nm show transient particle-protein associations with lifetimes shorter than gel band resolution times in gel assays as a consequence of repetitive binding and unbinding processes. 31 As Boselli et al pointed out, 31 this transient effect is extremly size dependent.…”