Nanoparticles (NPs) can be designed to interact controllably with proteins and regulate protein function. Owing to their small size (comparable to proteins), colloidal stability and protein-like surface chemistries, ultrasmall gold nanoparticles (AuNPs) can provide attractive possibilities in the creation of novel synthetic protein receptors. Herein we investigate the molecular interactions of α-thrombin with ultrasmall AuNPs, and verify the impact of these interactions on α-thrombin’s enzymatic activity.<b> </b>AuNPs had a core diameter of 2 nm and were passivated with negatively charged 4-mercaptobenzoic acid ligands. All experiments were carried out in Tris buffer supplemented with 100 mM NaCl. Fluorescence titration quenching revealed the AuNPs interacted with α-thrombin. Moreover, through the use of competing exosite-binding ligands, the major sites of NP binding on α-thrombin were found to be exosites I and II. Thrombin activity in the presence of NPs decreased by only ~ half, despite saturating concentrations of NPs. Taken together, these results support electrostatic-based interactions between the negatively charged NPs and the positively charged exosites. Most strikingly, the AuNPs were able to allosteric regulate – without fully inhibiting – thrombin activity. The partial inhibition observed is likely the result of subtle NP-induced conformational changes propagated from α-thrombin’s exosites towards its active site, which, presumably, only partly disturb α-thrombin’s catalytic efficiency.<b> </b>In conclusion, ultrasmall AuNPs were shown to bind selectively to α-thrombin’s exosites, and to perform as synthetic receptors for modulating enzyme activity.

Regulation of α-thrombin enzymatic activity through interactions with gold nanoparticles

254th National Meeting (2017)

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