| Full text | |
| Author(s): |
Total Authors: 3
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| Affiliation: | [1] Univ Fed Sao Paulo, Dept Biochem, BR-04044020 Sao Paulo, SP - Brazil
Total Affiliations: 1
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| Document type: | Journal article |
| Source: | NANOSCALE; v. 12, n. 37, p. 19230-19240, OCT 7 2020. |
| Web of Science Citations: | 1 |
| Abstract | |
To date, extensive effort has been devoted toward the characterization of protein interactions with synthetic nanostructures. However, much remains to be understood, particularly concerning microscopic mechanisms of interactions. Here, we have conducted a detailed investigation of the kinetics of nanoparticle-protein complexation to gain deeper insights into the elementary steps and molecular events along the pathway for complex formation. Toward that end, the binding kinetics betweenp-mercaptobenzoic acid-coated ultrasmall gold nanoparticles (AuMBA) and fluorescently-labeled ubiquitin was investigated at millisecond time resolution using stopped-flow spectroscopy. It was found that both the association and dissociation kinetics consisted of multiple exponential phases, hence suggesting a complex, multi-step reaction mechanism. The results fit into a picture where complexation proceeds through the formation of a weakly-bound first-encounter complex with an apparent binding affinity (K-D) of similar to 9 mu M. Encounter complex formation is followed by unimolecular tightening steps of partial desolvation/ion removal and conformational rearrangement, which, collectively, achieve an almost 100-fold increase in affinity of the final bound state (apparentK(D)similar to 0.1 mu M). The final state is found to be weakly stabilized, displaying an average lifetime in the range of seconds. Screening of the electrostatic forces at high ionic strength weakens the AuMBA-ubiquitin interactions by destabilizing the encounter complex, whereas the average lifetime of the final bound state remains largely unchanged. Overall, our rapid kinetics investigation has revealed novel quantitative insights into the molecular-level mechanisms of ultrasmall nanoparticle-protein interactions. (AU) | |
| FAPESP's process: | 19/04372-6 - Interactions of ultrasmall gold nanoparticles with proteins and cells |
| Grantee: | Alioscka Augusto Sousa |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 16/25535-2 - Biointeractions of gold nanoparticles: influence of macromolecular crowding and soft interactions |
| Grantee: | Rodrigo da Silva Ferreira |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |