Exploring the equilibrium competition binding assay and ligand-induced current noise at the single-molecule level
Date of Award
Doctor of Philosophy (PhD)
Ion Channel;Protein Dynamics;Protein Engineering;Single-Molecule Electrophysiology
Physical Sciences and Mathematics | Physics
This dissertation details a structurally directed strategy for using FhuA, an engineered membrane protein nanopore, as a membrane transporter for real-time single-molecule stochastic sensing of protein-protein interactions (PPIs). For this proof-of-concept investigation, the Barnase-Barstar (Bn-Bs) PPI system serves as a case in point. In our work, we investigated the frequency-domain analysis of current noise fluctuations obtained using a synthetic protein receptor. On the extramembranous side, the receptor was incorporated to the engineered transmembrane protein pore, a uniform and silent single-channel electrical current was easily detected. Nonetheless, a flexible and unstructured polypeptide extension engineered at the receptor's N terminus resulted in a significant amplification in the amplitude of current noise fluctuations. We also discovered that adding a protein ligand to the buffer solution considerably reduced the large-amplitude current fluctuations. We infer that the affinity of receptor-ligand interactions influences the regulation of current noise by an exogenous protein ligand. This result is consistent with theoretical expectations of protein channel-ligand interaction noise spectra. Our research on the Barstar mutants E76A Bs and D39A Bs allowed us to further evaluate the biosensor's sensitivity and selectivity. Our findings revealed that the biophysical approach can study medium and weak affinity PPI as well as detect two Bs variants interacting with the same binding site. We have challenged the selectivity and sensitivity of the nanopore sensor by simultaneously detecting both Bs WT and E76A Bs, or Bs WT and D39A Bs, later in the study, the roles of the inhibitor ligand and the competitor ligand were switched. We discussed the interaction of the inhibitor with a receptor, and the competitor ligand with the same receptor. We quantitatively investigated how the frequency of inhibitor ligand binding events with the receptor is influenced by the concentration of competitor ligands. In this study, we were able to separate two variants and confirm them by performing a competition by receptor occupancy type assay. We were also able to explain the dynamic behavior of competitive PPIs at single-event resolution involving ligand mixtures in a more mathematical and theoretical way.
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Sun, Jiaxin, "Exploring the equilibrium competition binding assay and ligand-induced current noise at the single-molecule level" (2024). Dissertations - ALL. 1844.