Developing membrane protein based nanopore for real-time sensing of transient protein-protein interaction (PPI) at the single molecule level
Date of Award
Doctor of Philosophy (PhD)
Nanopore, Single molecule
Physical Sciences and Mathematics
Understanding the real-time kinetics of protein-protein interactions (PPI) at the single-molecule level is essential for both a better mechanistic and quantitative knowledge of cellular regulatory processes, as well as for biomedical applications including drug design, biosensors, and clinical molecular diagnostics. To this end, the chapters in this thesis describe the structurally guided approach to employ FhuA, an engineered membrane protein pore, as an electrical transducer for real-time stochastic sensing and analysis of PPI at the single-molecule level. Herein, we used barnase-barstar (Bn-Bs) as a model PPI system for such proof-of-concept study. We first show that FhuA can tolerate genetic engineering and retain its pore-forming ability after genetically incorporating the Bn to the FhuA polypeptide chain. We then genetically incorporated a negatively charged unstructured peptide to this newly designed nanopore to convert electrically silent Bs binding signal into an active visible signal. This new and unique approach of increasing the temporal resolution of detection is different from the traditional method of charge mutation on protein nanopore. We then confirmed Bn-Bs binding by performing dose response test with no effect on the signal-to-noise ratio of the nanopore sensor. To further confirm the binding interface and test the sensitivity of the sensor, we studied Bs mutant, D39A Bs. This Bn-D39A Bs interaction turns out to be an extremely weak PPI, yielding a koff of 281 s-1. This finding is important as none of the available biophysical approach can study weak PPI with such high koff value without a major limitation. In Appendix I, it is also shown that we can detect medium affinity PPI by studying another Bs mutant, E76A Bs. Later in the studies, we have challenged the sensitivity and selectivity of the sensor by simultaneously detecting both Bs variants and confirming it by performing a competition by receptor occupancy type assay. After this, we took a big leap and performed the same Bn-Bs interaction study, except, this time in the 5% fetal bovine serum (FBS). The goal was to assess not just the strength and weakness of our nanopore sensor but for the whole sensing platform in general. Surprisingly, we were not only able to quantitate the specific interaction (Bn-Bs) but also the non-specific interaction. This is advantageous, because it determines to what accuracy one can quantify the kinetic parameter for a PPI with koff greater than 1 s-1 using this sensing approach. In this part of the study, we also show how one can use voltage and serum heat inactivation as part of the experimental design to control the non-specific interaction, thereby boosting the overall signal-to-noise ratio of the nanopore sensor. In Chapter Four, we explored the FhuA nanopore and its inner dimension to conductance relation. Here, we show that the presence of hexahistidine tag at the terminus of FhuA can affect its conformation. We confirm the discovery of new conductance state of FhuA, which is in agreement with its actual dimension estimated from the crystal structure of wt FhuA. Such a change in the confirmation of a membrane protein could not be studied and detected using any other biochemical and biophysical technique. Overall, we have explored FhuA, a β-barrel membrane protein scaffold, as a nanopore sensor for numerous applications and made a significant contribution to the field of membrane protein engineering.
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Thakur, Avinash Kumar, "Developing membrane protein based nanopore for real-time sensing of transient protein-protein interaction (PPI) at the single molecule level" (2019). Dissertations - ALL. 1017.