Date of Award
Doctor of Philosophy (PhD)
Nanopore, Crowding, peptide, single molecule
Physical Sciences and Mathematics
In the last three decades nanopore sensing has emerged as a powerful technique for DNA sequencing and bio-molecular sensing. One of the great appeals of this technique is that it usually does not require any labeling or chemical modification. Additionally, its ability to examine a single molecule in real time is a rare feature which is not achievable with most of other techniques. In recent years, great efforts have been made to design a nanopore sensor for sampling peptide-protein and protein- protein interaction. To pave the way for designing a high-throughput wearable nanopore sensor, we will investigate a few of the parameters that can potentially influence the sensitivity and reproducibility of a sensor and ultimately the obtained results.
The first parameter to consider is the cellular crowded environment. Although, cellular crowding is known to have significant implications in the kinetics and equilibrium of biopolymer interactions, it has been poorly investigated in nanopore sensing. Here, we show that the presence of polyethylene glycol (PEG) as an inert molecule affects the polypeptide-protein interaction. We provide an experimental evidence showing that less partitioning PEG above a critical value amplifies the association rate constant and reduces the dissociation rate constant. Our data is consistent with the lower diffusion rate and enhanced depletion-attraction force between a polypeptide and transmembrane protein pore at an elevated crowding concentration.
The second factor to investigate, is how the structural design of a nanopore sensor can affect its reproducibility and sensitivity. In the designed sensor, which is capable of sampling a biomolecule of interest outside a nanopore, a folded protein is genetically attached to the transmembrane protein. Therefore, the first question is, how far this protein must be positioned to remain in the sensing region of a nanopore and how it alters the conformational state of the sensor. The second question is, how one can control the orientation of the folded receptor domain, so that it remains accessible to the solution. We experimentally address these questions in chapter 3.
Ghahari Larimi, Motahareh, "Investigating the parameters affecting the sensitivity of a protein nanopore sensor" (2020). Dissertations - ALL. 1214.