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Author ORCID Identifier
https://orcid.org/0000-0001-9874-8567
AccessType
Open Access Dissertation
Document Type
dissertation
Degree Name
Doctor of Philosophy (PhD)
Degree Program
Chemistry
Year Degree Awarded
2022
Month Degree Awarded
September
First Advisor
Min Chen
Subject Categories
Biochemistry | Biophysics | Biotechnology
Abstract
Protein nanopores are pore-forming proteins which have been developed as single-molecule biosensors. Due to the high sensitivity, selectivity, label-free and real-time detection methodology, protein nanopores have been used for a wide variety of applications. In this dissertation, we use ClyA nanopore to investigate kinase conformational dynamics and develop a kinase/nanopore system for the specific detection of kinase allosteric inhibitors. Besides, we engineer OmpG nanopore to be a sensor for nucleic acid detection. Protein kinases play essential roles in cellular regulation by catalyzing the phosphorylation of target proteins and are promising drug targets. The conformational dynamics are critical for kinase functions. A comprehensive description of kinase dynamics requires the quantification of relative probabilities of different conformational states (thermodynamics) and transition rates between the states (kinetics). First, we use ClyA nanopore tweezers to assess the conformational dynamics of Abl kinase domain. Analysis of kinase-substrate and kinase-inhibitor interactions uncovers the functional roles of relevant conformational states. Furthermore, we obtain the energy landscape of Abl kinase by quantifying the population and transition rates of the conformational states. These results extend the view on the dynamic nature of Abl kinase. Second, we develop a generalizable label-free assay based on the Abl/ClyA nanopore system for the specifical detection of Abl allosteric inhibitors. Third, we further test the robustness of the ClyA nanopore in kinase dynamics studies with another kinase, Src kinase. Src can also be trapped inside the pore and shows different conformational states (the functional roles need to be further studied), suggesting nanopore tweezers can be used as an efficient tool for other members of the kinome. Nucleic acids are biopolymers which are essential to all know forms of life. Many nucleic acids have been implicated as important biomarkers. To assist disease diagnosis, we design an OmpG-based sensor by conjugating a short ssDNA as the probe to OmpG nanopore and show the OmpG-ssDNA sensor can form functional pore and successfully detect ssDNA analytes baring a complementary region with the ssDNA probe, although the system needs to be further optimized to resolve ssDNA targets vary in only several nucleotides.
DOI
https://doi.org/10.7275/31032142
Recommended Citation
LI, FANJUN, "Investigation of kinase conformational dynamics and analytes detection with protein nanopore" (2022). Doctoral Dissertations. 2650.
https://doi.org/10.7275/31032142
https://scholarworks.umass.edu/dissertations_2/2650
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.