Thesis
Stochastic kinetic analysis of single fluorescent ATP molecules binding to P2X1: Markov models containing photophysical corrections
Washington State University
Master of Science (MS), Washington State University
08/2020
DOI:
https://doi.org/10.7273/000000070
Handle:
https://hdl.handle.net/2376/118966
Abstract
Measuring the kinetics that govern ligand-receptor interactions is fundamental to our
understanding of pharmacology. For ligand-gated ion channels, such as the P2X family, binding
of an agonist triggers allosteric motion and opens an integral ion-permeable pore that allows for
the flow of ions. By mathematically modelling stochastic electrochemical responses with high
temporal resolution (ms), previous studies have been able to infer the rate constants of ligands
binding to the receptor. However, to date, no one has reported a direct measurement of singlemolecule binding events which are vital to how agonists exert their functional effects. For the first
time, we report the direct measurement of the rate constants between various binding states using
ATTO-700 labeled ATP (ATP*) and P2X1 receptors embedded in supported lipid bilayer. This
study used super resolution nanoscale topography (PAINT) to confirm that ATP* specifically
binds to the native sites on P2X1 and single molecule fluorescence time traces to measure
elementary rate constants and thermodynamic driving forces that are specific to the ligand driven
allosteric motion within the receptor. Accounting for the intrinsic dynamics of the ATTO 700 label was critical for measuring the biophysical rate constants. In order to account for blinking and
bleaching of the probe molecule a constrained hidden Markov analysis was developed and
validated through simulation.
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Details
- Title
- Stochastic kinetic analysis of single fluorescent ATP molecules binding to P2X1: Markov models containing photophysical corrections
- Creators
- Kui Ting Lam
- Contributors
- JAMES ALAN BROZIK (Degree Supervisor) - Washington State University, Chemistry, Department ofKERRY W HIPPS (Committee Member) - Washington State University, Chemistry, Department ofURSULA M MAZUR (Committee Member) - Washington State University, Chemistry, Department of
- Awarding Institution
- Washington State University
- Academic Unit
- Chemistry, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University
- Format
- pdf
- Number of pages
- 48
- Identifiers
- 99900590963601842
- Language
- English
- Resource Type
- Thesis