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The Equilibrium Binding Constant and Binding Strength02:18

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:

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High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy
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Direct Measurement of Single-Molecule Ligand-Receptor Interactions.

K-T Lam1, E L Taylor1, A J Thompson2

  • 1Department of Chemistry, Washington State University, PO Box 644630, Pullman, Washington 99164-4630United States.

The Journal of Physical Chemistry. B
|August 14, 2020
PubMed
Summary
This summary is machine-generated.

This study directly measures single-molecule binding events for ligand-gated ion channels, revealing key kinetic rates and free energy changes for ATP binding to P2X1 receptors. These findings advance our understanding of receptor pharmacology and protein interactions.

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Area of Science:

  • Pharmacology and biophysics
  • Molecular and cellular biology
  • Structural biology

Background:

  • Ligand-receptor interactions are crucial in pharmacology, with agonist binding to ligand-gated ion channels inducing conformational changes that open ion pores.
  • Previous studies inferred binding kinetics from electrophysiological data, but direct measurement of single-molecule binding events remained elusive.

Purpose of the Study:

  • To directly measure single-molecule binding events and kinetics of ligand-receptor interactions.
  • To determine rate constants and free energy changes associated with ligand binding and allosteric motion.
  • To establish the location and stochastic nature of ligand binding states on the P2X1 receptor.

Main Methods:

  • Utilized super-resolution Points Accumulation for Imaging in Nanoscale Topography (PAINT) to visualize ATP binding to P2X1 receptors.
  • Employed time-resolved single-molecule interaction analysis to quantify elementary rate constants and thermodynamic forces.
  • Calculated forward and reverse rates between ligand-binding states linked to allosteric motion and pore formation.

Main Results:

  • Reported the first direct measurements of single-molecule binding events for ligand-gated ion channels.
  • Quantified forward (kα = 1.41 sec⁻¹) and reverse (kβ = 0.32 sec⁻¹) rates for transitions between weak and strong ligand-binding states.
  • Determined the free energy change (3.7 kJ/mol) for the critical step linking binding to allosteric motion and ion pore formation.

Conclusions:

  • Direct single-molecule measurements provide unprecedented insights into ligand-receptor binding kinetics and thermodynamics.
  • The methods are applicable to various ligand-gated ion channels and other membrane proteins.
  • This work establishes the location of binding states and the stochastic nature of ligand interaction with the P2X1 receptor.