Structure and dynamics underlying elementary ligand binding events in human pacemaking channels.

Area of Science:

• Biophysics
• Molecular Biology
• Cardiology

Background:

• Cellular signaling relies on molecular recognition, but ensemble studies obscure underlying mechanisms.
• Single-molecule studies are limited by diffraction resolution at relevant concentrations.

Purpose of the Study:

• To directly observe binding dynamics at individual cyclic nucleotide-binding domains (CNBDs) of human pacemaker ion channels.
• To resolve the distinct mechanistic steps in cyclic nucleotide regulation of these channels.

Main Methods:

• Combined zero-mode waveguides with fluorescence resonance energy transfer (FRET).
• Observed single CNBDs from human pacemaker ion channels.

Main Results:

• Resolved dynamics of multiple binding steps: initial selection of a receptive conformation and ligand-induced isomerization.
• X-ray structure and simulations showed isomerization involves local and global transitions.

Conclusions:

• Revealed fundamental mechanisms of ligand regulation in pacemaker channels.
• The approach is applicable to studying weak-binding interactions in various signaling processes.