The cardiac Ca2+-sensitive regulatory switch, a system in dynamic equilibrium.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Cardiac muscle regulation involves a Ca(2+)-sensitive switch, a complex of troponin C (TnC) and troponin I (TnI).
• Understanding the allosteric communication within this complex is crucial for cardiac function.
• Previous studies suggest a two-step activation mechanism involving Ca(2+) and TnI.

Purpose of the Study:

• To elucidate the mechanistic role of troponin I (TnI) in the allosteric activation of the cardiac muscle regulatory switch.
• To quantitatively model the dynamic transitions and energy landscape of the TnC-TnI complex.
• To determine the thermokinetic parameters governing the opening of the regulatory switch.

Main Methods:

• Time-resolved equilibrium Förster resonance energy transfer (FRET) measurements.
• Stopped-flow FRET measurements for activation and deactivation kinetics.
• Global analysis of time-resolved, stopped-flow, and Ca(2+)-titration data using a quantitative dynamic model.
• Temperature-dependent FRET experiments to determine thermodynamic parameters.

Main Results:

• Ca(2+)-induced priming is the rate-limiting step in activation, preceding TnI-dependent opening.
• Closing is the rate-limiting step during deactivation.
• TnI actively promotes the opening of the regulatory switch.
• An incompletely deactivated state creates an accessory activation pathway.
• Ca(2+)-bound states exhibit a mixture of open and primed-closed conformers.

Conclusions:

• The study provides a comprehensive dynamic model of TnC-TnI allostery, detailing distance and free energy changes.
• The rate-limiting steps for both activation (priming) and deactivation (closing) have been identified.
• The findings reveal complex intermediate states and pathways influencing cardiac muscle regulation.
• Complete thermokinetic parameters for the opening process were determined, offering insights into the energy landscape.