Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation.

Area of Science:

• Biophysics
• Structural Biology
• Molecular Biology

Background:

• The Per-Arnt-Sim (PAS) domain is a conserved protein module crucial for cellular signaling and sensory functions.
• Activation mechanisms are often linked to conformational changes in helices adjacent to the PAS domain.

Purpose of the Study:

• To investigate the mechanical properties and conformational changes of the PAS domain in photoactive yellow protein (PYP) upon activation at the single-molecule level.
• To determine if the PAS domain itself undergoes unfolding during light-induced activation.

Main Methods:

• Utilized atomic force microscopy (AFM) to mechanically unfold single molecules of Cys-linked PYP multimers.
• Employed steered molecular dynamics (SMD) simulations on PYP mutants to analyze regional stability and structural changes.
• Measured protein extension and stability changes under illumination versus dark conditions.

Main Results:

• Illumination of PYP led to a ~3 nm extension and ~30% destabilization of the PAS domain.
• AFM and SMD analyses demonstrated anisotropic stability and local structural changes within the PAS domain upon activation.
• Confirmed partial unfolding of the PAS domain itself, challenging previous assumptions.

Conclusions:

• The PAS domain undergoes significant mechanical unfolding upon activation, serving as a direct signaling mechanism.
• Developed a versatile single-molecule technique to map functional conformational changes in proteins.
• Stimulus-induced partial protein unfolding is a viable biological signaling strategy.