Three-State Unfolding of Telomeric G-Quadruplexes through Conformational Switching in Crowded Cell-like Conditions

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Telomeric G-quadruplexes (G4s) are crucial for telomere elongation and regulation.
• The crowded intracellular environment significantly impacts G4 stability and dynamics.
• The molecular mechanisms of G4 unfolding in cellular conditions are not well understood.

Purpose of the Study:

• Investigate the thermal unfolding of human telomeric G4 sequences.
• Elucidate the influence of molecular crowders on G4 unfolding pathways.
• Understand G4 structural dynamics in cell-like environments.

Main Methods:

• Temperature-dependent circular dichroism (CD) spectroscopy.
• Singular value decomposition and multivariate curve resolution alternating least-squares (MCR-ALS).
• Well-tempered metadynamics simulations.

Main Results:

• In KCl, telomeric G4s show two-state unfolding (hybrid to random coil).
• Hydrophobic crowders induce a three-state unfolding pathway with a parallel G4 intermediate.
• Hydrophilic crowders have minimal impact on the unfolding pathway.

Conclusions:

• Hydrophobic crowders stabilize the parallel G4 topology through preferential interactions.
• Findings provide molecular insights into G4 unfolding in crowded cellular environments.
• This research aids in understanding complex telomere elongation processes.