Single molecule fluorescence studies of surface-adsorbed fibronectin

Area of Science:

• Biophysics
• Cell Biology
• Materials Science

Background:

• The extracellular matrix protein fibronectin regulates crucial cell functions like adhesion and migration.
• Previous studies focused on average protein conformations, potentially overlooking the significance of outliers.

Purpose of the Study:

• To investigate the conformational states and dynamics of individual fibronectin molecules adsorbed to a surface.
• To understand how conformational outliers and fluctuations influence cell behavior.

Main Methods:

• Single molecule imaging and spectroscopy techniques were employed.
• Single-step photobleaching confirmed single-molecule sensitivity.
• Single-pair fluorescence resonance energy transfer (FRET) and fluorescence polarization measured conformational dynamics.

Main Results:

• Fibronectin molecules displayed conformational fluctuations in peak wavelength and FRET efficiency upon surface adsorption.
• A fraction of molecules exhibited folded conformations allowing for energy transfer between labeled sites.
• Fluctuations were attributed to both fluorophore orientation changes and fibronectin conformational dynamics on the second timescale.

Conclusions:

• Individual fibronectin molecules exist in a range of conformational states with significant temporal fluctuations.
• These dynamic conformational properties are likely crucial for fibronectin's role in cell adhesion and migration.
• The study highlights the importance of considering single-molecule dynamics beyond average properties.