Conformational distribution of surface-adsorbed fibronectin molecules explored by single molecule localization...

Area of Science:

• Biomaterials Science
• Surface Chemistry
• Molecular Biophysics

Background:

• Cell adhesion to biomaterials is mediated by adsorbed proteins.
• Protein conformation changes upon adsorption, affecting function.
• Understanding protein behavior at interfaces is crucial for biomaterial design.

Purpose of the Study:

• To investigate the conformational changes of individual fibronectin molecules at the glass-water interface.
• To determine how surface chemistry and solution conditions influence protein conformation during adsorption.
• To extend high-resolution microscopy techniques for probing biomolecule conformation.

Main Methods:

• Site-specific labeling of fibronectin with Cy3B at four cysteine residues.
• Utilizing stepwise photobleaching in localization microscopy to measure distances between labels.
• Analyzing the conformational distribution of single fibronectin molecules under varying adsorption conditions.

Main Results:

• Fibronectin molecules showed non-uniform and non-linear arrangements at the interface.
• Adsorption conditions, such as denaturants (4 M guanidinium HCl) and surface hydrophobicity, significantly altered fibronectin conformation.
• Denaturing conditions during adsorption increased average distances to 43 nm, while hydrophobic surfaces shifted median distances to 49 nm.

Conclusions:

• The conformation of adsorbed fibronectin is determined during the adsorption process and 'locked-in'.
• Surface chemistry and solution conditions during adsorption critically influence protein structure and function.
• Localization microscopy is a powerful tool for characterizing biomolecules at interfaces.