Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force...

Area of Science:

• Biophysics
• Molecular Biology
• Biosensing

Background:

• Avidin-based proteins are engineered for specific ligand binding.
• Testosterone binders are crucial for various diagnostic and research applications.
• Understanding molecular binding dynamics is key to developing sensitive biosensors.

Purpose of the Study:

• To characterize the binding and dissociation properties of two novel testosterone-binding proteins, sbAvd-1 and sbAvd-2.
• To compare the molecular recognition capabilities of sbAvd-1 and sbAvd-2 towards testosterone and biotin.
• To evaluate the potential of these proteins in biosensing applications based on their binding behavior.

Main Methods:

• Utilized molecular recognition force spectroscopy, a biosensing atomic force microscopy technique.
• Tethered biotin or testosterone to an atomic force microscopy probe and immobilized the binding proteins on a surface.
• Analyzed ligand-receptor complex formation and rupture at varying pulling velocities to determine dissociation rates (k(off)) and energy landscape distances (x(β)).

Main Results:

• Kinetic off-rates (k(off)) for both proteins and ligands were found to be similar.
• Energy landscape distances (x(β)) and complex formation probabilities varied significantly between the protein-ligand pairs.
• Competitive binding experiments indicated decreased cross-reactivity for sbAvd-2 with testosterone and biotin.

Conclusions:

• The study elucidates the distinct binding characteristics of sbAvd-1 and sbAvd-2.
• sbAvd-2 exhibits promising reduced cross-reactivity, enhancing its potential for specific testosterone sensing.
• These findings are expected to advance the development of testosterone-based biosensing technologies.