Mechanistic Insights into Nanobody-Based Quenchbody Sensing via Structural Modeling and Molecular Simulations

Area of Science:

• Biochemistry
• Structural Biology
• Biotechnology

Background:

• Nanobody-based quenchbodies are fluorescent biosensors utilizing tryptophan quenching for target detection.
• Interleukin-6 (IL-6) targeting quenchbodies show inconsistent fluorescence responses, necessitating mechanistic investigation.

Purpose of the Study:

• To elucidate the structural basis for variable fluorescence responses in IL-6 quenchbodies.
• To guide the rational design of improved nanobody-based fluorescent biosensors.

Main Methods:

• Benchmarking HelixFold3 and AlphaFold3 for nanobody structure prediction.
• Utilizing HelixFold3 for data-driven structural prediction of nanobody-IL-6 complexes.
• Employing molecular dynamics simulations to analyze protein structures and dynamics.

Main Results:

• HelixFold3 demonstrated higher accuracy than AlphaFold3 in predicting nanobody-lysozyme complexes.
• Low and non-responding IL-6 quenchbodies retain solvent-exposed tryptophan residues after antigen binding, causing continued quenching.
• High-responding IL-6 quenchbodies bury tryptophan residues upon antigen binding, reducing quenching and enhancing fluorescence signal.

Conclusions:

• The study decodes the mechanism behind variable quenchbody fluorescence responses.
• Integrating structural prediction and molecular dynamics simulations is effective for understanding and optimizing biosensor function.
• Findings provide a basis for designing more sensitive and reliable nanobody-based fluorescent biosensors.