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

Nanobody-based quenchbodies are emerging as powerful tools for the sensitive and specific detection of biomolecular targets. These fluorescent sensors contain tryptophan residues in the antigen-binding site that can quench (darken) a fluorophore attached via a flexible linker. Upon antigen binding the fluorophore is displaced, resulting in an increase in intensity. Recent quenchbodies developed against the inflammatory cytokine interleukin-6 (IL-6) exhibit variable fluorescence responses despite comparable antigen binding. To investigate the molecular basis of this discrepancy, we combined data-driven structural prediction with physics-based molecular dynamics simulations. Using a benchmark dataset of nanobody–lysozyme complexes, we first established that HelixFold3 provides more accurate structural predictions than AlphaFold3. Leveraging this finding, we used HelixFold3 to model the structures of the IL6-binding quenchbodies. Our analyses revealed that poor responders exhibit solvent-exposed tryptophan residues upon antigen binding, notably tryptophan 110, allowing fluorophore quenching and reduced fluorescence. In contrast, good responders adopt conformations in which these tryptophan residues are largely buried, resulting in a more pronounced fluorescence increase upon antigen binding. Together, these results illustrate that integrating structural predictions with molecular dynamics simulations offer a powerful framework for decoding quenchbody mechanisms and advancing the rational development of highly responsive fluorescent sensors.