Binding-Induced Stabilization Measured on the Same Molecular Protein Substrate Using Single-Molecule Magnetic Tweezers and Heterocovalent Attachments
| Autor: | Narayan Dahal, Ionel Popa, Annie Eis, Joel Nowitzke |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Protein Folding
Magnetic tweezers Ligands 010402 general chemistry Mechanotransduction Cellular 01 natural sciences 0103 physical sciences Materials Chemistry Nanotechnology Molecule Physical and Theoretical Chemistry Mechanotransduction Mechanical Phenomena 010304 chemical physics biology Chemistry Magnetic Phenomena Force spectroscopy Adhesion 0104 chemical sciences Surfaces Coatings and Films Dissociation constant Protein L Biophysics biology.protein Protein folding |
| Zdroj: | The Journal of Physical Chemistry B. 124:3283-3290 |
| ISSN: | 1520-5207 1520-6106 |
| DOI: | 10.1021/acs.jpcb.0c00167 |
| Popis: | Binding-induced mechanical stabilization plays key roles in proteins involved in muscle contraction, cellular mechanotransduction, or bacterial adhesion. Because of the vector nature of force, single-molecule force spectroscopy techniques are ideal for measuring the mechanical unfolding of proteins. However, current approaches are still prone to calibration errors between experiments and geometrical variations between individual tethers. Here, we introduce a single-molecule assay based on magnetic tweezers and heterocovalent attachment, which can measure the binding of the substrate-ligand using the same protein molecule. We demonstrate this approach with protein L, a model bacterial protein which has two binding interfaces for the same region of kappa-light chain antibody ligands. Engineered molecules with eight identical domains of protein L between a HaloTag and a SpyTag were exposed to repeated unfolding-refolding cycles at forces up to 100 pN for several hours at a time. The unfolding behavior of the same protein was measured in solution buffers with different concentrations of antibody ligands. With increasing antibody concentration, an increasing number of protein L domains became more stable, indicative of ligand binding and mechanical reinforcement. Interestingly, the dissociation constant of the mechanically reinforced states coincides with that measured for the low-avidity binding interface of protein L, suggesting a physiological role for the second binding interface. The molecular approach presented here opens the road to a new type of binding experiments, where the same molecule can be exposed to different solvents or ligands. |
| Databáze: | OpenAIRE |
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