Protein force spectroscopy using magnetic tweezers: Slow and steady wins the race?

Figures

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  Figure 1. Comparison of single-molecule force spectroscopy techniques and capabilities of magnetic tweezers. (a–d) Common single-molecule force spectroscopy techniques and typical force ranges accessible to them. Schematics of magnetic tweezers (a), optical tweezers (b), and AFM-based (c) single-molecule measurements of a protein of interest. The protein shown is the mechano-protein ddFLN4. (d) Force ranges accessible to the techniques are shown schematically in (a)–(c). Partially adapted from (11). (e and f) Schematic representations of the types of data that are obtained in constant force measurements (such as in magnetic tweezers, e) and in constant loading rate measurements (in particular in AFM-based force spectroscopy, e). Example schematic data are shown for a SARS-CoV-2 tethered ligand construct (12) (Fig. 3). Constant force measurements record molecular extension vs. time at different levels of force and can monitor repeated reversible transitions or irreversible unfolding events. Data are usually summarized as the fraction dissociated or unfolded and as lifetimes vs. force. Constant loading rate measurements record force vs. extension. In the resulting saw-tooth pattern, each…

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  Figure 2. Early pioneering MT work from the Sackmann lab. (a) Central component of the magnetic bead rheometer. A soft iron-core electromagnet (1200-turn coil) delivers forces up to 10,000 pN on a 4.5-μm bead, with the pole tip positioned 10–100 μm from the sample. The assembly is mounted on a microscope stage for precise control. (b) Schematic illustration of a fibronectin-coated bead linked to the cell cytoskeleton through integrin-mediated adhesion. (c) Representative creep and relaxation response of a 4.5-μm bead bound to the membrane of a 3T3 fibroblast via fibronectin-integrin interactions. Although this setup probes collective, cellular-scale mechanics rather than single molecules, it effectively interrogates mechanoprotein responses within the adhesion network, thereby establishing the conceptual foundation for later single-molecule MT studies. Force pulses of 2000 pN were applied for 2.5 s. Reprinted (adapted) with permission from Biophys. J. 1998, 75, 2038–2049 (52). Copyright 1998, The Biophysical Society. Published by Elsevier Inc.

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  Figure 3. Protein force spectroscopy in MTs. (a) Schematic of a vWF dimer in magnetic tweezers. vWF is tethered to the flow cell via an elastin-like polypeptide (ELP) linker and coupled to a paramagnetic bead using a biotin–streptavidin bond. Reference beads correct for drift, and forces are applied via two permanent magnets. Right: three-state transitions in vWF dimers are observed regardless of A2, D4, or A1 domain presence. Extension-time traces at constant forces (7.5–9.0 pN) show distinct transitions, confirmed by histograms fitted with three-term Gaussians, indicating domain-independent conformational switching. Reprinted (adapted) with permission from Blood Adv. 2022, 6 (17), 5198–5209 (142). Copyright 2022, American Society of Hematology (ASH). (b) Left: schematic of the experimental setup. A talin rod construct is tethered between a cover glass and a paramagnetic bead via NTA–His and streptavidin–biotin linkages. Right: force is applied with permanent magnets, and constructs are stretched with or without vinculin VD1. The unfolding force distributions of wild-type talin R1–R3 (top) and the IVVI mutant (bottom) at a 5 pN s−1 loading rate are ≤23 pN. Reprinted (adapted) with…

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  Figure 4. Nanoscale magnetic torquer (m-Torquer) for in vivo neuromodulation of mice. (a) The m-Torquer system combines a circular magnet array (CMA) producing a uniform rotating magnetic field (∇B ≈ 0) with a nanoparticle torque generator that acts as a nanoscale magnetic compass to deliver torque (Fτ) for magnetomechanical signaling. (b) Time-resolved Ca2+ influx measurements show repetitive stimulation of Piezo1 in neurons treated with m-Torquers (red) or WGA-m-Torquers (blue), using 0.5-s pulses at 0.5 Hz with 90° rotations. Results were consistent across three independent experiments. (c) Left: locomotion trajectories of five mice with m-Torquer in the right premotor cortex during CMA rotation (15 cycles, 2 s on/off). Right: control mice without CMA. Reprinted (adapted) with permission from Nat. Mater. 2021, 20, 1029–1036 (183). Copyright 2021, Springer Nature Limited.