Single-molecule force spectroscopy (SMFS) is a powerful tool for studying protein folding, as the application of force tilts the folding energy landscape in a controlled manner that can be theoretically modeled. However, traditional SMFS assays offer temporal resolution >50 μs, significantly slower than a typical globular protein’s transition path time (1-10 μs)—the time it takes to cross the activation barrier. Achieving SMFS with 1-μs temporal resolution therefore opens the door to quantifying a protein’s folding trajectory as it crosses an activation barrier. Additionally, improved temporal resolution enhances the ability to resolve fleetingly populated intermediates. Recent theoretical work also raises the concern that slowly responding force probes obscure true molecular trajectories. A recent study demonstrates atomic force microscopy (AFM)-based SMFS with 1-μs temporal resolution using ultrashort (L = 9 µm) cantilevers on a custom-built Ando-style AFM. Notwithstanding the pioneering nature of this work, ultrashort cantilevers are not optimized for force spectroscopy. First, their high stiffness results in significant force drift. Further, these cantilevers are underdamped (Q > 0.5) in liquid near a surface, leading to a high-frequency force modulation that is not accounted for in standard force spectroscopy analysis. In order to overcome these limitations, we used a focused-ion beam (FIB) to micro-machine ultrashort cantilevers, generating softer cantilevers that maintained a 1-μs response time but offered improved force stability and precision. Moreover, these modifications led to overdamped or critically damped cantilever motion. To leverage the ease-of-use of a commercial AFM, we retrofitted our commercial instrument with a custom detection module that featured a 3-μm circular spot size to detect modified ultrashort cantilevers. We then used these cantilevers to mechanically unfold a polyprotein of NuG2, a well characterized α/β protein, with a state-of-the-art combination of temporal resolution and force precision. Ongoing work is measuring the computationally designed protein α3D, which has a reported transition path time of 12 µs. More generally, we expect that these enhancements to AFM-based SMFS on a commercial AFM will accelerate high precision studies of protein folding on the 1-µs timescale.

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