Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope

Abstract

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50–1000 μs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-μs temporal resolution was achieved using an ultrashort (L = 9 μm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-μm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-μs response time while eliminating cantilever ringing (Q ≅ 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.

Communication: Transition-path velocity as an experimental measure of barrier crossing dynamics

“…Indeed, the thermal velocity of, say, a 20-200 nucleotide DNA construct is √ k B T/M ∼ 6-20 m/s (where M is its mass). During a time interval of ∆t ∼ 1 µs, comparable to the best time resolution achieved in force spectroscopy studies, 32 the total trajectory length is 6000-20 000 nm, which is nearly 3-4 orders of magnitude longer than a typical …”

Force spectroscopy unveils hidden protein-folding states

“…So they engineered a new detection-laser module for their AFM system to focus the laser to a sufficiently small spot. 5 …”

Perspective: Mechanochemistry of biological and synthetic molecules

“…Of course, D, partially or fully originating from solvent friction, is expected to be viscosity dependent even in the absence of memory effects; however, nonlinear viscosity dependence, if observed, could be a signature of memory. 124,125 Improvements in the force stability and the time resolution of single-molecule force spectroscopy [126][127][128][129][130][131] enable more direct approaches to measuring molecular dynamics. Indeed, the information contained in the temporal evolution of a molecule's extension x(t) (cf.…”

“…1(b)) is not limited to the transition rate k(F). For example, recent designs of the atomic force microscope cantilevers allow temporal resolution of ∼1 microsecond, 127 more than an order of magnitude shorter than the typical duration of the transition path, a short segment of a molecular trajectory where the molecule is caught in the act of crossing over from the initial (reactant) state to the final (product) state (shown in Fig. 1(b)); a typical transition path time for the folding of single-domain proteins is tens of microseconds.…”