Probing Single Individual Proteins Unfold and Refold with 1-μs Resolution: Improved AFM-Based Single Molecule Force Spectroscopy

Abstract

Protein folding is understood as a set of transitions between states. Yet, an over-simplified view of the folding process emerges if briefly populated states remain undetected due to ensemble averaging and/or limited instrumental precision. 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 cantilever is 50-1,000 μs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-μs temporal resolution was achieved using ultrashort (L=9 µm) cantilevers on a custom-built, high-speed AFM. By modifying such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected these cantilevers on a commercial AFM retrofitted with a detection laser featuring a 3-μm spot size. The improved capabilities of the modified cantilevers were demonstrated in two biophysical experiments. First, we unfolded a polyprotein, a popular assay, where these cantilevers maintained a 1-μs response time while eliminating cantilever ringing (Q≈0.5). In particular, these cantilever had improved short-term precision by avoiding periods of 30-90 pN (peak-to-peak) force modulation exhibited by unmodified ultrashort cantilevers undergoing underdamped motion at ∼500 kHz. In the second assay, we unfolded bacteriorhodopsin (bR), a model membrane protein. The resulting force-extension curves show unprecedented detail, increasing the number of intermediates resolved while unfolding a pair of transmembrane helices from 2 to 14. Equilibrium measurements revealed the cooperative folding of a 3-amino-acid structural element, resolved those states in <15-μs, and deduced the transition's underlying energy landscape. These bR results sharpen the picture of membrane protein folding and, more broadly, the instrumental enhancements demonstrate a new experimental regime: studying the equilibrium folding and unfolding of individual proteins with 1-µs resolution.