Keynote Talk  - Wednesday, 15 September I 14:25 PM (CEST)

Single-molecule force spectroscopy (SMFS) has become a critical tool in unraveling the behavior of mechanoresponsive proteins. High-precision SMFS studies of nucleic acids and globular proteins have leveraged the stability and precision of custom-built optical traps, yielding kinetic rate constants, energetics, intermediate states, folding pathways, and finally a full 1D projection of the underlying free-energy landscape. Recently, we have developed a set of modified cantilevers for atomic force microscopy (AFM) that has enabled high-precision studies on a commercial AFM. For example, we achieved sub-pN stability over ~80 s coupled with 2-μs temporal resolution. To demonstrate broad applicability, we performed equilibrium and non-equilibrium assays on 3 classes of bio-molecules (membrane proteins, globular proteins, and structured RNA). Importantly, these studies were not limited to high forces—the traditional strength of AFM—but also characterized low-force transitions (5–30 pN). Concurrent advances in data acquisition and site-specific bio-conjugation allowed the same individual molecule to be unfolded and refolded more than 1,000 times in 15 min. The resulting increased data quantity and quality enabled free-energy landscape reconstruction from both equilibrium and non-equilibrium data for all three classes of bio-molecules. Moreover, improved constant-force assays of a protein-ligand complex yielded the height of the transition state in addition to the two traditional SMFS parameters (e.g., zero-force dissociation rate constant and the distance to the transition state). Looking forward, we anticipate that the ease-of-use of such advanced assays on a commercial AFM will accelerate the high-precision characterization of a broad range of mechano-responsive systems.