Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

Single-molecule force spectroscopy with the atomic force microscope provides molecular level insights into protein function, allowing researchers to reconstruct energy landscapes and understand functional mechanisms in biology. With steadily advancing methods, this technique has greatly accelerated our understanding of force transduction, mechanical deformation, and mechanostability within single- and multi-domain polyproteins, and receptor-ligand complexes. In this focused review, we summarize the state of the art in terms of methodology and highlight recent methodological improvements for AFM-SMFS experiments, including developments in surface chemistry, considerations for protein engineering, as well as theory and algorithms for data analysis. We hope that by condensing and disseminating these methods, they can assist the community in improving data yield, reliability, and throughput and thereby enhance the information that researchers can extract from such experiments. These leading edge methods for AFM-SMFS will serve as a groundwork for researchers cognizant of its current limitations who seek to improve the technique in the future for in-depth studies of molecular biomechanics.