Abstract
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.
Highlights
Single-molecule force spectroscopy (SMFS) is a well-established method that directly probes structural changes of macromolecules under the influence of mechanical force
Since mechanical forces are ubiquitous in biology, insights gleaned from SMFS experiments shed light onto fundamentally important molecular mechanisms by which biological systems are able to sense, transduce and generate mechanical forces in vivo
Several native biological systems where mechanical forces play a significant role have been investigated with SMFS, including examples from muscle (Rief et al, 1997; Puchner et al, 2008a; Rivas-Pardo et al, 2016; Eckels et al, 2019), hearing (Lee et al, 2006; Bartsch et al, 2019; Hazra et al, 2019; Mulhall et al, 2019; Oroz et al, 2019), blood coagulation (Kim et al, 2010; Zhmurov et al, 2011; Müller et al, 2016), cell adhesion (Zhang et al, 2002; Marshall et al, 2003; Evans and Calderwood, 2007), the extracellular matrix
Summary
Single-molecule force spectroscopy (SMFS) is a well-established method that directly probes structural changes of macromolecules under the influence of mechanical force. We describe bioconjugation strategies for immobilizing proteins with sitespecific attachment to surfaces and cantilevers for AFM-SMFS and describe recent approaches to protein-ligation which can facilitate novel measurement formats.
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