Single-Molecule Force Spectroscopy in Biomolecular Interactions and Dynamics

Single-molecule force spectroscopy (SMFS) has emerged as a powerful approach to probe the mechanics, kinetics and energetics of individual biomolecules as they undergo folding, binding and chemical reactions. By applying controlled forces via tools such as atomic force microscopy (AFM), optical tweezers or magnetic tweezers, it is possible to record force–extension profiles, measure bond rupture forces and reconstruct free-energy landscapes underlying conformational transitions. This capability has greatly advanced our understanding of mechanotransduction in proteins, the mechanical stability of receptor–ligand complexes, and force-induced chemistry in metalloproteins. Beyond fundamental insight, SMFS informs the design of biomaterials, reveals pathways of protein unfolding and refolding under physiological forces, and guides the development of novel mechanobiological therapeutics. The technique’s sensitivity to single molecular events affords a direct view of heterogeneity in molecular response, enabling the deconvolution of parallel reaction pathways and the quantification of transient intermediates that remain obscured in ensemble measurements.

Research from Nature Portfolio

Recent studies have revealed the extraordinary mechanical resilience of biological adhesion complexes and the intricate coupling between mechanical force and chemical reactivity. One investigation of a bacterial cellulosome anchoring complex employed AFM-based SMFS and molecular dynamics simulations to show that specific receptor–ligand pairs withstand forces approaching 700 pN, uncovering a force-activated stabilisation mechanism that preserves network integrity under shear. Another work combined chemical denaturants, mechanical pulling and site-directed mutations to demonstrate that even simple two-state proteins can unfold through multiple transition barriers; this chemo-mechanical unfolding approach elucidated how small changes in sequence or environment shift the flux between structurally distinct pathways. A third study used AFM-induced partial unfolding to expose a buried FeS₄ centre in a rubredoxin domain, thereby enabling the direct measurement of protonation and ligand-exchange reactions under tension; the results linked force-induced structural destabilisation to altered metal-centre reactivity, illustrating a novel mechanochemical dimension of metalloprotein function.

Single-Molecule Force Spectroscopy in Biomolecular Interactions and Dynamics publication trend

The graph below shows the total number of articles in single-molecule force spectroscopy in biomolecular interactions and dynamics across all publications each year (not limited to Nature Index journals).

Technical terms

Single-molecule force spectroscopy: A set of techniques that apply and measure forces on individual biomolecules to probe their mechanical and kinetic properties.

Atomic force microscopy (AFM): An imaging and manipulation method that uses a cantilever with a sharp tip to apply piconewton-scale forces and record deflections at the single-molecule level.

Optical tweezers: A technique employing focused laser beams to trap and exert forces on dielectric particles or beads attached to biomolecules, allowing force-extension measurements.

Free-energy landscape: A multidimensional description of the energetic states and barriers a biomolecule traverses during conformational change or chemical reaction.

Receptor–ligand interaction: The specific binding event between a protein receptor and its molecular ligand, often characterised by a rupture force and kinetic rates under load.

References

1. Dynamics of Equilibrium Folding and Unfolding Transitions of Titin Immunoglobulin Domain under Constant Forces. Journal of the American Chemical Society (2015).
2. Ultrastable cellulosome-adhesion complex tightens under load. Nature Communications (2014).
3. Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein. Nature Communications (2015).
4. Force-induced chemical reactions on the metal centre in a single metalloprotein molecule. Nature Communications (2015).
5. Streptavidin/biotin: Tethering geometry defines unbinding mechanics. Science Advances (2020).
6. Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes. Frontiers in Molecular Biosciences (2020).