Structural and mechanistic insights into mechanoactivation of focal adhesion kinase.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal.

DNA-free directed assembly in single-molecule cut-and-paste.

Single-molecule cut-and-paste facilitates bottom-up directed assembly of nanoscale biomolecular networks in defined geometries and enables analysis with spatio-temporal resolution. However, arrangement of diverse molecules of interest requires versatile handling systems. The novel DNA-free, genetically encodable scheme described here utilises an orthogonal handling strategy to promote arrangement of enzymes and enzyme networks.

Force-Induced Unravelling of DNA Origami.

The mechanical properties of DNA nanostructures are of widespread interest as applications that exploit their stability under constant or intermittent external forces become increasingly common. We explore the force response of DNA origami in comprehensive detail by combining AFM single molecule force spectroscopy experiments with simulations using oxDNA, a coarse-grained model of DNA at the nucleotide level, to study the unravelling of an iconic origami system: the Rothemund tile. We contrast the force-induced melting of the tile with simulations of an origami 10-helix bundle.

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality.

Single-molecule force spectroscopy on polyproteins and receptor-ligand complexes: The current toolbox.

Single-molecule force spectroscopy sheds light onto the free energy landscapes governing protein folding and molecular recognition. Since only a single molecule or single molecular complex is probed at any given point in time, the technique is capable of identifying low-probability conformations within a large ensemble of possibilities. It furthermore allows choosing certain unbinding pathways through careful selection of the points at which the force acts on the protein or molecular complex.

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force.

Ultrastable cellulosome-adhesion complex tightens under load.

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force.

From genes to protein mechanics on a chip.

Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever.

Investigating receptor-ligand systems of the cellulosome with AFM-based single-molecule force spectroscopy.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods.