We directly measured at the single-molecule level the forces and lifetimes of DNA base-pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual-beam optical tweezers with DNA origami components to allow positioning of blunt-end DNA helices so that the weak stacking force could be isolated. Base-pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free-energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from -0.8 to -3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA-based nanoscale devices according to user specifications.

Actin-binding proteins (ABPs) regulate the assembly of actin filaments (F-actin) into networks and bundles that provide the structural integrity of the cell. Two of these ABPs, filamin and ␣-actinin, have been extensively used to model the mechanical properties of actin networks grown in vitro; however, there is a lack in the understanding of how the molecular interactions between ABPs and F-actin regulate the dynamic properties of the cytoskeleton. Here, we present a native-like assay geometry to test the rupture force of a complex formed by an ABP linking two quasiparallel actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin and ␣-actinin. For filamin/actin and ␣-actinin/actin, we measured similar rupture forces of 40 -80 pN for loading rates between 4 and 50 pN/s. Both ABP unfolding and conformational transition events were observed, demonstrating that both are important and may be a significant mechanism for the temporal regulation of the mechanical properties of the actin cytoskeleton. With this modular, singlemolecule assay, a wide range of ABP/actin interactions can be studied to better understand cytoskeletal and cell dynamics.␣-actinin ͉ filamin ͉ optical tweezers ͉ single-molecule force spectroscopy

Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20-21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.optical tweezers | mechanosensing

Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high‐resolution single‐molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.

The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90's chaperone cycle. A unique linker, called charged linker (CL), connects the N-and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 k B T (2.7 kJ/mol) through binding of the CL to the Ndomain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90's chaperone cycle governing client activation, cell viability, and stress tolerance. T he molecular chaperone Hsp90 (heat shock protein 90) is essential for the folding, maturation, and activation of approximately 10% of the yeast proteome. The set of substrate proteins is structurally and functionally diverse and ranges from telomerase to kinases and transcription factors (1-3). Processing of these substrates requires ATP turnover and large conformational rearrangements within Hsp90 (4-6). Interestingly, these conformational states of yeast Hsp90 are not strictly coupled to the binding of nucleotides (7) but are recognized and regulated by the interaction with cochaperones (8) and substrate proteins (9).Hsp90 is a dimer with each monomer consisting of three domains (N, M, and C). The N-terminal domain comprises the nucleotide binding site, whereas the M-domain is important for the binding of many substrates. The C-terminal domain is mainly responsible for the dimerization of the protein. A long charged linker (CL) region, amino acids 211-272 in yeast, connects the N-and M-domain in eukaryotes (Fig. 1A). The crystal structure of yeast Hsp90 was obtained by partly deleting the CL region and shows a closed, compact conformation in the presence of AMP-PNP [Adenosine 5′-(β,γ-imido)triphosphate] and Sba1/p23 (10). The fact that the CL region is difficult to map structurally led to the assumption that this region is disordered and flexible, thereby enabling the conformational rearrangements of Hsp90 (11,12). Besides the structural indetermination of the CL, its ultimate function remains elusive as well (13). Early studies show that parts of the CL region (amino acids 211-259) are dispensable in yeast (14), whereas more extended deletions affect cell viability, substrate maturation, and regulation by cochaperones (11). These deficiencies can be partially rescued by a short linker consisting of an artificial sequence (11), but its specific amino acid sequence is associated with a gain of function, probably an additional Hsp90 regulatory mechanism (15).Single-molecule experiments have recently provided detailed insight int...