Double-stranded DNA (dsDNA) unconstrained by torsion undergoes an overstretching transition at about 65 pN, elongating the to 1.7-fold. Three possible structural transitions have been debated for nature of overstretching: ( i ) “peeling” apart dsDNA produce a peeled ssDNA strand under tension while other coils, ii “inside-strand separation” two parallel strands that share (melting bubbles), and iii “B-to-S” novel dsDNA, termed S-DNA. Here we overstretched end-opened (with one open end allow...

Formins, an important family of force-bearing actin-polymerizing factors, function as homodimers that bind with the barbed end actin filaments through a ring-like structure assembled from dimerized FH2 domains. It has been hypothesized force applied to formin may facilitate transition ring inhibitory closed conformation permissive open conformation, speeding up polymerization. We confirm this hypothesis for mDia1 dependent polymerization by stretching single-actin filament in absence...

Abstract Titin N2B unique sequence (N2B-us) is a 572 amino acid that acts as an elastic spring to regulate muscle passive elasticity. It thought lack stable tertiary structures and force-bearing region regulated by mechanical stretching. In this study, the conformation of N2B-us its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions single-molecule experimental validation. Surprisingly, alpha/beta structural predicted confirmed in can...

α-Actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework cytoskeleton through crosslinking actin filaments, well focal adhesion maturation. However, molecular mechanisms underlying its functions are unclear. Here, by mechanical manipulation single human α-actinin 1 using magnetic tweezers, we determined stability and kinetics functional domains 1. Moreover, identified force-dependence...

Dystrophin is the largest protein isoform (427 kDa) expressed from gene defective in Duchenne muscular dystrophy, a lethal muscle-wasting and genetically inherited disease. Dystrophin, localized within cytoplasmic lattice termed costameres, connects intracellular cytoskeleton of myofiber through cell membrane (sarcolemma) to surrounding extracellular matrix. In spite its mechanical regulation roles stabilizing sarcolemma during muscle contraction, underlying molecular mechanism still...

Abstract The giant protein titin plays a critical role in regulating the passive elasticity of muscles, mainly through stochastic unfolding and refolding its numerous immunoglobulin domains I‐band sarcomeres. dynamics at force range greater than 100 pN has been studied by atomic microscopy, while that smaller physiological forces not measured before. By using magnetic tweezers, it is found I27 domain unfolds surprising non‐monotonic force‐dependent manner pN, with slowest rate occurring...

Talin and vinculin are part of a multicomponent system involved in mechanosensing cell-matrix adhesions. Both exist autoinhibited forms, activation requires binding to mechanically activated talin, yet how forces affect talin's interaction with has not been investigated. Here by quantifying the kinetics force-dependent talin-vinculin interactions using single-molecule analysis, we show that mechanical exposure single site (VBS) talin is sufficient relieve autoinhibition vinculin, resulting...

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response force, talin undergoes switch-like behavior its multiple rod domains modulate interactions with binding partners. Cyclin-dependent kinase-1 (CDK1) key regulator cell cycle, exerting effects through synchronized phosphorylation large number protein targets. CDK1 activity maintains during interphase, and inhibition prerequisite for tightly choreographed...

Mechanotransduction, the process by which cells respond to tension transmitted through various supramolecular linkages, is important for understanding cellular behavior. Tension gauge tethers (TGTs), short fragments of double-stranded DNA that irreversibly break under shear-stretch conditions, have been used in live cell experiments study mechanotransduction. However, our current TGTs' mechanical responses limited, limits information can be gleaned from experimental observations. In this...

The differential structural–elastic properties of molecules between their transition and initial (native or denatured) states determine force-dependent rates.

Abstract Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding that serves to protect ssDNA from degradation and annealing as template for recruitment of many downstream factors in virtually all transactions cell. During these transactions, tethered likely subject force. Previous studies RPA's behavior on were conducted the absence force; therefore RPA-ssDNA conformations regulated by force remain unclear. Here, using combination atomic microscopy imaging...

Single-molecule manipulation reveals high mechanical stability of vinculin—talin and vinculin—α-catenin protein-protein interfaces.

The cold shock domain (CSD) protein acts as a nucleic acid-binding and exhibits strong affinity for the Y-box core motif ATTGG segment on gene promoter regions, playing significant role in regulation. Given moderate folding unfolding rates of from Bacillus caldolyticus (BcCsp) observed previous magnetic tweezers studies, it is suitable choice investigating interaction between CSD single-stranded DNA containing ATTGG. While earlier studies utilizing single molecule force spectroscopy delved...

All-α proteins typically fold rapidly and are unable to withstand high forces. Acyl-coenzyme A binding protein (ACBP), a four-α-helix bundle protein, serves as model for studying the folding dynamics of all-α proteins. In previous biochemistry single molecule force spectroscopy experiments, controversy exists pathway conformation transition state. this article, we investigate unfolding ACBP in range 4-10 pN using magnetic tweezers, revealing anomalous force-dependent rates. The rate remains...

Abstract Spectrin domains, characterized by a distinctive triple helix structure, are crucial in physiological processes, particularly maintaining membrane shape and crosslinking cytoskeletons. Previous research on the 16th domain of α -spectrin repeats (R16) has yielded conflicting results: bulk experiments showed an unfolding rate approximately two orders magnitude faster than zero-force result extrapolated from single-molecule force spectroscopy using Atomic Force Microscopy (AFM). To...

Titin, essential for mechano-homeostasis in cardiac and skeletal sarcomere, contains numerous mechanosensitive immunoglobulin-like (Ig) domains its I-band region. However, how proline isomerization cysteine-mediated disulfide bond collectively regulate Ig domain dynamics within the physiological force range remains unclear. Here, we use single-molecule spectroscopy to quantify proximal Ig1 domain, revealing that leads two native states–trans cis states–with distinct mechanical thermal...

Self-assembling actin filaments not only form the basis of cytoskeleton network in cells but also are utilized as nanosized building blocks to make novel active matter which dynamic polymerization and depolymerization play a key role. Formins belong main family nucleation factors that bind barbed end regulate through an interaction with profilin. Due actomyosin contractility relative rotation between formin filaments, formin-dependent is subject force constraints. However, it remains unclear...

Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions DNA proteins. An important aspect such is obtain dynamics interactions; however initial binding often difficult due large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid exchange method for magnetic tweezers single-molecule experiments, which achieved by tethering molecules inside microwells (typical dimensions-diameter...

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with integrin/actin-binding protein talin several components of microtubule-stabilizing cortical complexes. Because actomyosin contractility, talin-KANK complex is likely under mechanical force, its stability expected be a critical determinant recruitment focal adhesions. Here, we quantified lifetime rod domain R7 KN KANK1 shear-force geometry found that it...

The adhesions between Gram-positive bacteria and their hosts are exposed to varying magnitudes of tensile forces. Here, using an ultrastable magnetic tweezer-based single-molecule approach, we show the catch-bond kinetics prototypical adhesion complex SD-repeat protein G (SdrG) a peptide from fibrinogen β (Fgβ) over physiologically important force range piconewton (pN) tens pN, which was not technologically accessible previous studies. At 37 °C, lifetime exponentially increases seconds at...

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational repair. Although ssDNA-binding protein (SSB) plays a complicated role in reactions vivo, much our understanding mechanism based binding directly to ssDNA. Here we investigate SSB regulation polymerization ssDNA, differential force responses single 576-nucleotide-long ssDNA associated with and SSB. We find that outcompetes higher concentrations RecA, resulting inhibition...

αT (Testes)-catenin, a critical factor regulating cell-cell adhesion in the heart, directly couples cadherin-catenin complex to actin cytoskeleton at intercalated disk (ICD), unique junction that cardiomyocytes. Loss of αT-catenin mice reduces plakophilin2 and connexin 43 recruitment ICD. Since is subjected mechanical stretch during actomyosin contraction cardiomyocytes, its activity could be regulated by force. To provide insight how force regulates function, we investigated stability...

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal plasmid transformation. We showed that in the rATP·Mg2+ bound form (RecA·ATP), could not compete RecX, SsbA or SsbB assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA-SsbA mediator reversed negative effect on RecA filament extension, SsbB. In...