Anomalous Force-Dependent Transition Rates Unveil Dual Pathways in Folding and Unfolding Dynamics of Acyl-coenzyme A Binding Protein.

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 a model protein for studying the folding dynamics of all-α proteins. In previous biochemistry and single molecule force spectroscopy experiments, a controversy exists for the folding pathway and the conformation of the transition state.

Effect of ssDNA ligand in modulating the folding and unfolding dynamics of cold shock protein BcCsp.

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

Folding and Misfolding Dynamics of Irisin Protein Revealed by Single-Molecule Magnetic Tweezers.

Irisin, a fibronectin III protein secreted by muscles during physical exercise, plays a significant role in the browning of white fat and cell adhesion, highlighting the importance of its conformational transitions. In this study, we investigated the folding and unfolding dynamics of a single irisin domain using a single-molecule manipulation technique known as magnetic tweezers. In addition to the native state, irisin can also fold transiently into a misfolded state.

Single-molecule force spectroscopy reveals intra- and intermolecular interactions of HMP-1 during mechanotransduction.

The HMP-2/HMP-1 complex, akin to the mammalian [Formula: see text]-catenin-[Formula: see text]-catenin complex, serves as a critical mechanosensor at cell-cell adherens junctions, transducing tension between HMR-1 (also known as cadherin in mammals) and the actin cytoskeleton. Essential for embryonic development and tissue integrity in , this complex experiences tension from both internal actomyosin contractility and external mechanical microenvironmental perturbations. While offering a valuable evolutionary comparison to its mammalian counterpart, the impact of tension on the mechanical stability of HMP-1 and HMP-2/HMP-1 interactions remains unexplored.

Structural domain in the Titin N2B-us region binds to FHL2 in a force-activation dependent manner.

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

Exploring the free energy landscape of proteins using magnetic tweezers.

Proteins fold to their native states by searching through the free energy landscapes. As single-domain proteins are the basic building block of multiple-domain proteins or protein complexes composed of subunits, the free energy landscapes of single-domain proteins are of critical importance to understand the folding and unfolding processes of proteins. To explore the free energy landscapes of proteins over large conformational space, the stability of native structure is perturbed by biochemical or mechanical means, and the conformational transition process is measured.

single-molecule investigations of the impacts of biochemical perturbations on conformational intermediates of monomeric α-synuclein.

α-Synuclein aggregation is a common trait in synucleinopathies, including Parkinson's disease. Being an unstructured protein, α-synuclein exists in several distinct conformational intermediates, contributing to both its function and pathogenesis. However, the regulation of these monomer conformations by biochemical factors and potential drugs has remained elusive.

Free Energy Landscape of Type III Fibronectin Domain with Identified Intermediate State and Hierarchical Symmetry.

The tenth domain of type III fibronectin (FNIII_{10}) mediates cell adhesion to the extracellular matrix. Despite its structural similarity to immunoglobulin domains, FNIII_{10} exhibits unique unfolding behaviors. We employed magnetic tweezers to investigate the unfolding and folding dynamics of FNIII_{10} under physiological forces (4-50 pN).

A Mechanical Assay for the Quantification of Anti-RBD IgG Levels in Finger-Prick Whole Blood.

A large portion of the global population has been vaccinated with various vaccines or infected with SARS-CoV-2, the virus that causes COVID-19. The resulting IgG antibodies that target the receptor binding domain (RBD) of SARS-CoV-2 play a vital role in reducing infection rates and severe disease outcomes. Different immune histories result in the production of anti-RBD IgG antibodies with different binding affinities to RBDs of different variants, and the levels of these antibodies decrease over time.

Tension Gauge Tethers as Tension Threshold and Duration Sensors.

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 to study mechanotransduction. However, our current understanding of TGTs' mechanical responses is limited, which limits the information that can be gleaned from experimental observations.

Mechanical Stabilization of a Bacterial Adhesion Complex.

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 of the prototypical adhesion complex of SD-repeat protein G (SdrG) to a peptide from fibrinogen β (Fgβ) over a physiologically important force range from piconewton (pN) to tens of pN, which was not technologically accessible to previous studies. At 37 °C, the lifetime of the complex exponentially increases from seconds at several pN to ∼1000 s as the force reaches 30 pN, leading to mechanical stabilization of the adhesion.

Force-Dependent Interactions between Talin and Full-Length Vinculin.

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

Unexpected Low Mechanical Stability of Titin I27 Domain at Physiologically Relevant Temperature.

The extensively studied immunoglobulin (Ig) domain I27 of the giant force-bearing protein titin has provided a basis for our current understanding of the structural stability, dynamics, and function of the numerous mechanically stretched Ig domains in the force-bearing I-band of titin. The current consensus is that titin I27 has a high mechanical stability characterized by very low unfolding rate (<10 s) in physiological force range and high unfolding forces (>100 pN) at typical physiological force loading rates from experiments at typical laboratory temperatures. Here, we report that when the temperature is increased from 23 to 37 °C, the unfolding rate of I27 drastically increases by ∼100-fold at the physiological level of forces, indicating a low mechanical stability of I27 at physiological conditions.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1.

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

Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells.

Vinculin, a mechanotransducer associated with both adherens junctions (AJs) and focal adhesions (FAs), plays a central role in force transmission through cell-cell and cell-substratum contacts. We generated the conditional knockout (cKO) of vinculin in murine skin that results in the loss of bulge stem cell (BuSC) quiescence and promotes continual cycling of the hair follicles. Surprisingly, we find that the AJs in vinculin cKO cells are mechanically weak and impaired in force generation despite increased junctional expression of E-cadherin and α-catenin.

Fungal Wound Healing through Instantaneous Protoplasmic Gelation.

Multicellular organisms employ fluid transport networks to overcome the limit of diffusion and promote essential long-distance transport. Connectivity and pressurization render these networks especially vulnerable to wounding. To mitigate this risk, animals, plants, and multicellular fungi independently evolved elaborate clotting and plugging mechanisms.

Modulating mechanical stability of heterodimerization between engineered orthogonal helical domains.

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices.

Direct single-molecule quantification reveals unexpectedly high mechanical stability of vinculin-talin/α-catenin linkages.

The vinculin-mediated mechanosensing requires establishment of stable mechanical linkages between vinculin to integrin at focal adhesions and to cadherins at adherens junctions through associations with the respective adaptor proteins talin and α-catenin. However, the mechanical stability of these critical vinculin linkages has yet to be determined. Here, we developed a single-molecule detector assay to provide direct quantification of the mechanical lifetime of vinculin association with the vinculin binding sites in both talin and α-catenin, which reveals a surprisingly high mechanical stability of the vinculin-talin and vinculin-α-catenin interfaces that have a lifetime of >1000 s at forces up to 10 pN and can last for seconds to tens of seconds at 15 to 25 pN.

Mechanical regulation of formin-dependent actin polymerization.

The actomyosin cytoskeleton network plays a key role in a variety of fundamental cellular processes such as cell division, migration, and cell adhesion. The functions of cytoskeleton rely on its capability to receive, generate, respond to and transmit mechanical signals throughout the cytoskeleton network within the cells and throughout the tissue via cell-extracellular matrix and cell-cell adhesions. Crucial to the cytoskeleton's functions is actin polymerization that is regulated by many cellular factors.

Single-molecule manipulation quantification of site-specific DNA binding.

The execution of functions on DNA relies on complex interactions between DNA and proteins in a sequence and structure dependent manner. Accurate quantification of the affinity and kinetics of these interactions is critical for understanding the molecular mechanisms underlying their corresponding biological functions. The development of single-molecule manipulation technologies in the last two decades has made it possible to apply a mechanical constraint to a single DNA molecule and measure the end-to-end extension changes with nanometer resolution in realtime.

Phosphorylation Reduces the Mechanical Stability of the α-Catenin/ β-Catenin Complex.

The α-catenin/β-catenin complex serves as a critical molecular interface involved in cadherin-catenin-based mechanosensing at the cell-cell adherence junction that plays a critical role in tissue integrity, repair, and embryonic development. This complex is subject to tensile forces due to internal actomyosin contractility and external mechanical micro-environmental perturbation. However, the mechanical stability of this complex has yet to be quantified.

Label-free Single-Molecule Quantification of Rapamycin-induced FKBP-FRB Dimerization for Direct Control of Cellular Mechanotransduction.

Chemically induced dimerization (CID) has been applied to study numerous biological processes and has important pharmacological applications. However, the complex multistep interactions under various physical constraints involved in CID impose a great challenge for the quantification of the interactions. Furthermore, the mechanical stability of the ternary complexes has not been characterized; hence, their potential application in mechanotransduction studies remains unclear.

Force-Dependent Regulation of Talin-KANK1 Complex at Focal Adhesions.

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions.