Single-Molecule Investigation of Load-Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin.

Myosins are ATP-powered, force-generating motor proteins involved in cardiac and muscle contraction. The external load experienced by the myosins modulates and coordinates their function in vivo. Here, this study investigates the tension-sensing mechanisms of rabbit native ß-cardiac myosin (ßM-II) and slow skeletal myosins (SolM-II) that perform in different physiological settings. Using mobile optical tweezers with a square wave-scanning mode, a range of external assisting and resisting loads from 0 to 15 pN is exerted on single myosin molecules as they interact with the actin filament. Influenced of load on specific strongly-bound states in the cross-bridge cycle is examined by adjusting the [ATP]. The results implies that the detachment kinetics of actomyosin ADP.Pi strongly-bound force-generating state are load sensitive. Low assisting load accelerates, while the resisting load hinders the actomyosin detachment, presumably, by slowing both the Pi and ADP release. However, under both high assisting and resisting load, the rate of actomyosin dissociation decelerates. The transition from actomyosin ADP.Pi to ADP state appears to occur with a higher probability for ßM-II than SolM-II. This study interpret that dissociation of at least three strongly-bound actomyosin states are load-sensitive and may contribute to functional diversity among different myosins.

Spin-State Switching of Spin-Crossover Complexes on Cu(111) Evidenced by Spin-Flip Spectroscopy.

Spin-crossover compounds can be switched between two stable states with different magnetic moments, conformations, electronic, and optical properties, which opens appealing perspectives for technological applications including miniaturization down to the scale of single molecules. Although control of the spin states is crucial their direct identification is challenging in single-molecule experiments. Here we investigate the spin-crossover complex [Fe(HB(1,2,4-triazol-1-yl)3)2] on a Cu(111) surface with scanning tunneling microscopy and density functional theory calculations. Spin crossover of single molecules in dense islands is achieved via electron injection. Spin-flip excitations are resolved in scanning tunneling spectra in a magnetic field enabling the direct identification of the molecular spin state, and revealing the existence of magnetic anisotropy in the HS molecules.

Eukaryotic yeast V1-ATPase rotary mechanism insights revealed by high-resolution single-molecule studies.

Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V1 complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the Vo complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V1 without C, and VO. ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V1 is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position of Saccharomyces cerevisiae V1-ATPase lacking subunits H and C (V1ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120° power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V1ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring ∼45° after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell, ∼2-fold longer occurring 112° that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112° and MgADP released at ∼45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.

Single-Molecule Characterization and Super-Resolution Imaging of Alzheimer's Disease-Relevant Tau Aggregates in Human Samples.

Hyperphosphorylation and aggregation of the protein tau play key roles in the development of Alzheimer's disease (AD). While the molecular structure of the filamentous tau aggregates has been determined to atomic resolution, there is far less information available about the smaller, soluble aggregates, which are believed to be more toxic. Traditional techniques are limited to bulk measures and struggle to identify individual aggregates in complex biological samples. To address this, we developed a novel single-molecule pull-down-based assay (MAPTau) to detect and characterize individual tau aggregates in AD and control post-mortem brain and biofluids. Using MAPTau, we report the quantity, as well as the size and circularity of tau aggregates measured using super-resolution microscopy, revealing AD-specific differences in tau aggregate morphology. By adapting MAPTau to detect multiple phosphorylation markers in individual aggregates using two-color coincidence detection, we derived compositional profiles of the individual aggregates. We find an AD-specific phosphorylation profile of tau aggregates with more than 80 % containing multiple phosphorylations, compared to 5 % in age-matched non-AD controls. Our results show that MAPTau is able to identify disease-specific subpopulations of tau aggregates phosphorylated at different sites, that are invisible to other methods and enable the study of disease mechanisms and diagnosis.

Single-molecular diffusivity and long jumps of large organic molecules: CoPc on Ag(100).

Energy dissipation and the transfer rate of adsorbed molecules do not only determine the rates of chemical reactions but are also a key factor that often dictates the growth of organic thin films. Here, we present a study of the surface dynamical motion of cobalt phthalocyanine (CoPc) on Ag(100) in reciprocal space based on the helium spin-echo technique in comparison with previous scanning tunnelling microscopy studies. It is found that the activation energy for lateral diffusion changes from 150 meV at 45-50 K to ≈100 meV at 250-350 K, and that the process goes from exclusively single jumps at low temperatures to predominantly long jumps at high temperatures. We thus illustrate that while the general diffusion mechanism remains similar, upon comparing the diffusion process over widely divergent time scales, indeed different jump distributions and a decrease of the effective diffusion barrier are found. Hence a precise molecular-level understanding of dynamical processes and thin film formation requires following the dynamics over the entire temperature scale relevant to the process. Furthermore, we determine the diffusion coefficient and the atomic-scale friction of CoPc and establish that the molecular motion on Ag(100) corresponds to a low friction scenario as a consequence of the additional molecular degrees of freedom.

2,3-Dichloroanthracene crystal, a new rigid matrix for single molecule optical investigations.

Absorption and emission spectra of single crystals of 2,3-dichloroathracene (23DCA) and 23DCA dispersed in n-nonane matrix were studied at 5 K. Singlet and triplet excitonic bands in the crystal were estimated to be at about 415 nm and at wavelengths shorter than 700 nm, respectively. Thus, from the spectroscopic point of view, these crystals satisfy all criteria for a transparent and rigid matrix for low temperature optical studies of single molecules of dibenzoterrylene, which have their purely electronic S0→S1 transition at around 785 nm. Quantum-chemistry calculations were used to analyze the spectra.

Probing the in-plane dipole moment vector between ground and excited state of single molecules by the Stark effect.

Single molecules, embedded inside a well-defined insertion site of a single-crystalline host matrix, are sensitive probes of electric field via the induced Stark shift on their lifetime-limited electronic transition. Though the response of molecules to electric field has been shown to be relatively homogeneous, crystal symmetry allows for several, spectroscopically-indistinguishable, orientations of the net permanent dipole moment between the ground and excited state - the dipole vector - and this is problematic for measuring field orientation and magnitude. In this work, we measure for each terrylene molecule, embedded inside a new host matrix, the dipole vector independently by an electric field that we can rotate in the plane of the crystal. This single crystal host matrix, called [1]BenzoThieno[3,2-b]BenzoThiophene, induces a moderate symmetry breaking of the embedded centrosymmetric terrylene molecule, and gives rise to a net dipole moment of 0.28±0.09 Debye. Based on quantum chemistry calculations we propose an insertion site that best matches the experimental findings.

Molecular Adhesion of a Pilus-Derived Peptide Involved in Pseudomonas aeruginosa Biofilm Formation on Non-Polar ZnO-Surfaces.

Bacterial colonization and biofilm formation on abiotic surfaces are initiated by the adhesion of peptides and proteins. Understanding the adhesion of such peptides and proteins at a molecular level thus represents an important step toward controlling and suppressing biofilm formation on technological and medical materials. This study investigates the molecular adhesion of a pilus-derived peptide that facilitates biofilm formation of Pseudomonas aeruginosa, a multidrug-resistant opportunistic pathogen frequently encountered in healthcare settings. Single-molecule force spectroscopy (SMFS) was performed on chemically etched ZnO 11 2 ‾ 0 ${\left(11\bar{2}0\right)}$ surfaces to gather insights about peptide adsorption force and its kinetics. Metal-free click chemistry for the fabrication of peptide-terminated SMFS cantilevers was performed on amine-terminated gold cantilevers and verified by X-ray photoelectron spectroscopy (XPS) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Atomic force microscopy (AFM) and XPS analyses reveal stable topographies and surface chemistries of the substrates that are not affected by SMFS. Rupture events described by the worm-like chain model (WLC) up to 600 pN were detected for the non-polar ZnO surfaces. The dissociation barrier energy at zero force ΔG(0), the transition state distance xb and bound-unbound dissociation rate at zero force koff (0) for the single crystalline substrate indicate that coordination and hydrogen bonds dominate the peptide/surface interaction.

Kinetic Referencing Allows Identification of Epigenetic Cytosine Modifications by Single-Molecule Hybridization Kinetics and Superresolution DNA-PAINT Microscopy.

We develop a DNA origami-based internal kinetic referencing system with a colocalized reference and target molecule to provide increased sensitivity and robustness for transient binding kinetics. To showcase this, we investigate the subtle changes in binding strength of DNA oligonucleotide hybrids induced by cytosine modifications. These cytosine modifications, especially 5-methylcytosine but also its oxidized derivatives, have been increasingly studied in the context of epigenetics. Recently revealed correlations of epigenetic modifications and disease also render them interesting biomarkers for early diagnosis. Internal kinetic referencing allows us to probe and compare the influence of the different epigenetic cytosine modifications on the strengths of 7-nucleotide long DNA hybrids with one or two modified nucleotides by single-molecule imaging of their transient binding, revealing subtle differences in binding times. Interestingly, the influence of epigenetic modifications depends on their position in the DNA strand, and in the case of two modifications, effects are additive. The sensitivity of the assay indicates its potential for the direct detection of epigenetic disease markers.

Connecting the dots: key insights on ParB for chromosome segregation from single-molecule studies.

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome segregation during the bacterial cell cycle. Over the past decades, research has greatly advanced our knowledge of the ParABS system. However, many intricate details of the mechanism of ParB proteins were only recently uncovered using in vitro single-molecule techniques. These approaches allowed the exploration of ParB proteins in precisely controlled environments, free from the complexities of the cellular milieu. This review covers the early developments of this field but emphasizes recent advances in our knowledge of the mechanistic understanding of ParB proteins as revealed by in vitro single-molecule methods. Furthermore, we provide an outlook on future endeavors in investigating ParB, ParB-like proteins, and their interaction partners.

Hierarchical Mechanical Transduction of Precision-Engineered DNA Hydrogels with Sacrificial Bonds.

Engineering the response to external signals in mechanically switchable hydrogels is important to promote smart materials applications. However, comparably little attention has focused on embedded precision mechanisms for autonomous nonlinear response in mechanical profiles in hydrogels, and we lack understanding of how the behavior from the molecular scale transduces to the macroscale. Here, we design a nonlinear stress-strain response into hydrogels by engineering sacrificial DNA hairpin loops into model network hydrogels formed from star-shaped building blocks. We characterize the force-extension response of single DNA hairpins and are able to describe how the specific topology influences the nonlinear mechanical behavior at different length scales. For this purpose, we utilize force spectroscopy as well as microscopic and macroscopic deformation tests. This study contributes to a better understanding of designing nonlinear strain-adaptive features into hydrogel materials.

Plasmon-Molecule Interactions in Single-Molecule Junctions.

Single-molecule optoelectronics offers opportunities for advancing integrated photonics and electronics, which also serves as a tool to elucidate the underlying mechanism of light-matter interaction. Plasmonics, which plays pivotal role in the interaction of photons and matter, have became an emerging area. A comprehensive understanding of the plasmonic excitation and modulation mechanisms within single-molecule junctions (SMJs) lays the foundation for optoelectronic devices. Consequently, this review primarily concentrates on illuminating the fundamental principles of plasmonics within SMJs, delving into their research methods and modulation factors of plasmon-exciton. Moreover, we underscore the interaction phenomena within SMJs, including the enhancement of molecular fluorescence by plasmonics, Fano resonance and Rabi splitting caused by the interaction of plasmon-exciton. Finally, by emphasizing the potential applications of plasmonics within SMJs, such as their roles in optical tweezers, single-photon sources, super-resolution imaging, and chemical reactions, we elucidate the future prospects and current challenges in this domain.

Spin-Selective Electron Transport Through Single Chiral Molecules.

The interplay between chirality and magnetism is a source of fascination among scientists for over a century. In recent years, chirality-induced spin selectivity (CISS) has attracted renewed interest. It is observed that electron transport through layers of homochiral molecules leads to a significant spin polarization of several tens of percent. Despite the abundant experimental evidence gathered through mesoscopic transport measurements, the exact mechanism behind CISS remains elusive. This study reports spin-selective electron transport through single helical aromatic hydrocarbons that are sublimed in vacuo onto ferromagnetic cobalt surfaces and examined with spin-polarized scanning tunneling microscopy (SP-STM) at a temperature of 5 K. Direct comparison of two enantiomers under otherwise identical conditions revealed magnetochiral conductance asymmetries of up to 50% when either the molecular handedness is exchanged or the magnetization direction of the STM tip or Co substrate is reversed. Importantly, the results rule out electron-phonon coupling and ensemble effects as primary mechanisms responsible for CISS.

A Review of the Current State of Magnetic Force Microscopy to Unravel the Magnetic Properties of Nanomaterials Applied in Biological Systems and Future Directions for Quantum Technologies.

Magnetism plays a pivotal role in many biological systems. However, the intensity of the magnetic forces exerted between magnetic bodies is usually low, which demands the development of ultra-sensitivity tools for proper sensing. In this framework, magnetic force microscopy (MFM) offers excellent lateral resolution and the possibility of conducting single-molecule studies like other single-probe microscopy (SPM) techniques. This comprehensive review attempts to describe the paramount importance of magnetic forces for biological applications by highlighting MFM's main advantages but also intrinsic limitations. While the working principles are described in depth, the article also focuses on novel micro- and nanofabrication procedures for MFM tips, which enhance the magnetic response signal of tested biomaterials compared to commercial nanoprobes. This work also depicts some relevant examples where MFM can quantitatively assess the magnetic performance of nanomaterials involved in biological systems, including magnetotactic bacteria, cryptochrome flavoproteins, and magnetic nanoparticles that can interact with animal tissues. Additionally, the most promising perspectives in this field are highlighted to make the reader aware of upcoming challenges when aiming toward quantum technologies.

Looping the Genome with SMC Complexes.

SMC (structural maintenance of chromosomes) protein complexes are an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. Here, we describe the roles of SMCs in chromosome biology and particularly review in vitro single-molecule studies that have recently advanced our understanding of SMC proteins. We describe the mechanistic biophysical aspects of loop extrusion that govern genome organization and its consequences.

Electrical Actuation of DNA-Based Nanomechanical Systems.

DNA nanotechnology provides efficient methods for the sequence-programmable construction of mechanical devices with nanoscale dimensions. The resulting nanomachines could serve as tools for the manipulation of macromolecules with similar functionalities as mechanical tools and machinery in the macroscopic world. In order to drive and control these machines and to perform specific tasks, a fast, reliable, and repeatable actuation mechanism is required that can work against external loads. Here we describe a highly effective method for actuating DNA structures using externally applied electric fields. To this end, electric fields are generated with controllable direction and amplitude inside a miniature electrophoresis device integrated with an epifluorescence microscope. With this setup, DNA-based nanoelectromechanical devices can be precisely controlled. As an example, we demonstrate how a DNA-based nanorobotic system can be used to dynamically position molecules on a molecular platform with high speeds and accuracy. The microscopy setup also described here allows simultaneous monitoring of a large number of nanorobotic arms in real time and at the single nanomachine level.

Driving a Third Generation Molecular Motor with Electrons Across a Surface.

Excitation of single molecules with electrons tunneling between a sharp metallic tip of a scanning tunneling microscope and a metal surface is one way to study and control dynamics of molecules on surfaces. Electron tunneling induced dynamics may lead to hopping, rotation, molecular switching, or chemical reactions. Molecular motors that convert rotation of subgroups into lateral movement on a surface can in principle also be driven by tunneling electrons. For such surface-bound motor molecules the efficiency of motor action with respect to electron dose is still not known. Here, the response of a molecular motor containing two rotor units in the form of overcrowded alkene groups to inelastic electron tunneling has been examined on a Cu(111) surface in ultrahigh vacuum at 5 K. Upon vibrational excitation, switching between different molecular conformations is observed, including conversion of enantiomeric states of chiral conformations. Tunneling at energies in the range of electronic excitations causes activation of motor action and movement across the surface. The expected unidirectional rotation of the two rotor units causes forward movements but with a low degree of translational directionality.

Expanded Conformations of Monomeric Tau Initiate Its Amyloidogenesis.

Understanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well-characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single-molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tau-RD244-368 ). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro-amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti-amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau-RD244-368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies.

Effects of Gly Residue and Cholesterol on the GXXXG-Mediated Parallel Association of Transmembrane Helices: A Single-Pair FRET Study.

Small residue-mediated interhelical packing is ubiquitous in helical membrane proteins: however, the lipid dependence of its stability remains unclear. We previously demonstrated that the introduction of a GXXXG sequence in the middle of de novo-designed (AALALAA)3 helices (AALALAA AGLALGA AALALAA) facilitated their dimerization, which was abolished by cholesterol. Here single-pair FRET measurements revealed that a longer GXXXGXXXG segment (AALALAA A GLALGA AAGALAA) promoted helix dimerization in POPC/cholesterol bilayers, but not without cholesterol. The predicted dimer structures and degrees of helix packing suggested that helix dimers with small (∼10°) and large (∼55°) crossing angles were only stabilized in POPC and POPC/cholesterol membranes, respectively. A steric hindrance in the dimer interface and the large flexibility of helices prevented the formation of stable dimers. Therefore, amino acid sequences and lipid compositions distinctively constrain stable dimer structures in membranes.

Single-molecule investigations of single-chain cellulose biosynthesis.

Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.