Single-molecule Behavior Research Articles

Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.

SUMMARYKinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Microtubule-associated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

Supramolecular Chirality in Porphyrin Self-Assembly Systems in Aqueous Solution

Abstract: The self-assembly process appears as a powerful and attractive strategy for constructing complex supramolecules by the spontaneous organization of appropriate building blocks. In this scenario, water-soluble porphyrinoids lend themselves as ideal paradigms to disclose the self-assembly phenomenon by exploiting their well-known tendency to build aggregates in aqueous media via weak non-covalent forces. Nevertheless, the spontaneous organization of achiral porphyrins can result in a final chiral superstructure moving away from single- molecule behaviour to supramolecular chirality. Therefore, over the years numerous attempts have been implemented to investigate how a porphyrin aggregate, made up of achiral monomers, becomes not-symmetric and which processes govern the bias for a certain enantiomeric assembly rather than another. Thus, in this mini-review, we exclusively discuss the main strategies for designing and building chiral aggregates in water from achiral porphyrin monomers, with particular regard to their chiroptical features.

Single-Molecule Junction: A Reliable Platform for Monitoring Molecular Physical and Chemical Processes.

Monitoring and manipulating the physical and chemical behavior of single molecules is an important development direction of molecular electronics that aids in understanding the molecular world at the single-molecule level. The electrical detection platform based on single-molecule junctions can monitor physical and chemical processes at the single-molecule level with a high temporal resolution, stability, and signal-to-noise ratio. Recently, the combination of single-molecule junctions with different multimodal control systems has been widely used to explore significant physical and chemical phenomena because of its powerful monitoring and control capabilities. In this review, we focus on the applications of single-molecule junctions in monitoring molecular physical and chemical processes. The methods developed for characterizing single-molecule charge transfer and spin characteristics as well as revealing the corresponding intrinsic mechanisms are introduced. Dynamic detection and regulation of single-molecule conformational isomerization, intermolecular interactions, and chemical reactions are also discussed in detail. In addition to these dynamic investigations, this review discusses the open challenges of single-molecule detection in the fields of physics and chemistry and proposes some potential applications in this field.

A deep learning-based approach to model anomalous diffusion of membrane proteins: the case of the nicotinic acetylcholine receptor.

We present a concatenated deep-learning multiple neural network system for the analysis of single-molecule trajectories. We apply this machine learning-based analysis to characterize the translational diffusion of the nicotinic acetylcholine receptor at the plasma membrane, experimentally interrogated using superresolution optical microscopy. The receptor protein displays a heterogeneous diffusion behavior that goes beyond the ensemble level, with individual trajectories exhibiting more than one diffusive state, requiring the optimization of the neural networks through a hyperparameter analysis for different numbers of steps and durations, especially for short trajectories (<50 steps) where the accuracy of the models is most sensitive to localization errors. We next use the statistical models to test for Brownian, continuous-time random walk and fractional Brownian motion, and introduce and implement an additional, two-state model combining Brownian walks and obstructed diffusion mechanisms, enabling us to partition the two-state trajectories into segments, each of which is independently subjected to multiple analysis. The concatenated multi-network system evaluates and selects those physical models that most accurately describe the receptor's translational diffusion. We show that the two-state Brownian-obstructed diffusion model can account for the experimentally observed anomalous diffusion (mostly subdiffusive) of the population and the heterogeneous single-molecule behavior, accurately describing the majority (72.5 to 88.7% for α-bungarotoxin-labeled receptor and between 73.5 and 90.3% for antibody-labeled molecules) of the experimentally observed trajectories, with only ~15% of the trajectories fitting to the fractional Brownian motion model.

Comparing Transient Oligonucleotide Hybridization Kinetics Using DNA-PAINT and Optoplasmonic Single-Molecule Sensing on Gold Nanorods

We report a comparison of two photonic techniques for single-molecule sensing: fluorescence nanoscopy and optoplasmonic sensing. As the test system, oligonucleotides with and without fluorescent labels are transiently hybridized to complementary docking strands attached to gold nanorods. Comparing the measured single-molecule kinetics helps to examine the influence of fluorescent labels as well as factors arising from different sensing geometries. Our results demonstrate that DNA dissociation is not significantly altered by the fluorescent label, while DNA association is affected by geometric factors in the two techniques. These findings open the door to exploiting plasmonic sensing and fluorescence nanoscopy in a complementary fashion, which will aid in building more powerful sensors and uncovering the intricate effects that influence the behavior of single molecules.

Untethering Single Molecule Spectroscopy with 3D-SMART

Measuring the behavior of single molecules enables the discovery of states and dynamics obscured by bulk measurements. However, molecules in solution rapidly diffuse in three dimensions, precluding long-duration and high-temporal resolution measurement. To overcome this hurdle, we have developed 3D single-molecule active real-time tracking (3D-SMART) which enables active feedback tracking of rapidly diffusing and lowly emitting fluorescent particles. 3D-SMART uses a 3D flying laser focus to tag each arriving photon with 3D position information and a piezoelectric nanopositioner to lock the diffusing object in the objective focal volume, at diffusive speeds exceeding 10 μm2/sec and emission rates of 10 kHz or less. Here we demonstrate the application of 3D-SMART to a range targets, from single virus-like particles and quantum dots in water, all the way down to single fluorophores in viscous solution. This new single molecule tracking can be applied to continuously monitor single proteins and nucleic acids, including real-time measurement of transcription on a freely diffusing, single-dye labelled DNA strand. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.

Collective Force Generator Model of Muscle Contraction

Muscle contraction results from cyclic interactions of actin-myosin binding coupled to the catalyzed hydrolysis of ATP. Although the mechanics and kinetics of muscle myosins are well established, it is less evident how these single molecule behaviors scale up to the collective behaviors of many myosin molecules working together. We have developed a computer simulation of collective force generation by myosin molecules based on well-established kinetic and mechanical properties of single myosin molecules and use an in-vitro motility assay to test model predictions. A collective force model predicts that even in in vitro motility assays myosin heads collectively generate force in common compliant elements and at sufficiently high numbers, N, reach a stall force Consistent with our collective force model, we show that ATPase activity in a motility assay increases linearly at low myosin density, saturating with actin sliding velocities, V, at high myosin densities. We show that activation of V upon addition of inorganic phosphate, Pi, results from Pi decreasing the auto-inhibiting collective force, which in turn activates the ATPase activity. Collective force generation by myosin results in unexpected emergent mechanical behaviors that offer new interpretations for how muscle works.

Multidimensional Perovskite Cesium Lead Halide Nanocrystal for Light Conversion Applications

Zero-dimensional (0D) inorganic perovskites have recently emerged as a new class of material for optoelectronics owing to their outstanding excitonic properties, strong photoluminescence (PL), and high exciton binding energy. These materials have a unique quantum-confined structure, which originates from the presence of fully isolated octahedra exhibiting single-molecule behavior. In this work, we probed the optical behavior of single molecule-like isolated octahedra in 0D Cs4PbX6 (X = Cl, Br/Cl) perovskite through isovalent (Mn2+) doping at Pb sites. The incorporation of Mn2+ stabilizes the Cs4PbX6 phase by lowering the symmetry of PbX6 via enhanced octahedral distortion (preventing the high-symmetry cubic CsPbX3 impurity) and controlling the compositional variation of Cs-Pb salts. This strategy enabled the synthesis of CsPbX3 free Cs4PbX6 nanocrystals. A high PL quantum yield (QY) of Mn2+ emission was obtained in the colloidal (29%) and solid (21%, powder) forms. These performances can be attributed to structure-induced confinement effects, which enhance the energy transfer from localized host exciton states to Mn2+ dopant within the isolated octahedra of 0D perovskite. The present findings could lead to designing of low-dimensional perovskites as efficient light emitters with photo and chemical stability for high-performance optoelectronic devices.

Selective Fabrication of Single-Molecule Junctions by Interface Engineering.

Recent progress in addressing electrically driven single-molecule behaviors has opened up a path toward the controllable fabrication of molecular devices. Herein, the selective fabrication of single-molecule junctions is achieved by employing the external electric field. For molecular junctions with methylthio (-SMe), thioacetate (-SAc), amine (-NH2 ), and pyridyl (-PY), the evolution of their formation probabilities along with the electric field is extracted from the plateau analysis of individual single-molecule break junction traces. With the increase of the electric field, the SMe-anchored molecules show a different trend in the formation probability compared to the other molecular junctions, which is consistent with the density functional theory calculations. Furthermore, switching from an SMe-anchored junction to an SAc-anchored junction is realized by altering the electric field in a mixed solution. The results in this work provide a new approach to the controllable fabrication and modulation of single-molecule junctions and other bottom-up nanodevices at molecular scales.

A Course of Hands-On Nanopore Experiments for Undergraduates: Single-Molecule Detection with Portable Electrochemical Instruments

The prompt introduction of single-molecule science into undergraduates’ curricula is an urgent but challenging task. However, the research experiments are often too complicated and the instruments are too expensive to be widely transferred into a compulsory course of undergraduate students. Herein, we introduce a recently developed nanopore technique into an accessible laboratory course for students with the advantages of simple operation, high repeatability, and low cost. The nanopore course serves to help undergraduates in practice to understand single-molecule behaviors, think at a single molecule level, and gradually develop single-molecule solutions for scientific issues. The learning activities for undergraduate students include setup of the instrument, fabrication of a biological nanopore on an artificial lipid bilayer membrane, detection of individual oligonucleotides, and data analysis. An experiment example is single-molecule detection of the model DNA sample poly(dA)n (n = 4, 5) using the aerolysin nanopore. As our developed nanopore sensing system is simple, portable, and easy to handle, the experiment course could be implemented and popularized in undergraduates’ laboratory education. For the undergraduate students who have taken general chemistry and/or biochemistry courses, this hands-on experience of nanopore experiments would benefit them with a better understanding of the chemical world at a single-molecule level and further using single-molecule tools in various applications including DNA and/or protein sequencing.

Gaining insight into cellular cardiac physiology using single particle tracking

Single particle tracking (SPT) is a robust technique to monitor single-molecule behaviors in living cells directly. By this approach, we can uncover the potential biological significance of particle dynamics by statistically characterizing individual molecular behaviors. SPT provides valuable information at the single-molecule level, that could be obscured by simple averaging that is inherent to conventional ensemble measurements. Here, we give a brief introduction to SPT including the commonly used optical implementations, fluorescence labeling strategies, and data analysis methods. We then focus on how SPT has been harnessed to decipher myocardial function. In this context, SPT has provided novel insight into the lateral diffusion of signal receptors and ion channels, the dynamic organization of cardiac nanodomains, subunit composition and stoichiometry of cardiac ion channels, myosin movement along actin filaments, the kinetic features of transcription factors involved in cardiac remodeling, and the intercellular communication by nanotubes. Finally, we speculate on the prospects and challenges of applying SPT to future questions regarding cellular cardiac physiology using SPT.

Transient Spectral Fluctuations of Single Molecules Revealed using an Optical Antenna

Fluorescence spectra and lifetimes of single molecules reflect their specific local environments, which is not available from the ensemble-averaged measurements. Usually, a low signal-to-noise ratio of single-molecule spectroscopy impedes access to transient emission spectra of single molecules at room temperature, especially for the molecules with a low quantum yield. Here, we realize detection of transient single-molecule spectra with an effective integration time of ∼2.14 milliseconds at room temperature of methylene blue (MB) or crystal violet (CV) molecules. In the experiments, the transient emission spectra detection of single molecules is achieved through the enhanced signal using a plasmonic antenna, the suppressed background noise due to a low quantum yield, and the short dwelling time of molecular adsorption on the antenna surface. The transient spectra show apparent fluctuation in the emission intensity and shifts of the peak maximum. Interestingly, the emission spectrum presents a narrow band which can be fitted well with a single Lorentzian lineshape. The specific molecule configuration should dominate the transient spectral lineshape mainly, which is supported by the numerical ab initio simulations. These spectral fluctuations should attribute to the transitions between metastable minima in the molecular potential-energy surface. These findings provide us more understanding of the transient behaviors of single molecules at the solid-aqueous interfaces.

Binding Behavior of Carbonmonoxide to Gold Atoms on Ag(001)

The adsorption behavior of single CO molecules at 4 K bound to Au adatoms on a Ag(001) metal surface is studied with scanning tunneling microscopy (STM) and inelastic electron tunneling spectroscopy (IETS). In contrast to earlier observations two different binding configurations are observed—one on top of a Au adatom and the other one adsorbed laterally to Au on Ag(001). Moreover, IETS reveals different low-energy vibrational energies for the two binding sites as compared to the one for a single CO molecule bound to Ag(001). Density functional theory (DFT) calculations of the adsorption energies, the diffusion barriers, and the vibrational frequencies of the CO molecule on the different binding sites rationalize the experimental findings.

On the competition between interface energy and temperature in phase transition phenomena

Phase nucleation and propagation phenomena can be characterized by a cooperative behavior regulated by non local interactions between the multistable domains and with the loading device. Cooperativity is often macroscopically witnessed by a stress-peak, distinguishing the nucleation from the propagation stress, and by a larger size of the first nucleated domain. When low dimensional scales are considered, both in nanostructures or single molecule behaviors, the interfacial energy can compete with entropic effects, leading to the experimental observation of a temperature dependent phase transition strategy. We propose a fully analytical model, in the framework of equilibrium Statistical Mechanics, measuring such energetic competition and temperature dependent behavior, that well reproduces important experimental evidences. The effectiveness of the model is successfully tested in predicting the temperature dependent phase transition behavior of shape memory nanowires.

Nanomechanical Differences between Inactive and Active States of Rhodopsin from Molecular-Scale Simulation

Several membrane proteins, including G protein-coupled receptors (GPCRs), present a challenge in studying their structural and dynamical properties under physiological conditions. Moreover, to better understand the activity of proteins requires examination of single molecule behaviors rather than ensemble averaged behaviors. In this work we report the Force-distance curve-based AFM (FD-AFM) which was utilized to directly probe and localize the conformational states of a GPCR within an artificial membrane at nanoscale resolution. We have further validated the experimental results by molecular scale coarse-grained (CG) simulations of rhodopsin biomolecules. In the past, our CG model has been applied successfully for the study of the mechanical properties of large biological assemblies such as β-amyloid and α-synuclein fibrils. Both FD-AFM experimental results and the computational force profiles revealed that the active state of the receptor has a higher Young's modulus compared to the inactive state of the receptor. We show how the deformation of the hydrogen bond network triggers this difference and by the statistical analysis of the native contacts we highlight the underlying mechanism. Hence, the inactive and active states of rhodopsin could be differentiated based on the stiffness of the receptor. Our work paves the route towards the molecular characterization of protein states based on the Young's modulus, which is clear indication of the mechanochemical interplay of proteins within the cell membrane.

Analyzing Single-Molecule Behavior of a Small Protein in Confined Nanospace of a Biological Nanopore

Analyzing Single-Molecule Behavior of a Small Protein in Confined Nanospace of a Biological Nanopore

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy.

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

Giant Conductance Enhancement of Intramolecular Circuits through Interchannel Gating

Summary For neutral intramolecular circuits with two constitutionally identical branches, a maximum 4-fold increase in total conductance can be obtained according to constructive quantum interference (CQI). For charged intramolecular circuits, however, the strong electrostatic interactions entangle the quantum states of these two parallel pathways, thus introducing complicated transport behavior that warrants experimental investigation of the intramolecular circuit rules. Here, we report that a tetracationic cyclophane with parallel channels exhibits a 50-fold conductance enhancement compared with that of a single-channel control, an observation that supplements intramolecular circuit law in systems with strong Coulombic interactions. Flicker noise measurements and theoretical calculations show that strong electrostatic interactions between charged parallel channels—serving as the chemical gate to promote the effective conductance of each channel—and CQI boosts the total conductance of the two-channel circuit. The molecular design presented herein constitutes a proof-of-principle approach to charged intramolecular circuits that are desirable for quantum circuits and devices.

Lanthanide complexes based on a conjugated pyridine carboxylate ligand: structures, luminescence and magnetic properties.

Three lanthanide compounds have been synthesized, namely, {[Dy2(bpda)3(H2O)3]4·2H2O}(Dy-1), {[Sm(bpda)2·(H2O)]·H2O}n (Sm-2) and {[Tb2(bpda)3(H2O)3]4·2H2O} (Tb-3) (H2bpda = 2,2′-bipyridine-6,6′-dicarboxylic acid). Their structures were determined by single crystal X-ray diffraction and characterized by elemental analysis, infrared spectroscopy and thermogravimetric analysis. Dy-1 and Tb-3 are isostructural with a conjugate bimolecular four-nuclear cluster structure constructed with intramolecular hydrogen bonds and they form a 3D supramolecular structure with intermolecular hydrogen bonding. Sm-2 is a one-dimensional chain structure and is further connected by intricate hydrogen bonds into a three-dimensional supramolecular structure. These three compounds exhibit significant characteristic luminescence from the ligand to the central Ln(iii) ion, which is found by solid-state photoluminescence measurement. Sm-2 exhibits a long luminescence lifetime and high fluorescence quantum yield. A slow relaxation phenomenon is observed for the dysprosium compound by measuring the alternating-current susceptibility at low temperature and the underlying mechanism was further confirmed by theoretical calculations.

Multiple kinesins induce tension for smooth cargo transport.

How cargoes move within a crowded cell-over long distances and at speeds nearly the same as when moving on unimpeded pathway-has long been mysterious. Through an in vitro force-gliding assay, which involves measuring nanometer displacement and piconewtons of force, we show that multiple mammalian kinesin-1 (from 2 to 8) communicate in a team by inducing tension (up to 4 pN) on the cargo. Kinesins adopt two distinct states, with one-third slowing down the microtubule and two-thirds speeding it up. Resisting kinesins tend to come off more rapidly than, and speed up when pulled by driving kinesins, implying an asymmetric tug-of-war. Furthermore, kinesins dynamically interact to overcome roadblocks, occasionally combining their forces. Consequently, multiple kinesins acting as a team may play a significant role in facilitating smooth cargo motion in a dense environment. This is one of few cases in which single molecule behavior can be connected to ensemble behavior of multiple motors.