Single-molecule biophysics

  • Article
    | Open Access

    Bacterial surface adhesion proteins are characterized by unusual mechanical properties. Here, the authors use atomic force microscopy-based technique to study a surface-anchoring protein Cpe0147 from Clostridium perfringens and show that an ester bond can withstand considerable mechanical forces and prevent complete protein unfolding.

    • Hai Lei
    • , Quan Ma
    •  & Yi Cao
  • Article
    | Open Access

    Variants of the extracellular chaperone Clusterin are associated with Alzheimer’s disease (AD) and Clusterin levels are elevated in AD patient brains. Here, the authors show that Clusterin binds to oligomeric Tau, which enhances the seeding capacity of Tau aggregates upon cellular uptake. They also demonstrate that Tau/Clusterin complexes enter cells via the endosomal pathway, resulting in damage to endolysosomes and entry into the cytosol, where they induce the aggregation of endogenous, soluble Tau.

    • Patricia Yuste-Checa
    • , Victoria A. Trinkaus
    •  & F. Ulrich Hartl
  • Article
    | Open Access

    High-speed atomic force microscopy height spectroscopy and single channel electrophysiology recordings are used to correlate conformational and functional dynamics of the model membrane protein, outer membrane protein G (OmpG). These techniques show that both states coexist and rapidly interchange in all conditions supported by molecular dynamics simulations.

    • Raghavendar Reddy Sanganna Gari
    • , Joel José Montalvo‐Acosta
    •  & Simon Scheuring
  • Article
    | Open Access

    Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs) but only few specific interactions are known. Using TIRF microscopy based assays, functional studies and an experimentally validated prediction algorithm, the authors show that specific PIP binding is widespread among human PH domains.

    • Nilmani Singh
    • , Adriana Reyes-Ordoñez
    •  & Jie Chen
  • Article
    | Open Access

    Fly Dicer-2 is thought to use two distinct – processive or distributive – modes of cleavage by distinguishing the terminal structures of double-stranded RNA (dsRNA) substrates with the help of its cofactor LoquaciousPD (Loqs-PD). Here the authors show by single-molecule imaging that dsRNA terminal structures and Loqs-PD change the probability for Dicer to initiate processive cleavage but not the mode of cleavage action per se.

    • Masahiro Naganuma
    • , Hisashi Tadakuma
    •  & Yukihide Tomari
  • Article
    | Open Access

    miRNA profiling from patient blood can be used for cancer diagnosis. Here the authors present an electro-optical nanopore sensing platform which allows sensitive and specific miRNA detection directly in human serum and apply to monitoring of miR-141-3p and miR-375-3p in different stage of prostate cancer.

    • Shenglin Cai
    • , Thomas Pataillot-Meakin
    •  & Joshua B. Edel
  • Article
    | Open Access

    Ring ATPase translocases that operate on disordered substrates adopt lockwasher architectures and use a hand-over-hand mechanism. By challenging the dsDNA packaging motor of bacteriophage ϕ29 with hybrid and dsRNA, the authors propose that the motor cycles between planar and lock-washer structures.

    • Juan P. Castillo
    • , Alexander B. Tong
    •  & Carlos Bustamante
  • Article
    | Open Access

    Nanopores have been used for direct observation of RNA structure in native environments but have limited RNA differentiation capabilities. Here, the authors report on the use of Mycobacterium smegmatis porin A nanopores for the trapping and translocation identification of microRNA, siRNA, tRNA and ribosomal RNA.

    • Yuqin Wang
    • , Xiaoyu Guan
    •  & Shuo Huang
  • Article
    | Open Access

    During bacterial cell division, the protein FtsZ is the main component of the contractile ring, though how precisely FtsZ treadmilling and its ability to deform membranes cooperate are unclear. Here, the authors show that dynamic FtsZ may deform lipid membranes via torsional stress that may provide sufficient force to constrict membranes in vivo and in vitro.

    • Diego A. Ramirez-Diaz
    • , Adrián Merino-Salomón
    •  & Petra Schwille
  • Article
    | Open Access

    Most insights on DNA-mediated allostery upon transcription factor (TF) binding were either based on artificial promoters or found to be short-ranged. Here authors use single-molecule FRET and cryo-EM to show that Bacillus subtilis bacteria utilize long-range allostery in a stochastic and reversible phenotype switch.

    • Gabriel Rosenblum
    • , Nadav Elad
    •  & Hagen Hofmann
  • Article
    | Open Access

    Molecular dynamics (MD) techniques enable atomic-level observations, but simulations of “slow” biomolecular processes are challenging because of current computer speed limitations. Here, the authors develop a method to accelerate MD simulations by high-frequency ultrasound perturbation and reveal binding events between the protein CDK2 and its small-molecule inhibitors.

    • Mitsugu Araki
    • , Shigeyuki Matsumoto
    •  & Yasushi Okuno
  • Article
    | Open Access

    N-Methyl-D-aspartate receptors (NMDARs) activation involves closure of the GluN1 and GluN2 subunit ligand binding domains, which is regulated allosterically by the amino-terminal domain (ATD). Here, smFRET, used to monitor conformational rearrangements of the NMDAR ATD, reveals that glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation that are regulated by protons.

    • Vojtech Vyklicky
    • , Cherise Stanley
    •  & Ehud Y. Isacoff
  • Article
    | Open Access

    In the genome, repetitive guanine-rich sequences have the potential to spontaneously fold into non-canonical DNA secondary structures known as G-quadruplex (G4). Using novel single-molecule imaging approaches, the authors reveal that G4 formation within active replication forks locally perturb replisome dynamics and damage response signaling, which require RPA and FANCJ for regulation.

    • Wei Ting C. Lee
    • , Yandong Yin
    •  & Eli Rothenberg
  • Article
    | Open Access

    Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their sensitivity and specificity are still poorly defined. Here, authors report a FRET-based sensor that informs directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level.

    • Janett Göhring
    • , Florian Kellner
    •  & Gerhard J. Schütz
  • Article
    | Open Access

    Relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here authors combine ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling.

    • Thomas Schlichthaerle
    • , Caroline Lindner
    •  & Ralf Jungmann
  • Article
    | Open Access

    Von Willebrand factor (VWF) is a large glycoprotein in the blood secreted from endothelial cells lining the blood vessel and activation of VWF leads to formation of VWF-platelet complexes or thrombi. Here authors use single-molecule force measurement, X-ray crystallography and functional measurements to monitor the activation of VWF via mechanical unfolding of the autoinhibitory module (AIM).

    • Nicholas A. Arce
    • , Wenpeng Cao
    •  & Renhao Li
  • Article
    | Open Access

    P450 oxidoreductase (POR) selectively activates numerous cytochromes P450 (CYP), crucial for metabolism of drugs, steroids and xenobiotics and natural product biosynthesis. Here, the authors identify ligands that bind POR and bias its specificity towards CYP redox partners, activating distinct metabolic cascades in cells.

    • Simon Bo Jensen
    • , Sara Thodberg
    •  & Nikos S. Hatzakis
  • Article
    | Open Access

    Eukaryotic DNA replication is regulated to ensure copying of the genome (only) once per cell cycle. Here the authors, using optical trapping and confocal microscopy, demonstrate the dynamics of the origin recognition complex and subsequent intermediates that lead up to the loading of an MCM helicase onto DNA.

    • Humberto Sánchez
    • , Kaley McCluskey
    •  & Nynke H. Dekker
  • Article
    | Open Access

    FUS/EWS/TAF15 (FET) fusion oncoproteins contain low complexity domain which forms biomolecular condensates that recruit RNA polymerase II. Here the authors develop a single-molecule assay to visualize this phenomenon providing in vitro evidence to support causative relationship between the formation of condensates on DNA and gene transcription. Furthermore, they also determine a threshold number of fusion-binding DNA satellite elements required for the formation of FET protein condensates.

    • Linyu Zuo
    • , Guanwei Zhang
    •  & Zhi Qi
  • Article
    | Open Access

    In cells, DNA is arranged into topologically-constrained (supercoiled) structures, but how this supercoiling affects the detailed double-helical structure of DNA remains unclear. Here authors use atomic force microscopy and atomistic molecular dynamics simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution.

    • Alice L. B. Pyne
    • , Agnes Noy
    •  & Sarah A. Harris
  • Article
    | Open Access

    The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link. Here authors show that a single tip-link bond is more mechanically stable relative to classic cadherins, and that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains.

    • Eric M. Mulhall
    • , Andrew Ward
    •  & Wesley P. Wong
  • Article
    | Open Access

    Our understanding of the molecular mechanisms underlying pathological protein aggregation remains incomplete. Here, single molecule infrared nanospectroscopy (AFM-IR) offers insight into the structure of Aβ42 oligomeric and fibrillar species and their interaction with an aggregation inhibitor, paving the way for single molecule drug discovery studies.

    • Francesco Simone Ruggeri
    • , Johnny Habchi
    •  & Tuomas P. J. Knowles
  • Article
    | Open Access

    While Cas9 outperforms TALENs in euchromatin, it is less efficient in heterochromatic regions. Here the authors, using single-molecule imaging, show that Cas9 uses a less efficient search strategy compared to TALENs in these regions.

    • Surbhi Jain
    • , Saurabh Shukla
    •  & Huimin Zhao
  • Article
    | Open Access

    Bacterial protein FtsZ polymerizes at mid-cell and exhibits treadmilling dynamics, driving the movement of enzymes that synthesize septal peptidoglycan. Here, McCausland et al. combine theoretical modelling with single-molecule imaging of bacteria to show that FtsZ treadmilling drives enzyme movement via a Brownian ratchet mechanism.

    • Joshua W. McCausland
    • , Xinxing Yang
    •  & Jian Liu
  • Article
    | Open Access

    Genome recoding with quadruplet codons requires a +1-frameshift-suppressor tRNA able to insert an amino acid at quadruplet codons of interest. Here the authors identify the mechanisms resulting in +1 frameshifting and the steps of the elongation cycle in which it occurs.

    • Howard Gamper
    • , Haixing Li
    •  & Ya-Ming Hou
  • Article
    | Open Access

    DNA three-way junctions are branched structures formed during replication, repair, and recombination, and are involved in models of repeat expansion. Here the authors use single-molecule Förster resonance energy transfer to reveal the dynamics of DNA three-way junctions containing slip-outs composed of CAG or CTG repeats.

    • Tianyu Hu
    • , Michael J. Morten
    •  & Steven W. Magennis
  • Article
    | Open Access

    The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology. Here, the authors use a combination of live cell imaging and in vitro single molecule binding measurements to show that tandem calponin homology domains (CH1–CH2) are sensitive to actin filament conformation, biasing their subcellular localization.

    • Andrew R. Harris
    • , Pamela Jreij
    •  & Daniel A. Fletcher
  • Article
    | Open Access

    Biochemistry combined with biophysical measurements and mathematical modeling offer insight into the mechanism by which the cotranslational chaperone, nascent polypeptide-associated complex (NAC), modulates substrate selection by signal recognition particle (SRP) and reduces aberrant, nonspecific targeting of ribosomes to the ER.

    • Hao-Hsuan Hsieh
    • , Jae Ho Lee
    •  & Shu-ou Shan
  • Article
    | Open Access

    Traces from single-molecule fluorescence microscopy (SMFM) experiments exhibit photophysical artifacts that typically make analysis time-consuming. Here, the authors have developed an easily accessible software, AutoSiM, for two distinct applications of deep learning to the efficient processing of SMFM time traces.

    • Jieming Li
    • , Leyou Zhang
    •  & Nils G. Walter
  • Article
    | Open Access

    The intrinsically disordered linker histone H1.0 and prothymosin α form a complex which exhibits slow exchange between bound and unbound populations at low protein concentrations and fast exchange at high concentrations. Here authors explain this observation by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange.

    • Andrea Sottini
    • , Alessandro Borgia
    •  & Benjamin Schuler
  • Article
    | Open Access

    Broad uptake of smFRET has been hindered by high instrument costs and a lack of open-source hardware and acquisition software. Here, the authors present the smfBox, a cost-effective open-source platform capable of measuring precise FRET efficiencies between dyes on freely diffusing single molecules.

    • Benjamin Ambrose
    • , James M. Baxter
    •  & Timothy D. Craggs
  • Article
    | Open Access

    Building regulatory networks often requires trade-offs between accuracy and speed. Here the authors show in a bistable network the transition from a slow decision making system to a rapid one dominated by small number fluctuations.

    • Ferdinand Greiss
    • , Shirley S. Daube
    •  & Roy Bar-Ziv
  • Article
    | Open Access

    The xrRNA1 RNA from ZIKA virus (ZIKV) forms a complex ring-like architecture and is known for its mechanical anisotropy, but the mechanism for its direction-dependent mechanical responses remain unclear. Here authors use a single-molecule nanopore sensing technique combined with molecular dynamics simulations and show that the anisotropy in ZIKV xrRNA1 depends on Mg2+ and the key tertiary interactions.

    • Xiaolin Niu
    • , Qiuhan Liu
    •  & Xianyang Fang
  • Article
    | Open Access

    During colonization of host tissues and biomaterials, staphylococci are exposed to extreme mechanical forces. Here authors use force-clamp spectroscopy and show that the dock, lock and latch interaction between staphylococcal surface protein SpsD and fibrinogen is extremely strong, and exhibits an unusual catch-slip transition.

    • Marion Mathelié-Guinlet
    • , Felipe Viela
    •  & Yves F. Dufrêne
  • Article
    | Open Access

    To overcome the limitation of FRET data being too sparse to cover all structural details, FRET experiments need to be carefully designed and complemented with simulations. Here the authors present a toolkit for automated design of FRET experiments, which determines how many and which FRET pairs should be used to maximize the accuracy, and for FRET-assisted structural modeling and refinement at the atomistic level.

    • Mykola Dimura
    • , Thomas-Otavio Peulen
    •  & Holger Gohlke
  • Article
    | Open Access

    In vivo experiments and optical tweezers force-spectroscopy measurements assessing the co-translational folding of the G-domain from bacterial elongation factor G reveal a sequential folding pathway initiating from the C-terminus. These results suggest that protein folding and synthesis proceed in opposite directions.

    • Xiuqi Chen
    • , Nandakumar Rajasekaran
    •  & Christian M. Kaiser
  • Article
    | Open Access

    Excitatory amino acid transporters (EAATs) are crucial for the removal of excitatory amino acids from the synaptic cleft. Here authors combined high-speed atomic force microscopy line-scanning with automated state assignment for the determination of transport dynamics of GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution.

    • Tina R. Matin
    • , George R. Heath
    •  & Simon Scheuring
  • Article
    | Open Access

    Determining molecular clustering in Photoactivated Localization Microscopy (PALM) experiments requires knowledge of the blinking properties of the fluorophore to prevent overcounting artefacts. Here the authors develop an experimental and analytical framework to determine the blinking parameters of fluorophores and incorporate this information into cluster analysis.

    • René Platzer
    • , Benedikt K. Rossboth
    •  & Mario Brameshuber
  • Article
    | Open Access

    Single molecule force measurements have shed light on dynamic biological events, but rare events escape notice owing to low throughput of the methods. Here, the authors combine an array of magnetic tweezers with lateral flow to increase throughput 100-fold, and detect rare DNA breaks induced by gyrase.

    • Rohit Agarwal
    •  & Karl E. Duderstadt
  • Article
    | Open Access

    Mechanically stable specific heterodimerization formed with reversible bonds are used as a molecular anchorage in single-molecule force spectroscopy studies with unique mechanical properties. Here authors develop a variety of heterodimerization molecular systems with a range of mechanical stability from a set of recently engineered helix-heterotetramers.

    • Miao Yu
    • , Zhihai Zhao
    •  & Jie Yan
  • Article
    | Open Access

    DNA-PAINT is a powerful super-resolution imaging method but is limited in speed due to slow exchange kinetics of the imaging strand. Here the authors present a method involving the addition of ethylene carbonate to the imaging buffer and modifications to the docking strand to improve the quality and speed of DNA-PAINT.

    • Fehmi Civitci
    • , Julia Shangguan
    •  & Xiaolin Nan
  • Article
    | Open Access

    Conformational dynamics during the early stage of transcription is crucial to understanding the regulation of transcription efficiency and fidelity. Here the authors, by single-molecule fluorescence resonance energy transfer approaches, examine the conformational dynamics of the two-component transcription system of yeast mitochondria with single-base resolution.

    • Byeong-Kwon Sohn
    • , Urmimala Basu
    •  & Hajin Kim
  • Article
    | Open Access

    The ATPase SecA drives Sec-dependent protein translocation across the bacterial plasma membrane. Here, the authors combine kinetic translocation measurements with single-molecule force spectroscopy and demonstrate that the SecA motor generates mechanical force to unfold and translocate preproteins.

    • Riti Gupta
    • , Dmitri Toptygin
    •  & Christian M. Kaiser