Single-molecule Force Measurements Research Articles

Joining forces: integrating the mechanical and optical single molecule toolkits

Combining single molecule force measurements with fluorescence detection opens up exciting new possibilities for the characterization of mechanoresponsive molecules in Biology and Materials Science.

Single Molecule Force Measurements of DNA and RNA Hairpin Structures

DNA and RNA hairpins contribute to fundamental mechanisms of molecular biology, for instance to the regulation of transcription and translation. Moreover, the hairpin secondary structure is a model system for studying the dynamic assembly of nucleic acid structures. Using a high-precision dual-trap optical tweezers setup, we performed a quantitative investigation of the folding dynamics of hairpins under mechanical load.A hairpin structure is composed of a double-helical stem and a single-stranded loop. Two different hairpins were studied, each one as a DNA and as an RNA molecule. They have the same stem of 13 basepairs, but exhibit loops of 10 and 18 nucleotides respectively. Significant differences are observed, not only between the two hairpins but also between DNA and RNA. As illustrated in the figure, hysteresis between unfolding and refolding curves is much stronger for RNA than for DNA. For narrow-loop hairpins, flipping between folded and unfolded states is observed for DNA and RNA. The wide-loop hairpins, however, show flips only for DNA. Altogether, our results show that the RNA molecule is more easily driven out-of-equilibrium by an external perturbation than DNA.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Polyarginine induces an antitumor immune response through binding to toll-like receptor 4.

A novel function of polyarginine as an activator of the immune system is reported. Single-molecule fluorescence imaging and single-molecule force measurements demonstrate that polyarginine binds specifically to the toll-like receptor 4 (TLR4), inducing receptor dimerization and activation. Moreover, polyarginine induced immune activation inhibits tumor growth. These results suggest the potential use ofpolyarginine for cancer immunotherapy.

Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome

BRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important serine/threonine-protein kinase. Two domains on BRaf can independently bind its upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain (RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers to compare the Ras binding process in two pathological mutants of BRaf responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two mutants differ in their kinetics of Ras-binding, though both bind Ras with similar increased overall affinity. BRaf (A246P) exhibits a slightly higher Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force versus the wild type. The contrary phenomenon is observed in the Q257R mutation. Simulations of the unstressed-off rate, koff (0), yield results in accordance with the changes revealed by the mean unbinding force. Our approach can be applied to rapidly assess other mutated proteins to deduce the effects of mutation on their kinetics compared to wild type proteins and to each other.

Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells

With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods.

P130Cas substrate domain is intrinsically disordered as characterized by single-molecule force measurements

P130Cas is a docking protein essentially coordinating tyrosine-kinase-based signaling pathways associated with cell adhesion and migration etc. Its central substrate domain (CasSD) can bind to Crk and includes 15 YxxP motifs, where most tyrosine phosphorylation happens. It has been shown that CasSD can be stretched to promote phosphorylation, the mechanism of which needs to be explored in detail. Thus, it is important to uncover the native structure(s) of CasSD and the structural changes associated with mechanical stretching, both of which are still unclear. Here, we used atomic force microscopy force mode and magnetic tweezers to stretch individual molecules of CasSD constructs. Our results showed that the CasSD domain was intrinsically disordered. Natively, CasSD domains took many conformations beside random coils, while most of these conformations possessed limited mechanical stability. In magnetic tweezers experiments, the intramolecular interactions stabilizing the varied native conformations of CasSD were found similar in strength. Such diversity in native conformations of CasSD domains, as discovered here, should play important role in their signaling functions and their limited strength should be relevant to the mechanical activation of those signaling pathways.

Identification of an Inhibitory Mechanism of Luteolin on the Insulin‐Like Growth Factor‐1 Ligand–Receptor Interaction

Using single-molecule force measurement and fluorescence imaging, we have demonstrated that luteolin has an inhibitory effect on IGF-1 ligand-receptor binding, the initial step in IGF-1 signaling. This inhibition mechanism, which was confirmed by flow cytometry and molecular docking, could play a role in cancer therapy.

Denaturation transition of stretched DNA

In the last two decades, single-molecule force measurements using optical and magnetic tweezers and atomic force spectroscopy have dramatically expanded our knowledge of nucleic acids and proteins. These techniques characterize the force on a biomolecule required to produce a given molecular extension. When stretching long DNA molecules, the observed force-extension relationship exhibits a characteristic plateau at approximately 65 pN where the DNA may be extended to almost twice its B-DNA length with almost no increase in force. In the present review, I describe this transition in terms of the Poland-Scheraga model and summarize recent related studies.

A Novel Electromagnetic Apparatus for Rapid Multiplex Single Molecule Force Spectroscopy

Single-molecule force spectroscopy has revolutionized our ability to probe the details of molecular structures and interactions, but the numbers of individual measurements required for achieving a statistically reliable result can sometimes prove daunting. To overcome this problem, a number of instruments have recently been developed that are capable of monitoring the behavior of tens of individual biomolecules simultaneously. In this work, we have constructed a novel electromagnetic apparatus for multiplex single molecule force measurements utilizing magnetic microspheres. In this system, the magnetic field is generated with an electron-lens of an electron microscope mated with a high voltage flash light circuit to rapidly attain a stable magnetic field. We show that this instrument can generate a uniform magnetic force of up to -20 pN within 5 ms, over a region spanning 1 mm. The successful application of this apparatus to the force-dependent extension of dsDNA fully validates this approach. Furthermore, the lens-like design of the pole piece is fully compatible with optical imaging, thus allowing for the integration of single molecule fluorescence capabilities that should make this system a particularly powerful apparatus for multi-dimensional characterization of fast processes within interacting single molecules.

Resolving Cadherin Catch Bond Formation at the Single Molecule Level

Cadherins are Ca2+-dependent cell-cell adhesion proteins which play key roles in the formation and maintenance of tissues in multi-cellular organisms. We recently showed that cadherins withstand tensile force by forming catch bonds where their bond lifetimes increase with tensile force. Here we resolve the molecular mechanism of cadherin catch bond formation. Using Steered Molecular Dynamics (SMD) simulations, we show that catch bonds are formed because tensile force re-orients the cadherins such that they lock into a tighter contact. Our simulations also predict that cadherins are unable to lock into a tighter binding conformation if their extracellular regions are made more floppy by titrating Ca2+ ions from solution. Using single molecule force measurements with an Atomic Force Microscope (AFM), we confirm that catch bond formation is abolished as Ca2+ concentration is reduced. Based on these results we propose a molecular model by which cadherins withstand mechanical stress and strengthen cell-cell junctions.

Effect of Non-Canonical Structures on the DNA Duplex

DNA is constantly being modified by exogenous and endogenous agents, leading to covalent changes to its structure and, if left unrepaired, to permanent changes in sequence. We use a wide variety of biophysical techniques to examine the effect on DNA structure and dynamics of the common base lesion 8-oxoguanine, the cis-syn thymine dimer lesion, single-base mismatches, and bubbles. The stability of duplex formation in the presence of these disturbances is explored using UV melting thermodynamics, calorimetry, and single-molecule force measurements. Duplex structure is analyzed using small molecule reactive probes, circular dichroism, and NOESY spectroscopy. NMR proton exchange experiments reveal locations of dynamic changes to the DNA duplex, including base pair opening and flexibility. By bringing a diverse set of techniques to bear on each non-canonical structure, we have shown the 8oxoG lesion to be surprisingly stable, while the thymine dimer lesion is significantly more labile. Even Hoogsteen-binding wobble mismatches can effect the kinetics and thermodynamics of duplex formation in surprising ways. These results complement and extend the information obtained by crystallographic methods to include dynamic and thermodynamic factors.

Computational Studies on Physical Mechanisms of T7 RNA Polymerase Elongation and Nucleotide Selection

The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we study T7 RNAP as a model system for transcription elongation. Based on structural information and experimental data from single-molecule force measurements, we had shown that a small translocation free energy bias aids initial nucleotide selection during elongation [1]. The selection is conducted by a conserved residue Tyr639 next to the active site. At the post-translocated state under the energy bias, Tyr639 side chain is stabilized IN the active site in order to screen incoming nucleotides for pre-insertion. We further show kinetically that the screening at the early stage of nucleotide selection is particularly important for achieving both low error rate and high enough transcription speed. To test our hypotheses and examine the system with substantial details, atomistic molecular dynamics simulations are conducted to evaluate free energy changes accompanying essential conformational changes of the RNAP. We also study how exactly the nucleotide selection proceeds in T7 RNAP by examining binding affinities of different nucleotides to the elongation complex at both the pre-insertion and insertion checkpoints. Our studies intend to illustrate at multiple time and length scales the physical mechanisms of how a simplest RNAP moves and transcribes with high enough fidelity. [1] A small post-translocation energy bias aids nucleotide selection in T7 RNA polymerase transcription. Jin Yu, George Oster. Biophysical Journal, 102, 532-541,2012

Interactions of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes at cold and ambient temperatures—Surface morphology and single-molecule force measurements show phase separation, and reveal tertiary and quaternary associations

Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Here, we investigate the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We show using compression studies with a Langmuir–Blodgett trough that both dehydrins can stabilize lipid monolayers with a lipid composition mimicking the composition of the plant outer mitochondrial membrane, which had previously been shown to induce ordered secondary structures (disorder-to-order transitions) in the proteins. Ellipsometry of the monolayers during compression showed an increase in monolayer thickness upon introducing TsDHN-1 (acidic) at 4°C and TsDHN-2 (basic) at room temperature. Atomic force microscopy of supported lipid bilayers showed temperature-dependent phase transitions and domain formation induced by the proteins. These results support the conjecture that acidic dehydrins interact with and potentially stabilize plant outer mitochondrial membranes in conditions of cold stress. Single-molecule force spectroscopy of both proteins pulled from supported lipid bilayers indicated the induced formation of tertiary conformations in both proteins, and potentially a dimeric association for TsDHN-2.

Ideal, catch, and slip bonds in cadherin adhesion

Classical cadherin cell-cell adhesion proteins play key morphogenetic roles during development and are essential for maintaining tissue integrity in multicellular organisms. Classical cadherins bind in two distinct conformations, X-dimer and strand-swap dimer; during cellular rearrangements, these adhesive states are exposed to mechanical stress. However, the molecular mechanisms by which cadherins resist tensile force and the pathway by which they convert between different conformations are unclear. Here, we use single molecule force measurements with an atomic force microscope (AFM) to show that E-cadherin, a prototypical classical cadherin, forms three types of adhesive bonds: catch bonds, which become longer lived in the presence of tensile force; slip bonds, which become shorter lived when pulled; and ideal bonds that are insensitive to mechanical stress. We show that X-dimers form catch bonds, whereas strand-swap dimers form slip bonds. Our data suggests that ideal bonds are formed as X-dimers convert to strand-swap binding. Catch, slip, and ideal bonds allow cadherins to withstand tensile force and tune the mechanical properties of adhesive junctions.

Hesperetin: An inhibitor of the transforming growth factor-β (TGF-β) signaling pathway

We have identified a previously unknown function of the natural compound, hesperetin. Here, we demonstrate that this small molecular agent is able to inhibit the transforming growth factor-β (TGF-β) signaling pathway. Single-molecule force measurements and single-molecule fluorescence imaging show that hesperetin interferes with ligand–receptor interactions. Furthermore, by Western blot analysis, it was confirmed that hesperetin also inhibits the phosphorylation of Smad3, a down-stream target of the TGF-β pathway. In addition we demonstrated that this compound hinders TGF-β1-induced cancer cell migration and invasion. These results suggest a potential future application for hesperetin as a TGF-β inhibitor in cancer therapy.

The persistence length and length per base of single-stranded DNA obtained from fluorescence correlation spectroscopy measurements using mean field theory

A dynamical mean field theory is used to predict the end-monomer mean square displacement of single-stranded DNA and finally estimate two important parameters—the persistence length lp and the length per base ld. Both parameters are set free, and finally reach optimum values by fitting the theoretical data to the experimental data of Shusterman et al. [R. Shusterman, S. Alon, T. Gavrinyov, O. Krichevsky, Monomer dynamics in double- and single-stranded DNA polymers, Phys. Rev. Lett. 92 (2004) 048303]. Three optimization methods, global optimization, individual optimization and selected optimization are performed with the Monte Carlo method. All the optimization methods can faithfully reproduce the experimental data. In selected optimization for 2400 and 6700 bases ssDNA, lp=2.223nm and ld=0.676nm are obtained. The theoretical results show a larger persistence length for ssDNA than ordinary synthetic polymers, and the obtained length per base is larger than the reported value obtained from single molecule force measurements. The lp and ld obtained from mean field theory complement the current data previously measured for different salt concentrations in solution.

A Small Post-Translocation Energy Bias Aids Nucleotide Selection in T7 RNA Polymerase Transcription

The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we present a model of T7 RNAP translocation during elongation. Based on structural information and experimental data from single-molecule force measurements, we show that a small component of facilitated translocation or power stroke coexists with the Brownian-ratchet-driven motions, and plays a crucial role in nucleotide selection at pre-insertion. The facilitated translocation is carried out by the conserved Tyr639 that moves its side chain into the active site, pushing aside the 3′-end of the RNA, and forming a locally stabilized post-translocation intermediate. Pre-insertion of an incoming nucleotide into this stabilized intermediate state ensures that Tyr639 closely participates in selecting correct nucleotides. A similar translocation mechanism has been suggested for multi-subunit RNAPs involving the bridge-helix bending. Nevertheless, the bent bridge-helix sterically prohibits nucleotide binding in the post-transolocation intermediate analog; moreover, the analog is not stabilized unless an inhibitory protein factor binds to the enzyme. Using our scheme, we also compared the efficiencies of different strategies for nucleotide selection, and examined effects of facilitated translocation on forward tracking.

Single Molecule Measurements of Catch Bond Formation in Cadherin Cell-Adhesion Proteins

We resolve the molecular interactions by which cadherin cell adhesion proteins tune the mechanical properties of cell-cell junctions. Cells in multicellular organisms undergo constant rearrangements; during tissue formation and wound healing, cells divide, change positions and tug on their neighbors. While the genes that orchestrate these process have been studied extensively, we do not know nearly enough about how cell-cell contacts resist physical force. At the molecular level, cells are held together by adhesion proteins, most commonly by the cadherin family of proteins. Cadherins are essential for tissue formation and for maintaining tissue integrity. Using single molecule force measurements, we demonstrate that cadherin bonds become stronger as cells are pulled apart. This counter-intuitive data is the first demonstration that cadherin’s form catch bonds; the proteins grip strongly in the presence of an external force but detach when force is removed. Our results suggest a molecular mechanism for regulating cell-cell adhesion under conditions of variable mechanical stress.

Modelling Programmed Ribosomal Frameshifting with Elastic Network Model and Linear Response Theory

The dnaX gene in Escherichia coli encodes comparable amounts of τ and γ subunits of DNA polymerase III by programmed ribosomal frameshifting (PRF). An early termination codon in the −1 frame of dnaX produces the γ subunit, which is a truncated form of the τ subunit. PRF is also involved in protein expression in many viruses, such as the Gag-Pol polyprotein in HIV. Thus, PRF provides an intrinsic regulating pathway at the translation level from the same mRNA.In the dnaX mRNA, an upstream internal Shine-Dalgarno (SD) sequence, which lies within the open reading frame and forms base-pairing with the 3’-end of 16S rRNA, together with a downstream stem-loop structure promote PRF on the slippery site. However, the detailed mechanisms for ribosome stalling and backtracking during PRF remain to be elucidated. Here, we combine coarse-grained elastic network model (ENM) and linear response theory to model the intrinsic and perturbed dynamics of the ribosome; the latter results from the formation of SD-helix and the forces exerted by stem-loop hindrance during translation. We hypothesize that these opposing forces for ribosome progression, albeit from different RNA elements, induce similar conformational changes for the ribosome, possibly in an allosteric fashion. These changes in the ribosome are expected to promote the observed PRF efficiency. Ultimately, we wish to provide the mechanochemical bases of PRF through computational modelling and validations by single-molecule force measurements.

3C1058 リボソーム-SecM翻訳アレスト配列間相互作用の1分子顕微解析(核酸,口頭発表,日本生物物理学会第50回年会(2012年度))

3C1058 リボソーム-SecM翻訳アレスト配列間相互作用の1分子顕微解析(核酸,口頭発表,日本生物物理学会第50回年会(2012年度))