Rate limit of protein elastic response is tether dependent

Ronen Berkovich, Rodolfo I. Hermans, Ionel Popa, Guillaume Stirnemann, Sergi Garcia-Manyes, Bruce J. Berne, and Julio M. Fernandez

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient D_eff ∼ 1,200 nm²/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at D_eff ∼ 10⁸ nm²/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo withD_eff ∼ 10⁴–10⁶ nm²/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk.

DOI

Journal: Proceedings of the National Academy of Sciences

Interpreting the widespread nonlinear force spectra of intermolecular bonds

Raymond W. Friddle, Aleksandr Noy, and James J. De Yoreo

Single molecule force spectroscopy probes the strength, lifetime, and energetic details of intermolecular interactions in a simple experiment. A growing number of these studies have reported distinctly nonlinear trends in rupture force with loading rate that are typically explained in conventional models by invoking complex escape pathways. Recent analyses suggested that these trends should be expected even for simple barriers based on the basic assumptions of bond rupture dynamics and thus may represent the norm rather than the exception. Here we explore how these nonlinear trends reflect the two fundamental regimes of bond rupture: (i) a near-equilibrium regime, produced either by bond reforming in the case of a single bond or by asynchronized rupture of multiple individual bonds, and (ii) a kinetic regime produced by fast, non-equilibrium bond rupture. We analyze both single- and multi-bonded cases, describe the full evolution of the system as it transitions between near- and far-from-equilibrium loading regimes, and show that both interpretations produce essentially identical force spectra. Data from 10 different molecular systems show that this model provides a comprehensive description of force spectra for a diverse suite of bonds over experimentally relevant loading rates, removes the inconsistencies of previous interpretations of transition state distances, and gives ready access to both kinetic and thermodynamic information about the interaction. These results imply that single-molecule binding free energies for a vast number of bonds have already been measured.

DOI

Journal: Proceedings of the National Academy of Sciences

Transition Path Times for Nucleic Acid Folding Determined from Energy-Landscape Analysis of Single-Molecule Trajectories

Krishna Neupane, Dustin B. Ritchie, Hao Yu1, Daniel A. N. Foster, Feng Wang, and Michael T. Woodside

The duration of structural transitions in biopolymers is only a fraction of the time spent searching diffusively over the configurational energy landscape. We found the transition time, τTP, and the diffusion constant, D, for DNA and RNA folding using energy landscapes obtained from single-molecule trajectories under tension in optical traps. DNA hairpins, RNA pseudoknots, and a riboswitch all had τTP∼10  μs and D∼10-13–14  m2/s, despite widely differing unfolding rates. These results show how energy-landscape analysis can be harnessed to characterize brief but critical events during folding reactions.

DOI

Journal: Physical Review Letters

Flightless I is a focal adhesion-associated actin-capping protein that regulates cell migration

Ibrahim Mohammad, Pamma D. Arora, Yeganeh Naghibzadeh, Yongqiang Wang, Jeff Li, Wendall Mascarenhas, Paul A. Janmey, John F. Dawson and Christopher A. McCulloch


The role of adhesion-associated actin-binding proteins in cell migration is not well defined. In mouse fibroblasts we screened for focal adhesion-associated proteins that were isolated with collagen-coated beads and detected by tandem mass spectrometry. We identified flightless I (FliI) as an actin-binding protein in focal adhesion fractions, which was verified by immunoblotting. By confocal microscopy most FliI was distributed throughout the cytosol and in focal adhesions. By sedimentation assays and in vitro binding assays, we found that FliI associates with actin filaments and actin monomers. Assays using purified proteins showed that FliI inhibits actin polymerization and caps but does not sever actin filaments. Cells with FliI knockdown or cells overexpressing FliI migrated more or less rapidly, respectively, than wild-type controls. Compared with controls, cells with FliI knockdown were less adherent than wild-type cells, exhibited reduced numbers of focal adhesions containing activated β1 integrins and vinculin, and exhibited increased incorporation of actin monomers into nascent filaments at focal adhesions. These data indicate that FliI regulates cell migration through its localization to focal adhesions and its ability to cap actin filaments, which collectively affect focal adhesion maturation.—Mohammad, I., Arora, P. D., Naghibzadeh, Y., Wang, Y., Li, J., Mascarenhas, W., Janmey, P. A., Dawson, J. F., McCulloch, C. A. Flightless I is a focal adhesion-associated actin-capping protein that regulates cell migration.


DOI


Journal: The FASEB Journal

An α Helix to β Barrel Domain Switch Transforms the Transcription Factor RfaH into a Translation Factor

Björn M. Burmann, Stefan H. Knauer, Anastasia Sevostyanova, Kristian Schweimer, Rachel A. Mooney, Robert Landick, Irina Artsimovitch, Paul Rösch

NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a β barrel in NusG and an α hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-α to an all-β conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated.


DOI


Journal: Cell

Rocket Launcher Mechanism of Collaborative Actin Assembly Defined by Single-Molecule Imaging

Dennis Breitsprecher, Richa Jaiswal, Jeffrey P. Bombardier, Christopher J. Gould, Jeff Gelles, and Bruce L. Goode

Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single-molecule fluorescence microscopy to image the tumor suppressor adenomatous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers initiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly and then separate but retain independent associations with either end of the growing filament.

DOI

Journal: Science

Revealing the Angular Symmetry of Chemical Bonds by Atomic Force Microscopy

Joachim Welker, and Franz J. Giessibl


We have measured the angular dependence of chemical bonding forces between a carbon monoxide molecule that is adsorbed to a copper surface and the terminal atom of the metallic tip of a combined scanning tunneling microscope and atomic force microscope. We provide tomographic maps of force and current as a function of distance that revealed the emergence of strongly directional chemical bonds as tip and sample approach. The force maps show pronounced single, dual, or triple minima depending on the orientation of the tip atom, whereas tunneling current maps showed a single minimum for all three tip conditions. We introduce an angular dependent model for the bonding energy that maps the observed experimental data for all observed orientations and distances.


DOI


Journal: Science