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

Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy

Andrea Sorannoa,  Brigitte Buchlia,  Daniel Nettelsa,  Ryan R. Chengb, Sonja Müller-Spätha,  Shawn H. Pfeilc,  Armin Hoffmanna, Everett A. Lipmanc,  Dmitrii E. Makarovb, and  Benjamin Schuler

Internal friction, which reflects the “roughness” of the energy landscape, plays an important role for proteins by modulating the dynamics of their folding and other conformational changes. However, the experimental quantification of internal friction and its contribution to folding dynamics has remained challenging. Here we use the combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and microfluidic mixing to determine the reconfiguration times of unfolded proteins and investigate the mechanisms of internal friction contributing to their dynamics. Using concepts from polymer dynamics, we determine internal friction with three complementary, largely independent, and consistent approaches as an additive contribution to the reconfiguration time of the unfolded state. We find that the magnitude of internal friction correlates with the compactness of the unfolded protein: its contribution dominates the reconfiguration time of approximately 100 ns of the compact unfolded state of a small cold shock protein under native conditions, but decreases for more expanded chains, and approaches zero both at high denaturant concentrations and in intrinsically disordered proteins that are expanded due to intramolecular charge repulsion. Our results suggest that internal friction in the unfolded state will be particularly relevant for the kinetics of proteins that fold in the microsecond range or faster. The low internal friction in expanded intrinsically disordered proteins may have implications for the dynamics of their interactions with cellular binding partners.

DOI

Journal: Proceedings of the National Academy of Sciences

Functional Assembly of Aptamer Binding Sites by Single-Molecule Cut-and-Paste

Mathias Strackharn, Stefan W. Stahl, Elias M. Puchner, and Hermann E. Gaub

Bottom up assembly of functional molecular ensembles with novel properties emerging from composition and arrangement of its constituents is a prime goal of nanotechnology. By single-molecule cut-and-paste we assembled binding sites for malachite green in a molecule-by-molecule assembly process from the two halves of a split aptamer. We show that only a perfectly joined binding site immobilizes the fluorophore and enhances the fluorescence quantum yield by several orders of magnitude. To corroborate the robustness of this approach we produced a micrometer-sized structure consisting of more than 500 reconstituted binding sites. To the best of our knowledge, this is the first demonstration of one by one bottom up functional biomolecular assembly.


DOI


Journal: Nano Letters