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

Folding without charges

Martin Kurnik, Linda Hedberg, Jens Danielsson, and Mikael Oliveberg


Surface charges of proteins have in several cases been found to function as “structural gatekeepers,” which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.


DOI


Journal: Proceedings of the National Academy of Sciences

Long-range mechanical force enables self-assembly of epithelial tubular patterns

Chin-Lin Guo, Mingxing Ouyang, Jiun-Yann Yu, Jordan Maslov, Andrew Price, and Chih-Yu Shen



Enabling long-range transport of molecules, tubules are critical for human body homeostasis. One fundamental question in tubule formation is how individual cells coordinate their positioning over long spatial scales, which can be as long as the sizes of tubular organs. Recent studies indicate that type I collagen (COL) is important in the development of epithelial tubules. Nevertheless, how cell–COL interactions contribute to the initiation or the maintenance of long-scale tubular patterns is unclear. Using a two-step process to quantitatively control cell–COL interaction, we show that epithelial cells developed various patterns in response to fine-tuned percentages of COL in ECM. In contrast with conventional thoughts, these patterns were initiated and maintained by traction forces created by cells but not diffusive factors secreted by cells. In particular, COL-dependent transmission of force in the ECM led to long-scale (up to 600 μm) interactions between cells. A mechanical feedback effect was encountered when cells used forces to modify cell positioning and COL distribution and orientations. Such feedback led to a bistability in the formation of linear, tubule-like patterns. Using micro-patterning technique, we further show that the stability of tubule-like patterns depended on the lengths of tubules. Our results suggest a mechanical mechanism that cells can use to initiate and maintain long-scale tubular patterns.

DOI

Journal: Proceedings of the National Academy of Sciences