Superhelical Architecture of the Myosin Filament-Linking Protein Myomesin with Unusual Elastic Properties

Nikos Pinotsis, Spyros D. Chatziefthimiou Felix Berkemeier, Fabienne Beuron, Irene M. Mavridis, Petr V. Konarev, Dmitri I. Svergun, Edward Morris, Matthias Rief, Matthias Wilmanns


Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, to maintain an ordered state, they require molecular structures of extraordinary stability. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain arrays. Members of the myomesin protein family function as molecular bridges that connect major filament systems in the central M-band of muscle sarcomeres, which is a central locus of passive stress sensing. To unravel the mechanism of molecular elasticity in such filament-connecting proteins, we have determined the overall architecture of the complete C-terminal immunoglobulin domain array of myomesin by X-ray crystallography, electron microscopy, solution X-ray scattering, and atomic force microscopy. Our data reveal a dimeric tail-to-tail filament structure of about 360 Å in length, which is folded into an irregular superhelical coil arrangement of almost identical α-helix/domain modules. The myomesin filament can be stretched to about 2.5-fold its original length by reversible unfolding of these linkers, a mechanism that to our knowledge has not been observed previously. Our data explain how myomesin could act as a highly elastic ribbon to maintain the overall structural organization of the sarcomeric M-band. In general terms, our data demonstrate how repetitive domain modules such as those found in myomesin could generate highly elastic protein structures in highly organized cell systems such as muscle sarcomeres.


DOI


Journal: PLoS Biology

Nanomechanical detection of nuclear magnetic resonance using a silicon nanowire oscillator

John M. Nichol, Eric R. Hemesath, Lincoln J. Lauhon, and Raffi Budakian


The authors report the use of a radio frequency (rf) silicon nanowire mechanical oscillator as a low-temperature nuclear magnetic resonance force sensor to detect the statistical polarization of¹ H spins in polystyrene. To couple the ¹ H spins to the nanowire oscillator, a magnetic resonance force detection protocol was developed that utilizes a nanoscale current–carrying wire to produce large time-dependent magnetic field gradients as well as the rf magnetic field. Under operating conditions, the nanowire experienced negligible surface-induced dissipation and exhibited an ultralow force noise near the thermal limit of the oscillator.


DOI


Journal: Physical Review B

Chain Length Determines the Folding Rates of RNA

Changbong Hyeon and D. Thirumalai

We show that the folding rates (kFs) of RNA are determined by N, the number of nucleotides. By assuming that the distribution of free-energy barriers separating the folded and the unfolded states is Gaussian, which follows from central limit theorem arguments and polymer physics concepts, we show that . Remarkably, the theory fits experimental rates spanning over 7 orders of magnitude with . Our finding suggests that the speed limit of RNA folding is ∼1 ms, just as it is in the folding of globular proteins. RNA molecules are evolved biopolymers whose folding has attracted a great deal of attention  because of the crucial role they play in a number of cellular functions. The slightly branched polymeric nature of RNA implies that the shapes, relaxation dynamics, and even their folding rates must depend on N. In support of this assertion, it has been shown that the radius of gyration of the folded states, obtained with the use of data available in the Protein Data Bank, scales as  Å, where the Flory exponent ν varies from 0.33 to 0.40. Although this result is expected from the perspective of polymer physics, it is surprising from the viewpoint of structural biology because one might argue that the sequence and complexity of secondary and tertiary structure organization could lead to substantial deviations from the predictions based on Flory-like theory. Here, we show that the folding rates, kFs, of RNA are also primarily determined by N, thus adding to the growing evidence that it is possible to understand RNA folding by using polymer physics principles.


DOI


Journal: Biophysical Journal

Mechanical Force Can Fine-Tune Redox Potentials of Disulfide Bonds

Ilona B. Baldus and Frauke Gräter



Mechanical force applied along a disulfide bond alters its rate of reduction. We here aimed at quantifying the direct effect of force onto the chemical reactivity of a sulfur-sulfur bond in contrast to indirect, e.g., steric or mechanistic, influences. To this end, we evaluated the dependency of a disulfide bond's redox potential on a pulling force applied along the system. Our QM/MM simulations of cystine as a model system take conformational dynamics and explicit solvation into account and show that redox potentials increase over the whole range of forces probed here (303320 pN), and thus even in the absence of a significant disulfide bond elongation (<500 pN). Instead, at low forces, dihedrals and angles, as the softer degrees of freedom are stretched, contribute to the destabilization of the oxidized state. We find physiological forces to be likely to tune the disulfide's redox potentials to an extent similar to the tuning within proteins by point mutations.

DOI

Journal: Biophysical Journal

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

Tomoya Hino, Takatoshi Arakawa, Hiroko Iwanari, Takami Yurugi-Kobayashi, Chiyo Ikeda-Suno, Yoshiko Nakada-Nakura, Osamu Kusano-Arai, Simone Weyand, Tatsuro Shimamura, Norimichi Nomura, Alexander D. Cameron, Takuya Kobayashi, Takao Hamakubo, So Iwata, and Takeshi Murata

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A2AAR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A2AAR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β2-adrenergic receptor structure, but locks A2AAR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.


DOI


Journal: Nature

Cytoplasmic Dynein Moves Through Uncoordinated Stepping of the AAA+ Ring Domains

Mark A. DeWitt, Amy Y. Chang, Peter A. Combs, and Ahmet Yildiz

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos toward the microtubule minus end. How the two catalytic head domains interact and move relative to each other during processive movement is unclear. Here, we tracked the relative positions of both heads with nanometer precision and directly observed the heads moving independently along the microtubule. The heads remained widely separated, and their stepping behavior varied as a function of interhead separation. One active head was sufficient for processive movement, and an active head could drag an inactive partner head forward. Thus, dynein moves processively without interhead coordination, a mechanism fundamentally distinct from the hand-over-hand stepping of kinesin and myosin.


DOI


Journal: Science

Single-Molecule Lysozyme Dynamics Monitored by an Electronic Circuit

Yongki Choi, Issa S. Moody, Patrick C. Sims, Steven R. Hunt, Brad L. Corso, Israel Perez, Gregory A. Weiss, Philip G. Collins

Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stable, high-bandwidth transducer for protein motion. Electronic monitoring during 10-minute periods extended well beyond the limitations of fluorescence techniques to uncover dynamic disorder within a single molecule and establish lysozyme as a processive enzyme. On average, 100 chemical bonds are processively hydrolyzed, at 15-hertz rates, before lysozyme returns to its nonproductive, 330-hertz hinge motion. Statistical analysis differentiated single-step hinge closure from enzyme opening, which requires two steps. Seven independent time scales governing lysozyme’s activity were observed. The pH dependence of lysozyme activity arises not from changes to its processive kinetics but rather from increasing time spent in either nonproductive rapid motions or an inactive, closed conformation.

DOI

Journal: Science