Driving a Macroscopic Oscillator with the Stochastic Motion of a Hydrogen Molecule

Christian Lotze, Martina Corso, Katharina J. Franke, Felix von Oppen, Jose Ignacio Pascual

Energy harvesting from noise is a paradigm proposed by the theory of stochastic resonances. We demonstrate that the random switching of a hydrogen (H2) molecule can drive the oscillation of a macroscopic mechanical resonator. The H2 motion was activated by tunneling electrons and caused fluctuations of the forces sensed by the tip of a noncontact atomic force microscope. The stochastic molecular noise and the periodic oscillation of the tip were coupled in a concerted dynamic that drives the system into self-oscillation. This phenomenon could be a way for enhancing the transfer of energy from incoherent sources into coherent dynamics of a molecular engine.

DOI

Journal: Science

Protein Folding Drives Disulfide Formation

Pallav Kosuri, Jorge Alegre-Cebollada, Jason Feng, Anna Kaplan, Alvaro Inglés-Prieto, Carmen L. Badilla, Brent R. Stockwell, Jose M. Sanchez-Ruiz, Arne Holmgren, Julio M. Fernández

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.

DOI

Journal: Cell

Atomic Force Microscopy with Nanoscale Cantilevers Resolves Different Structural Conformations of the DNA Double Helix

Carl Leung, Aizhan Bestembayeva, Richard Thorogate, Jake Stinson, Alice Pyne, Christian Marcovich, Jinling Yang, Ute Drechsler, Michel Despont, Tilo Jankowski, Martin Tschöpe, and Bart W. Hoogenboom

Structural variability and flexibility are crucial factors for biomolecular function. Here we have reduced the invasiness and enhanced the spatial resolution of atomic force microscopy (AFM) to visualize, for the first time, different structural conformations of the two polynucleotide strands in the DNA double helix, for single molecules under near-physiological conditions. This is achieved by identifying and tracking the anomalous resonance behavior of nanoscale AFM cantilevers in the immediate vicinity of the sample.

DOI

Journal: Nano Letters

Regulation of the H4 tail binding and folding landscapes via Lys-16 acetylation

Davit A. Potoyan and Garegin A. Papoian

Intrinsically disordered proteins (IDP) are a broad class of proteins with relatively flat energy landscapes showing a high level of functional promiscuity, which are frequently regulated through posttranslational covalent modifications. Histone tails, which are the terminal segments of the histone proteins, are prominent IDPs that are implicated in a variety of signaling processes, which control chromatin organization and dynamics. Although a large body of work has been done on elucidating the roles of posttranslational modifications in functional regulation of IDPs, molecular mechanisms behind the observed behaviors are not fully understood. Using extensive atomistic molecular dynamics simulations, we found in this work that H4 tail mono-acetylation at LYS-16, which is a key covalent modification, induces a significant reorganization of the tail’s conformational landscape, inducing partial ordering and enhancing the propensity for alpha-helical segments. Furthermore, our calculations of the potentials of mean force between the H4 tail and a DNA fragment indicate that contrary to the expectations based on simple electrostatic reasoning, the Lys-16 mono-acetylated H4 tail binds to DNA stronger than the unacetylated protein. Based on these results, we propose a molecular mechanism for the way Lys-16 acetylation might lead to experimentally observed disruption of compact chromatin fibers.

DOI

Journal: Proceedings of the National Academy of Sciences

Direct Observation of Cotranscriptional Folding in an Adenine Riboswitch

Kirsten L. Frieda, Steven M. Block

Growing RNA chains fold cotranscriptionally as they are synthesized by RNA polymerase. Riboswitches, which regulate gene expression by adopting alternative RNA folds, are sensitive to cotranscriptional events. We developed an optical-trapping assay to follow the cotranscriptional folding of a nascent RNA and used it to monitor individual transcripts of the pbuE adenine riboswitch, visualizing distinct folding transitions. We report a particular folding signature for the riboswitch aptamer whose presence directs the gene-regulatory transcription outcome, and we measured the termination frequency as a function of adenine level and tension applied to the RNA. Our results demonstrate that the outcome is kinetically controlled. These experiments furnish a means to observe conformational switching in real time and enable the precise mapping of events during cotranscriptional folding.

DOI

Journal: Science

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

Andrea Soranno, Brigitte Buchli, Daniel Nettels, Ryan R. Cheng, Sonja Müller-Späth, Shawn H. Pfeil, Armin Hoffmann, Everett A. Lipman, Dmitrii E. Makarov, 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

Bond-Order Discrimination by Atomic Force Microscopy

Leo Gross, Fabian Mohn, Nikolaj Moll, Bruno Schuler, Alejandro Criado, Enrique Guitián, Diego Peña, André Gourdon, Gerhard Meyer

We show that the different bond orders of individual carbon-carbon bonds in polycyclic aromatic hydrocarbons and fullerenes can be distinguished by noncontact atomic force microscopy (AFM) with a carbon monoxide (CO)–functionalized tip. We found two different contrast mechanisms, which were corroborated by density functional theory calculations: The greater electron density in bonds of higher bond order led to a stronger Pauli repulsion, which enhanced the brightness of these bonds in high-resolution AFM images. The apparent bond length in the AFM images decreased with increasing bond order because of tilting of the CO molecule at the tip apex.

DOI

Journal: Science

Protein folding – simplicity in complexity

M.M. Lin, A.H. Zewail

Understanding and predicting protein folding would elucidate how misfolded proteins cause aggregation and amyloid formation, for example in Alzheimer's disease. Despite the seemingly bewildering complexity of protein biology, simple analytic models can still capture the basic physics and predict the fundamental limits for protein domain size and folding speed.

DOI

Journal: Annalen der Physik

Molecular Mechanics of Cardiac Myosin-Binding Protein C in Native Thick Filaments

M. J. Previs, S. Beck Previs, J. Gulick, J. Robbins, D. M. Warshaw

The heart’s pumping capacity results from highly regulated interactions of actomyosin molecular motors. Mutations in the gene for a potential regulator of these motors, cardiac myosin-binding protein C (cMyBP-C), cause hypertrophic cardiomyopathy. However, cMyBP-C’s ability to modulate cardiac contractility is not well understood. Using single-particle fluorescence imaging techniques, transgenic protein expression, proteomics, and modeling, we found that cMyBP-C slowed actomyosin motion generation in native cardiac thick filaments. This mechanical effect was localized to where cMyBP-C resides within the thick filament (i.e., the C-zones) and was modulated by phosphorylation and site-specific proteolytic degradation. These results provide molecular insight into why cMyBP-C should be considered a member of a tripartite complex with actin and myosin that allows fine tuning of cardiac muscle contraction.

DOI

Journal: Science

Initiation of transcription-coupled repair characterized at single-molecule resolution

Kévin Howan, Abigail J. Smith, Lars F. Westblade, Nicolas Joly, Wilfried Grange, Sylvain Zorman, Seth A. Darst, Nigel J. Savery, and Terence R. Strick

Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.

DOI

Journal: Nature

Collective many-body van der Waals interactions in molecular systems

Robert A. DiStasio, Jr., O. Anatole von Lilienfeld, and Alexandre Tkatchenko

Van der Waals (vdW) interactions are ubiquitous in molecules and condensed matter, and play a crucial role in determining the structure, stability, and function for a wide variety of systems. The accurate prediction of these interactions from first principles is a substantial challenge because they are inherently quantum mechanical phenomena that arise from correlations between many electrons within a given molecular system. We introduce an efficient method that accurately describes the nonadditive many-body vdW energy contributions arising from interactions that cannot be modeled by an effective pairwise approach, and demonstrate that such contributions can significantly exceed the energy of thermal fluctuations—a critical accuracy threshold highly coveted during molecular simulations—in the prediction of several relevant properties. Cases studied include the binding affinity of ellipticine, a DNA-intercalating anticancer agent, the relative energetics between the A- and B-conformations of DNA, and the thermodynamic stability among competing paracetamol molecular crystal polymorphs. Our findings suggest that inclusion of the many-body vdW energy is essential for achieving chemical accuracy and therefore must be accounted for in molecular simulations.

DOI

Journal: Proceedings of the National Academy of Sciences

Direct observation of a force-induced switch in the anisotropic mechanical unfolding pathway of a protein

Bharat Jagannathan, Phillip J. Elms, Carlos Bustamante, and Susan Marqusee

Many biological processes generate force, and proteins have evolved to resist and respond to tension along different force axes. Single-molecule force spectroscopy allows for molecular insight into the behavior of proteins under force and the mechanism of protein folding in general. Here, we have used src SH3 to investigate the effect of different pulling axes under the low-force regime afforded by an optical trap. We find that this small cooperatively folded protein shows an anisotropic response to force; the protein is more mechanically resistant to force applied along a longitudinal axis compared to force applied perpendicular to the terminal β strand. In the longitudinal axis, we observe an unusual biphasic behavior revealing a force-induced switch in the unfolding mechanism suggesting the existence of two parallel unfolding pathways. A site-specific variant can selectively affect one of these pathways. Thus, even this simple two-state protein demonstrates a complex mechanical unfolding trajectory, accessing multiple unfolding pathways under the low-force regime of the optical trap; the specific unfolding pathway depends on the perturbation axis and the applied force.

DOI

Journal: Proceedings of the National Academy of Sciences

OpenFovea: open-source AFM data processing software

Charles Roduit, Bhaskar Saha, Livan Alonso-Sarduy, Andrea Volterra, Giovanni Dietler, and Sandor Kasas

The atomic force microscope (AFM) is often used to detect specific molecules on the surfaces of specimens and to measure a specimen's mechanical properties. This information can be deduced from the force-distance curves generated by the deflection of the cantilever as it approaches, indents and recedes from the sample. Force-volume images can be generated from a series of successive force-distance curves recorded while scanning an area of the specimen. This imaging mode has been used since 1997 to image, among others, proteins at molecular resolution, molecular patterns, bacteria, fungi and living cells. Algorithms for processing force-volume data files have been published3, but there is no universal ready-to-use software for analysis of force-distance curves, and investigators must develop their own custom software.

DOI

Journal: Nature Methods

Key stabilizing elements of protein structure identified through pressure and temperature perturbation of its hydrogen bond network

Lydia Nisius, and Stephan Grzesiek

Hydrogen bonds are key constituents of biomolecular structures, and their response to external perturbations may reveal important insights about the most stable components of a structure. NMR spectroscopy can probe hydrogen bond deformations at very high resolution through hydrogen bond scalar couplings (HBCs). However, the small size of HBCs has so far prevented a comprehensive quantitative characterization of protein hydrogen bonds as a function of the basic thermodynamic parameters of pressure and temperature. Using a newly developed pressure cell, we have now mapped pressure- and temperature-dependent changes of 31 hydrogen bonds in ubiquitin by measuring HBCs with very high precision. Short-range hydrogen bonds are only moderately perturbed, but many hydrogen bonds with large sequence separations (high contact order) show greater changes. In contrast, other high-contact-order hydrogen bonds remain virtually unaffected. The specific stabilization of such topologically important connections may present a general principle with which to achieve protein stability and to preserve structural integrity during protein function.

DOI

Journal: Nature Chemistry

Experimental free-energy measurements of kinetic molecular states using fluctuation theorems

Anna Alemany, Alessandro Mossa, Ivan Junier, and Felix Ritort

Recent advances in non-equilibrium statistical mechanics and single-molecule technologies have made it possible to use irreversible work measurements to extract free-energy differences associated with the mechanical (un)folding of molecules. To date, free-energy recovery has been focused on native (or equilibrium) molecular states, but free-energy measurements of kinetic states have remained unexplored. Kinetic states are metastable, finite-lifetime states that are generated dynamically, and play important roles in diverse physical processes. In biophysics, there are many examples in which these states determine the fate of molecular reactions, including protein binding, enzymatic reactions, as well as the formation of transient intermediate states during molecular-folding processes. Here we demonstrate that it is possible to obtain free energies of kinetic states by applying extended fluctuation relations, using optical tweezers to mechanically unfold and refold deoxyribonucleic acid (DNA) structures exhibiting intermediate and misfolded kinetic states.

DOI

Journal: Nature Physics

A Whole-Cell Computational Model Predicts Phenotype from Genotype

Jonathan R. Karr, Jayodita C. Sanghvi, Derek N. Macklin, Miriam V. Gutschow, Jared M. Jacobs, Benjamin Bolival Jr., Nacyra Assad-Garcia, John I. Glass, Markus W. Covert

Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology that computational approaches are poised to tackle. We report a whole-cell computational model of the life cycle of the human pathogen Mycoplasma genitalium that includes all of its molecular components and their interactions. An integrative approach to modeling that combines diverse mathematics enabled the simultaneous inclusion of fundamentally different cellular processes and experimental measurements. Our whole-cell model accounts for all annotated gene functions and was validated against a broad range of data. The model provides insights into many previously unobserved cellular behaviors, including in vivo rates of protein-DNA association and an inverse relationship between the durations of DNA replication initiation and replication. In addition, experimental analysis directed by model predictions identified previously undetected kinetic parameters and biological functions. We conclude that comprehensive whole-cell models can be used to facilitate biological discovery.

DOI

Journal: Cell

Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae

Mohan Babu et. al

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases1. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein–protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.

DOI

Journal: Nature

How Do Proteins Unfold upon Adsorption on Nanoparticle Surfaces?

Hai Pan , Meng Qin , Wei Meng , Yi Cao, and Wei Wang

Owing to their many outstanding features, such as small size, large surface area, and cell penetration ability, nanoparticles have been increasingly used in medicine and biomaterials as drug carriers and diagnostic or therapeutic agents. However, our understanding of the interactions of biological entities, especially proteins, with nanoparticles is far behind the explosive development of nanotechnology. In typical protein–nanoparticle interactions, two processes (i.e., surface binding and conformational changes in proteins) are intermingled with each other and have not yet been quantitatively described. Here, by using a stopped-flow fast mixing technique, we were able to shed light on the kinetics of the adsorption-induced protein unfolding on nanoparticle surfaces in detail. We observed a biphasic denaturation behavior of protein GB1 on latex nanoparticle surfaces. Such kinetics can be adequately described by a fast equilibrium adsorption followed by a slow reversible unfolding of GB1. On the basis of this model, we quantitatively measured all rate constants that are involved in this process, from which the free-energy profile is constructed. This allows us to evaluate the effects of environmental factors, such as pH and ionic strength, on both the adsorption and the conformational change in GB1 on the latex nanoparticle surface. These studies provide a general physical picture of the adsorption-induced unfolding of proteins on nanoparticle surfaces and a quantitative description of the energetics of each transition. We anticipate that it will greatly advance our current understanding of protein–nanoparticle interactions and will be helpful for the rational control of such interactions in biomedical applications.

DOI

Journal: Langmuir

Covalent Mechanochemistry: Theoretical Concepts and Computational Tools with Applications to Molecular Nanomechanics

Jordi Ribas-Arino and Dominik Marx

DOI

Journal: Chemical Reviews

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates

Hao Yu, Amar Nath Gupta, Xia Liu,, Krishna Neupane, Angela M. Brigley, Iveta Sosova, and Michael T. Woodside

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.

DOI

Journal: Proceedings of the National Academy of Sciences

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

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

Evidence that a ‘dynamic knockout’ in Escherichia coli dihydrofolate reductase does not affect the chemical step of catalysis

E. Joel Loveridge, Enas M. Behiry, Jiannan Guo, and Rudolf K. Allemann


The question of whether protein motions play a role in the chemical step of enzymatic catalysis has generated much controversy in recent years. Debate has recently reignited over possible dynamic contributions to catalysis in dihydrofolate reductase, following conflicting conclusions from studies of the N23PP/S148A variant of the Escherichia coli enzyme. By investigating the temperature dependence of kinetic isotope effects, we present evidence that the reduction in the hydride transfer rate constants in this variant is not a direct result of impairment of conformational fluctuations. Instead, the conformational state of the enzyme immediately before hydride transfer, which determines the electrostatic environment of the active site, affects the rate constant for the reaction. Although protein motions are clearly important for binding and release of substrates and products, there appears to be no detectable dynamic coupling of protein motions to the hydride transfer step itself.

DOI

Journal: Nature Chemistry

Cavities determine the pressure unfolding of proteins

Julien Roche, Jose A. Caro, Douglas R. Norberto, Philippe Barthe, Christian Roumestand, Jamie L. Schlessman, Angel E. Garcia, Bertrand García-Moreno E., and Catherine A. Royer

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

DOI

Journal: Proceedings of the National Academy of Sciences

Single-molecule mechanical identification and sequencing

Fangyuan Ding, Maria Manosas, Michelle M Spiering, Stephen J Benkovic, David Bensimon, Jean-François Allemand, and Vincent Croquette



High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.

DOI

Journal: Nature Methods

Imaging the charge distribution within a single molecule

Fabian Mohn, Leo Gross, Nikolaj Moll, and Gerhard Meyer


Scanning tunnelling microscopy and atomic force microscopy can be used to study the electronic and structural properties of surfaces, as well as molecules and nanostructures adsorbed on surfaces, with atomic precision, but they cannot directly probe the distribution of charge in these systems. However, another form of scanning probe microscopy, Kelvin probe force microscopy, can be used to measure the local contact potential difference between the scanning probe tip and the surface, a quantity that is closely related to the charge distribution on the surface. Here, we use a combination of scanning tunnelling microscopy, atomic force microscopy and Kelvin probe force microscopy to examine naphthalocyanine molecules (which have been used as molecular switches) on a thin insulating layer of NaCl on Cu(111). We show that Kelvin probe force microscopy can map the local contact potential difference of this system with submolecular resolution, and we use density functional theory calculations to verify that these maps reflect the intramolecular distribution of charge. This approach could help to provide fundamental insights into single-molecule switching and bond formation, processes that are usually accompanied by the redistribution of charge within or between molecules 


DOI


Journal: Nature Nanotechnology

Piezo proteins are pore-forming subunits of mechanically activated channels

Bertrand Coste, Bailong Xiao, Jose S. Santos, Ruhma Syeda, Jörg Grandl, Kathryn S. Spencer, Sung Eun Kim, Manuela Schmidt, Jayanti Mathur, Adrienne E. Dubin, Mauricio Montal, and Ardem Patapoutian


Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ~1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.


DOI


Journal: Nature

Proton Grease: An Acid Accelerated Molecular Rotor

Brent E. Dial, Perry J. Pellechia, Mark D. Smith, and Ken D. Shimizu

A molecular rotor was designed that rotates 7 orders of magnitude faster upon protonation. The quinoline rotor is based on a rigid N-arylimide framework that displays restricted rotation due to steric interaction between the quinoline nitrogen and imide carbonyls. At rt (23 °C), the rotor rotates slowly (t1/2 = 26 min, ΔG‡ = 22.2 kcal/mol). However, upon addition of 3.5 equiv of acid the rotor rotates rapidly (t1/2 = 2.0 × 10–4 s, ΔG‡ = 12.9 kcal/mol). Mechanistic studies show that this dramatic acid catalyzed change is due to stabilization of the planar transition state by the formation of an intramolecular hydrogen bond between the protonated quinoline nitrogen (N+—H) and an imide carbonyl (O═C). The acid catalyzed acceleration is reversible and can be stopped by addition of base.

DOI


Journal: Journal of the American Chemical Society

Thermodynamic and Kinetic Analysis of an RNA Kissing Interaction and Its Resolution into an Extended Duplex

Nilshad Salim, Rajan Lamichhane, Rui Zhao, Tuhina Banerjee, Jane Philip, David Rueda, and Andrew L. Feig



Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.

DOI

Journal: Biophysical Journal

Mechanical Anisotropy of Ankyrin Repeats

Whasil Lee, Xiancheng Zeng, Kristina Rotolo, Ming Yang, Christopher J. Schofield, Vann Bennett, Weitao Yang, and Piotr E. Marszalek



Red blood cells are frequently deformed and their cytoskeletal proteins such as spectrin and ankyrin-R are repeatedly subjected to mechanical forces. While the mechanics of spectrin was thoroughly investigated in vitro and in vivo, little is known about the mechanical behavior of ankyrin-R. In this study, we combine coarse-grained steered molecular dynamics simulations and atomic force spectroscopy to examine the mechanical response of ankyrin repeats (ARs) in a model synthetic AR protein NI6C, and in the D34 fragment of native ankyrin-R when these proteins are subjected to various stretching geometry conditions. Our steered molecular dynamics results, supported by AFM measurements, reveal an unusual mechanical anisotropy of ARs: their mechanical stability is greater when their unfolding is forced to propagate from the N-terminus toward the C-terminus (repeats unfold at ∼60 pN), as compared to the unfolding in the opposite direction (unfolding force ∼ 30 pN). This anisotropy is also reflected in the complex refolding behavior of ARs. The origin of this unfolding and refolding anisotropy is in the various numbers of native contacts that are broken and formed at the interfaces between neighboring repeats depending on the unfolding/refolding propagation directions. Finally, we discuss how these complex mechanical properties of ARs in D34 may affect its behavior in vivo

DOI

Journal: Biophysical Journal

Peptide tag forming a rapid covalent bond to a protein

Bijan Zakeri, Jacob O. Fierer, Emrah Celik, Emily C. Chittock,Ulrich Schwarz-Linek, Vincent T. Moy, and Mark Howarth

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures. 

DOI

Journal: Proceedings of the National Academy of Sciences