Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

Sean Yu McLoughlin, Mark Kastantin, Daniel K. Schwartz, and Joel L. Kaar

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.

DOI

Journal: Proceedings of the National Academy of Sciences

High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations

Felix Rico, Laura Gonzalez, Ignacio Casuso, Manel Puig-Vidal, Simon Scheuring

The mechanical unfolding of the muscle protein titin by atomic force microscopy was a landmark in our understanding of single-biomolecule mechanics. Molecular dynamics simulations offered atomic-level descriptions of the forced unfolding. However, experiment and simulation could not be directly compared because they differed in pulling velocity by orders of magnitude. We have developed high-speed force spectroscopy to unfold titin at velocities reached by simulation (~4 millimeters per second). We found that a small β-strand pair of an immunoglobulin domain dynamically unfolds and refolds, buffering pulling forces up to ~100 piconewtons. The distance to the unfolding transition barrier is larger than previously estimated but is in better agreement with atomistic predictions. The ability to directly compare experiment and simulation is likely to be important in studies of biomechanical processes.

DOI

Journal: Science

Amyloid-β Dynamics Are Regulated by Orexin and the Sleep-Wake Cycle

Jae-Eun Kang, Miranda M. Lim, Randall J. Bateman, James J. Lee, Liam P. Smyth, John R. Cirrito, Nobuhiro Fujiki, Seiji Nishino, David M. Holtzman

Amyloid-β (Aβ) accumulation in the brain extracellular space is a hallmark of Alzheimer’s disease. The factors regulating this process are only partly understood. Aβ aggregation is a concentration-dependent process that is likely responsive to changes in brain interstitial fluid (ISF) levels of Aβ. Using in vivo microdialysis in mice, we found that the amount of ISF Aβ correlated with wakefulness. The amount of ISF Aβ also significantly increased during acute sleep deprivation and during orexin infusion, but decreased with infusion of a dual orexin receptor antagonist. Chronic sleep restriction significantly increased, and a dual orexin receptor antagonist decreased, Aβ plaque formation in amyloid precursor protein transgenic mice. Thus, the sleep-wake cycle and orexin may play a role in the pathogenesis of Alzheimer’s disease.

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Journal: Science

Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus

Robert H. Vass and Peter Chien

Chromosome replication relies on sliding clamps that are loaded by energy-dependent complexes. In Escherichia coli, the ATP-binding clamp loader subunit DnaX exists as both long (τ) and short (γ) forms generated through programmed translational frameshifting, but the need for both forms is unclear. Here, we show that in Caulobacter crescentus, DnaX isoforms are unexpectedly generated through partial proteolysis by the AAA+ protease casein lytic proteinase (Clp) XP. We find that the normally processive ClpXP protease partially degrades DnaX to produce stable fragments upon encountering a glycine-rich region adjacent to a structured domain. Increasing the sequence complexity of this region prevents partial proteolysis and generates a τ-only form of DnaX in vivo that is unable to support viability on its own. Growth is restored when γ is provided in trans, but these strains are more sensitive to DNA damage compared with strains that can generate γ through proteolysis. Our work reveals an unexpected mode of partial processing by the ClpXP protease to generate DnaX isoforms, demonstrates that both τ and γ forms of DnaX are required for Caulobacter viability, and identifies a role for clamp loader diversity in responding to DNA damage. The conservation of distinct DnaX isoforms throughout bacteria despite fundamentally different mechanisms for producing them suggests there may be a conserved need for alternate clamp loader complexes during DNA damaging conditions.

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Journal: Proceedings of the National Academy of Sciences

Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

Gabriel Žoldáka, Johannes Stigler, Benjamin Pelz, Hongbin Li, and Matthias Rief

In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.

DOI

Journal: Proceedings of the National Academy of Sciences

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Mahmood Haj-Yahya, Bruno Fauvet, Yifat Herman-Bachinsky, Mirva Hejjaoui, Sudhir N. Bavikara, Subramanian Vedhanarayanan Karthikeyan, Aaron Ciechanover, Hilal A. Lashuel, and Ashraf Brik

Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di- or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications.

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Journal: Proceedings of the National Academy of Sciences

Single-molecule fluorescence probes dynamics of barrier crossing

Hoi Sung Chung, and William A. Eaton

Kramers developed the theory on how chemical reaction rates are influenced by the viscosity of the medium1. At the viscosity of water, the kinetics of unimolecular reactions are described by diffusion of a Brownian particle over a free-energy barrier separating reactants and products. For reactions in solution this famous theory extended Eyring’s transition state theory, and is widely applied in physics, chemistry and biology, including to reactions as complex as protein folding. Because the diffusion coefficient of Kramers’ theory is determined by the dynamics in the sparsely populated region of the barrier top, its properties have not been directly measured for any molecular system. Here we show that the Kramers diffusion coefficient and free-energy barrier can be characterized by measuring the temperature- and viscosity-dependence of the transition path time for protein folding. The transition path is the small fraction of an equilibrium trajectory for a single molecule when the free-energy barrier separating two states is actually crossed. Its duration, the transition path time, can now be determined from photon trajectories for single protein molecules undergoing folding/unfolding transitions5. Our finding of a long transition path time with an unusually small solvent viscosity dependence suggests that internal friction as well as solvent friction determine the Kramers diffusion coefficient for α-helical proteins, as opposed to a breakdown of his theory, which occurs for many small-molecule reactions2. It is noteworthy that the new and fundamental information concerning Kramers’ theory and the dynamics of barrier crossings obtained here come from experiments on a protein rather than a much simpler chemical or physical system.

DOI

Journal: Nature

Nanomechanics of HaloTag Tethers

Ionel Popa , Ronen Berkovich, Jorge Alegre-Cebollada, Carmen L. Badilla, Jaime Andrés Rivas-Pardo, Yukinori Taniguchi , Masaru Kawakami , and Julio M. Fernandez 

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to 2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding–unfolding cycles at high forces, without interference from the HaloTag tether.

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Journal: Journal of the American Chemical Society

Reshaping of the conformational search of a protein by the chaperone trigger factor

Alireza Mashaghi, Günter Kramer, Philipp Bechtluft, Beate Zachmann-Brand, Arnold J. M. Driessen, Bernd Bukau, and Sander J. Tans

Protein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts as well as by rescuing misfolds after it ends. Although chaperones have long been speculated to also affect the conformational search itself—by reshaping the underlying folding landscape along the folding trajectory—direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome, with polypeptides released from the ribosome into the cytosol, and with fully folded proteins before their assembly into larger complexes. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.

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Journal: Nature

Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation

Joe Swift, Irena L. Ivanovska, Amnon Buxboim, Takamasa Harada, P. C. Dave P. Dingal, Joel Pinter, J. David Pajerowski, Kyle R. Spinler, Jae-Won Shin, Manorama Tewari, Florian Rehfeldt, David W. Speicher, Dennis E. Discher

Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.

DOI

Journal: Science

Switching of a coupled spin pair in a single-molecule junction

Stefan Wagner, Ferdinand Kisslinger, Stefan Ballmann, Frank Schramm, Rajadurai Chandrasekar, Tilmann Bodenstein, Olaf Fuhr, Daniel Secker, Karin Fink, Mario Ruben, and Heiko B. Weber

Single-molecule spintronics investigates electron transport through magnetic molecules that have an internal spin degree of freedom. To understand and control these individual molecules it is important to read their spin state. For unpaired spins, the Kondo effect has been observedas a low-temperature anomaly at small voltages. Here, we show that a coupled spin pair in a single magnetic molecule can be detected and that a bias voltage can be used to switch between two states of the molecule. In particular, we use the mechanically controlled break-junction technique to measure electronic transport through a single-molecule junction containing two coupled spin centres that are confined on two Co2+ ions. Spin–orbit configuration interaction methods are used to calculate the combined spin system, where the ground state is found to be a pseudo-singlet and the first excitations behave as a pseudo-triplet. Experimentally, these states can be assigned to the absence and occurrence of a Kondo-like zero-bias anomaly in the low-temperature conductance data, respectively. By applying finite bias, we can repeatedly switch between the pseudo-singlet state and the pseudo-triplet state.

DOI

Journal: Nature Nanotechnology

Nanomechanical Properties of Proteins and Membranes Depend on Loading Rate and Electrostatic Interactions

Izhar D. Medalsy and Daniel J. Müller 

Knowing the dynamic mechanical response of tissue, cells, membranes, proteins, nucleic acids, and carbohydrates to external perturbations is important to understand various biological and biotechnological problems. Atomic force microscopy (AFM)-based approaches are the most frequently used nanotechnologies to determine the mechanical properties of biological samples that range in size from microscopic to (sub)nanoscopic. However, the dynamic nature of biomechanical properties has barely been addressed by AFM imaging. In this work, we characterizethe viscoelastic properties of the native light-driven proton pump bacteriorhodopsin of the purple membrane of Halobacterium salinarum. Using force–distance curve (F–D)-based AFM we imaged purple membranes while force probing their mechanical response over a wide range of loading rates (from 0.5 to 100 μN/s). Our results show that the mechanical stiffness of protein and membrane increases with the loading rate up to a factor of 10 (from 0.3 to 3.2 N/m). In addition, the electrostatic repulsion between AFM tip and sample can alter the mechanical stiffness measured by AFM up to 60% (from 0.8 to 1.3 N/m).These findings indicate that the mechanical response of membranes and proteins and probably of other biomolecular systems should be determined at different loading rates to fully understand their properties.

DOI

Journal: ACS Nano

The length–tension curve in muscle depends on lattice spacing

C. David Williams, Mary K. Salcedo, Thomas C. Irving, Michael Regnier and Thomas L. Daniel

Classic interpretations of the striated muscle length–tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing is a significant force regulator, increasing the slope of muscle's force–length dependence.

DOI

Journal: Proceedings of the Royal Society B

DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate

Bian Liu, Ronald J. Baskin, and Stephen C. Kowalczykowski

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme–DNA complex by depleting Mg2+-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.

DOI

Journal: Nature

B Cells Use Mechanical Energy to Discriminate Antigen Affinities

Elizabeth Natkanski, Wing-Yiu Lee, Bhakti Mistry, Antonio Casal, Justin E. Molloy, Pavel Tolar

The generation of high-affinity antibodies depends on the ability of B cells to extract antigens from the surfaces of antigen-presenting cells. B cells that express high-affinity B cell receptors (BCRs) acquire more antigen and obtain better T cell help. However, the mechanisms by which B cells extract antigen remain unclear. Using fluid and flexible membrane substrates to mimic antigen-presenting cells, we showed that B cells acquire antigen by dynamic myosin IIa–mediated contractions that pull out and invaginate the presenting membranes. The forces generated by myosin IIa contractions ruptured most individual BCR-antigen bonds and promoted internalization of only high-affinity, multivalent BCR microclusters. Thus, B cell contractility contributes to affinity discrimination by mechanically testing the strength of antigen binding.

DOI

Journal: Science

Elastic energy storage in the shoulder and the evolution of high-speed throwing in Homo

Neil T. Roach,  Madhusudhan Venkadesan, Michael J. Rainbow, and Daniel E. Lieberman

Some primates, including chimpanzees, throw objects occasionally, but only humans regularly throw projectiles with high speed and accuracy. Darwin noted that the unique throwing abilities of humans, which were made possible when bipedalism emancipated the arms, enabled foragers to hunt effectively using projectiles. However, there has been little consideration of the evolution of throwing in the years since Darwin made his observations, in part because of a lack of evidence of when, how and why hominins evolved the ability to generate high-speed throws. Here we use experimental studies of humans throwing projectiles to show that our throwing capabilities largely result from several derived anatomical features that enable elastic energy storage and release at the shoulder. These features first appear together approximately 2 million years ago in the species Homo erectus. Taking into consideration archaeological evidence suggesting that hunting activity intensified around this time, we conclude that selection for throwing as a means to hunt probably had an important role in the evolution of the genus Homo.

DOI

Journal: Nature

Visualizing GroEL/ES in the Act of Encapsulating a Folding Protein

Dong-Hua Chen, Damian Madan, Jeremy Weaver, Zong Lin, Gunnar F. Schröder, Wah Chiu, Hays S. Rye

The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.

DOI

Journal: Cell

Stabilization of a protein conferred by an increase in folded state entropy

Shlomi Dagan, Tzachi Hagai, Yulian Gavrilov, Ruti Kapon, Yaakov Levy, and Ziv Reich

Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.

DOI

Journal: Proceedings of the National Academy of Sciences

Modulation of allostery by protein intrinsic disorder

Allan Chris M. Ferreon, Josephine C. Ferreon,  Peter E. Wright, and Ashok A. Deniz


Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical and empirical observations indicate that allostery is also manifest in intrinsically disordered proteins, which account for a substantial proportion of the proteome. Many intrinsically disordered proteins are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub intrinsically disordered protein. E1A can induce marked epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators such as the general transcriptional coactivator CREB binding protein (CBP), its paralogue p300, and the retinoblastoma protein (pRb; also called RB1). Little is known about the allosteric effects at play in E1A–CBP–pRb interactions, or more generally in hub intrinsically disordered protein interaction networks. Here we used single-molecule fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved to be essential for these studies, which are challenging owing to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A–CBP–pRb interactions have either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary versus binary E1A complexes, and may permit a context-specific tuning of associated downstream signalling outputs. Such a modulation of allosteric interactions is probably a common mechanism in molecular hub intrinsically disordered protein function.


DOI

Journal: Nature

Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy

Ionel Popa, Pallav Kosuri, Jorge Alegre-Cebollada, Sergi Garcia-Manyes, and Julio M Fernandez

Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.

DOI

Journal: Nature Protocols

A plasmonic ‘antenna-in-box’ platform for enhanced single-molecule analysis at micromolar concentrations

Deep Punj, Mathieu Mivelle, Satish Babu Moparthi, Thomas S. van Zanten, Hervé Rigneault, Niek F. van Hulst, María F. García-Parajó, and Jérôme Wenger

Single-molecule fluorescence techniques are key for a number of applications, including DNA sequencing, molecular and cell biology and early diagnosis. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes. Although these nanoantennas enhance fluorescence brightness, large background signals and/or unspecific binding to the metallic surface have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel ‘antenna-in-box’ platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules.

DOI

Journal: Nature Nanotechnology

Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

Ruifeng Qi, Evans Boateng Sarbeng, Qun Liu, Katherine Quynh Le,  Xinping Xu, Hongya Xu, Jiao Yang, Jennifer Li Wong, Christina Vorvis, Wayne A Hendrickson, Lei Zhou, and Qinglian Liu

The 70-kilodalton (kDa) heat-shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD), which binds and hydrolyzes ATP, and a substrate-binding domain (SBD), which binds extended polypeptides. NBD and SBD interact little when in the presence of ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact ATP-bound Hsp70 from Escherichia coli at 1.96-Å resolution. The ATP-bound NBD adopts a unique conformation, forming extensive interfaces with an SBD that has changed radically, having its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes, together with our biochemical assays, provide a structural explanation for allosteric coupling in Hsp70 activity.

DOI

Journal:Nature Structural & Molecular Biology

Defining Single Molecular Forces Required to Activate Integrin and Notch Signaling

Xuefeng Wang, Taekjip Ha

Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of about 40 piconewtons (pN) to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions.

DOI

Journal: Science

A Tissue-Like Printed Material

Gabriel Villar, Alexander D. Graham, and Hagan Bayley

Living cells communicate and cooperate to produce the emergent properties of tissues. Synthetic mimics of cells, such as liposomes, are typically incapable of cooperation and therefore cannot readily display sophisticated collective behavior. We printed tens of thousands of picoliter aqueous droplets that become joined by single lipid bilayers to form a cohesive material with cooperating compartments. Three-dimensional structures can be built with heterologous droplets in software-defined arrangements. The droplet networks can be functionalized with membrane proteins; for example, to allow rapid electrical communication along a specific path. The networks can also be programmed by osmolarity gradients to fold into otherwise unattainable designed structures. Printed droplet networks might be interfaced with tissues, used as tissue engineering substrates, or developed as mimics of living tissue.

DOI

Journal: Science

Non-optimal codon usage affects expression, structure and function of clock protein FRQ

Mian Zhou, Jinhu Guo, Joonseok Cha, Michael Chae, She Chen, Jose M. Barral, Matthew S. Sachs, and Yi Liu

Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes1, 2, 3. Codon usage is also implicated in the control of transcription, splicing and RNA structure4, 5, 6. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

DOI

Journal: Nature

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments

Helena Gradišar, Sabina Božič, Tibor Doles, Damjan Vengust, Iva Hafner-Bratkovič, Alenka Mertelj, Ben Webb, Andrej Šali, Sandi Klavžar, and Roman Jerala

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil–forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

DOI

Journal: Nature Chemical Biology

Torque Spectroscopy of DNA: Base-Pair Stability, Boundary Effects, Backbending, and Breathing Dynamics

Florian C. Oberstrass, Louis E. Fernandes, Paul Lebel, and Zev Bryant

Changes in global DNA linking number can be accommodated by localized changes in helical structure. We have used single-molecule torque measurements to investigate sequence-specific strand separation and Z-DNA formation. By controlling the boundary conditions at the edges of sequences of interest, we have confirmed theoretical predictions of distinctive boundary-dependent backbending patterns in torque-twist relationships. Abrupt torque jumps are associated with the formation and collapse of DNA bubbles, permitting direct observations of DNA breathing dynamics.

DOI

Journal: Physical Review Letters

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field

Kevin J. Freedman, S. Raza Haq, Joshua B. Edel, Per Jemth, and Min Jun Kim

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106 V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein..

DOI

Journal: Scientific Reports

Unraveling the Mechanism of Protein Disaggregation Through a ClpB-DnaK Interaction

Rina Rosenzweig, Shoeib Moradi, Arash Zarrine-Afsar, John R. Glover, and Lewis E. Kay

HSP-100 protein machines, such as ClpB, play an essential role in reactivating protein aggregates that can otherwise be lethal to cells. Although the players involved are known, including the DnaK/DnaJ/GrpE chaperone system in bacteria, details of the molecular interactions are not well understood. Using methyl–transverse relaxation–optimized nuclear magnetic resonance spectroscopy, we present an atomic-resolution model for the ClpB-DnaK complex, which we verified by mutagenesis and functional assays. ClpB and GrpE compete for binding to the DnaK nucleotide binding domain, with GrpE binding inhibiting disaggregation. DnaK, in turn, plays a dual role in both disaggregation and subsequent refolding of polypeptide chains as they emerge from the aggregate. On the basis of a combined structural-biochemical analysis, we propose a model for the mechanism of protein aggregate reactivation by ClpB.

DOI

Journal: Science

Why phosphate is a bad buffer for guanidinium chloride titrations

Alan R. Fersht and Miriana Petrovich

DOI

Journal: Proceedings of the National Academy of Sciences

Cooperativity in Thermal and Force-Induced Protein Unfolding: Integration of Crack Propagation and Network Elasticity Models

Amit Srivastava and Rony Granek

We investigate force-induced and temperature-induced unfolding of proteins using the combination of a Gaussian network model and a crack propagation model based on “bond”-breaking independent events. We assume the existence of threshold values for the mean strain and strain fluctuations that dictate bond rupture. Surprisingly, we find that this stepwise process usually leads to a few cooperative, first-order-like, transitions in which several bonds break simultaneously, reminiscent of the “avalanches” seen in disordered networks.

DOI

Journal: Physical Review Letters

Multistep protein unfolding during nanopore translocation

David Rodriguez-Larrea, and Hagan Bayley

Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.

DOI

Journal: Nature Nanotechnology

Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

Sithara S. Wijeratne, Eric Botello, Hui-Chun Yeh, Zhou Zhou, Angela L. Bergeron, Eric W. Frey, Jay M. Patel, Leticia Nolasco, Nancy A. Turner, Joel L. Moake, Jing-fei Dong, and Ching-Hwa Kiang

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

DOI

Journal: Physical Review Letters

A transgenic mouse for in vivo detection of endogenous labeled mRNA

Timothée Lionnet, Kevin Czaplinski, Xavier Darzacq, Yaron Shav-Tal, Amber L Wells, Jeffrey A Chao, Hye Yoon Park, Valeria de Turris, Melissa Lopez-Jones, and Robert H Singer

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3′ untranslated region of the essential ββ-actin gene. As β-actin–MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.

DOI

Journal: Nature Methods