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Single Molecule
Super-resolution microscopy with DNA-PAINT
May 18, 2017   Nature Protocols Add nature.com free-link Cancel
Schnitzbauer J, Strauss MT, Schlichthaerle T, Schueder F, Jungmann R
Super-resolution microscopy with DNA-PAINT
May 18, 2017
Nature Protocols
Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.
Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells
May 22, 2017   Nature Reviews. Genetics
Cuvier O, Fierz B
Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells
May 22, 2017
Nature Reviews. Genetics
The establishment and maintenance of chromatin states involves multiscale dynamic processes integrating transcription factor and multiprotein effector dynamics, cycles of chemical chromatin modifications, and chromatin structural organization. Recent developments in genomic technologies are emerging that are enabling a view beyond ensemble- and time-averaged properties and are revealing the importance of dynamic chromatin states for cell fate decisions, differentiation and reprogramming at the single-cell level. Concurrently, biochemical and single-molecule methodologies are providing key insights into the underlying molecular mechanisms. Combining results from defined in vitro and single-molecule studies with single-cell genomic approaches thus holds great promise for understanding chromatin-based transcriptional memory and cell fate. In this Review, we discuss recent developments in biochemical, single-molecule biophysical and single-cell genomic technologies and review how the findings from these approaches can be integrated to paint a comprehensive picture of dynamic chromatin states.
Single-molecule detection of dihydroazulene photo-thermal reaction using break junction technique
May 22, 2017   Nature Communications
Huang C, Jevric M, Borges A, Olsen ST, Hamill JM,   . . . . . .   , Wandlowski T, Mikkelsen KV, Solomon GC, Brøndsted Nielsen M, Hong W
Single-molecule detection of dihydroazulene photo-thermal reaction using break junction technique
May 22, 2017
Nature Communications
Charge transport by tunnelling is one of the most ubiquitous elementary processes in nature. Small structural changes in a molecular junction can lead to significant difference in the single-molecule electronic properties, offering a tremendous opportunity to examine a reaction on the single-molecule scale by monitoring the conductance changes. Here, we explore the potential of the single-molecule break junction technique in the detection of photo-thermal reaction processes of a photochromic dihydroazulene/vinylheptafulvene system. Statistical analysis of the break junction experiments provides a quantitative approach for probing the reaction kinetics and reversibility, including the occurrence of isomerization during the reaction. The product ratios observed when switching the system in the junction does not follow those observed in solution studies (both experiment and theory), suggesting that the junction environment was perturbing the process significantly. This study opens the possibility of using nano-structured environments like molecular junctions to tailor product ratios in chemical reactions.
Self-assembly of a supramolecular hexagram and a supramolecular pentagram
May 19, 2017   Nature Communications
Jiang Z, Li Y, Wang M, Song B, Wang K,   . . . . . .   , Moorefield CN, Newkome GR, Xu B, Li X, Wang P
Self-assembly of a supramolecular hexagram and a supramolecular pentagram
May 19, 2017
Nature Communications
Five- and six-pointed star structures occur frequently in nature as flowers, snow-flakes, leaves and so on. These star-shaped patterns are also frequently used in both functional and artistic man-made architectures. Here following a stepwise synthesis and self-assembly approach, pentagonal and hexagonal metallosupramolecules possessing star-shaped motifs were prepared based on the careful design of metallo-organic ligands (MOLs). In the MOL design and preparation, robust ruthenium-terpyridyl complexes were employed to construct brominated metallo-organic intermediates, followed by a Suzuki coupling reaction to achieve the required ensemble. Ligand LA (VRu2+X, V=bisterpyridine, X=tetraterpyridine, Ru=Ruthenium) was initially used for the self-assembly of an anticipated hexagram upon reaction with Cd2+ or Fe2+; however, unexpected pentagonal structures were formed, that is, [Cd5LA5]30+ and [Fe5LA5]30+. In our redesign, LB [V(Ru2+X)2] was synthesized and treated with 60° V-shaped bisterpyridine (V) and Cd2+ to create hexagonal hexagram [Cd12V3LB3]36+ along with traces of the triangle [Cd3V3]6+. Finally, a pure supramolecular hexagram [Fe12V3LB3]36+ was successfully isolated in a high yield using Fe2+ with a higher assembly temperature.
High conductance values in π-folded molecular junctions
May 18, 2017   Nature Communications
Carini M, Ruiz MP, Usabiaga I, Fernández JA, Cocinero EJ, Melle-Franco M, Diez-Perez I, Mateo-Alonso A
High conductance values in π-folded molecular junctions
May 18, 2017
Nature Communications
Folding processes play a crucial role in the development of function in biomacromolecules. Recreating this feature on synthetic systems would not only allow understanding and reproducing biological functions but also developing new functions. This has inspired the development of conformationally ordered synthetic oligomers known as foldamers. Herein, a new family of foldamers, consisting of an increasing number of anthracene units that adopt a folded sigmoidal conformation by a combination of intramolecular hydrogen bonds and aromatic interactions, is reported. Such folding process opens up an efficient through-space charge transport channel across the interacting anthracene moieties. In fact, single-molecule conductance measurements carried out on this series of foldamers, using the scanning tunnelling microscopy-based break-junction technique, reveal exceptionally high conductance values in the order of 10-1 G0 and a low length decay constant of 0.02 Å-1 that exceed the values observed in molecular junctions that make use of through-space charge transport pathways.
Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification
May 18, 2017   Nature Communications
Vernick S, Trocchia SM, Warren SB, Young EF, Bouilly D, Gonzalez RL, Nuckolls C, Shepard KL
Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification
May 18, 2017
Nature Communications
The study of biomolecular interactions at the single-molecule level holds great potential for both basic science and biotechnology applications. Single-molecule studies often rely on fluorescence-based reporting, with signal levels limited by photon emission from single optical reporters. The point-functionalized carbon nanotube transistor, known as the single-molecule field-effect transistor, is a bioelectronics alternative based on intrinsic molecular charge that offers significantly higher signal levels for detection. Such devices are effective for characterizing DNA hybridization kinetics and thermodynamics and enabling emerging applications in genomic identification. In this work, we show that hybridization kinetics can be directly controlled by electrostatic bias applied between the device and the surrounding electrolyte. We perform the first single-molecule experiments demonstrating the use of electrostatics to control molecular binding. Using bias as a proxy for temperature, we demonstrate the feasibility of detecting various concentrations of 20-nt target sequences from the Ebolavirus nucleoprotein gene in a constant-temperature environment.
Sub-nanometre control of the coherent interaction between a single molecule and a plasmonic nanocavity
May 19, 2017   Nature Communications
Zhang Y, Meng QS, Zhang L, Luo Y, Yu YJ, Yang B, Zhang Y, Esteban R, Aizpurua J, Luo Y, Yang JL, Dong ZC, Hou JG
Sub-nanometre control of the coherent interaction between a single molecule and a plasmonic nanocavity
May 19, 2017
Nature Communications
The coherent interaction between quantum emitters and photonic modes in cavities underlies many of the current strategies aiming at generating and controlling photonic quantum states. A plasmonic nanocavity provides a powerful solution for reducing the effective mode volumes down to nanometre scale, but spatial control at the atomic scale of the coupling with a single molecular emitter is challenging. Here we demonstrate sub-nanometre spatial control over the coherent coupling between a single molecule and a plasmonic nanocavity in close proximity by monitoring the evolution of Fano lineshapes and photonic Lamb shifts in tunnelling electron-induced luminescence spectra. The evolution of the Fano dips allows the determination of the effective interaction distance of ∼1 nm, coupling strengths reaching ∼15 meV and a giant self-interaction induced photonic Lamb shift of up to ∼3 meV. These results open new pathways to control quantum interference and field-matter interaction at the nanoscale.
Visualization and characterization of individual type III protein secretion machines in live bacteria
May 23, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Zhang Y, Lara-Tejero M, Bewersdorf J, Galán JE
Visualization and characterization of individual type III protein secretion machines in live bacteria
May 23, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.
Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk
May 23, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Lippert LG, Dadosh T, Hadden JA, Karnawat V, Diroll BT, Murray CB, Holzbaur ELF, Schulten K, Reck-Peterson SL, Goldman YE
Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk
May 23, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
The force-generating mechanism of dynein differs from the force-generating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.
Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change
May 23, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Mandal SS, Merz DR, Buchsteiner M, Dima RI, Rief M, Žoldák G
Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change
May 23, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.
Translation and folding of single proteins in real time
May 16, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Wruck F, Katranidis A, Nierhaus KH, Büldt G, Hegner M
Translation and folding of single proteins in real time
May 16, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.
Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair
May 15, 2017   PLoS Genetics
Claeys Bouuaert C, Keeney S
Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair
May 15, 2017
PLoS Genetics
Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutLγ complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.
The desmoplakin/intermediate filament linkage regulates cell mechanics
May 12, 2017   Molecular Biology Of The Cell
Broussard JA, Yang R, Huang C, Nathamgari SSP, Beese AM, Godsel LM, Lee S, Zhou F, Sniadecki NJ, Green KJ, Espinosa HD
The desmoplakin/intermediate filament linkage regulates cell mechanics
May 12, 2017
Molecular Biology Of The Cell
The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome/intermediate filament network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here, we modulated the interaction between desmosomes and intermediate filaments using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the desmosome/intermediate filament interaction increased cell-substrate and cell-cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction led to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in desmosome/intermediate filament network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems. © 2017 by The American Society for Cell Biology.
Methodologies for studying the spliceosome's RNA dynamics with single-molecule FRET
May 22, 2017   Methods (San Diego, Calif.)
van der Feltz C, Hoskins AA
Methodologies for studying the spliceosome's RNA dynamics with single-molecule FRET
May 22, 2017
Methods (San Diego, Calif.)
The spliceosome is an extraordinarily dynamic molecular machine in which significant changes in composition as well as protein and RNA conformation are required for carrying out pre-mRNA splicing. Single-molecule fluorescence resonance energy transfer (smFRET) can be used to elucidate these dynamics both in well-characterized model systems and in entire spliceosomes. These types of single-molecule data provide novel information about spliceosome components and can be used to identify sub-populations of molecules with unique behaviors. When smFRET is combined with single-molecule fluorescence colocalization, conformational dynamics can be further linked to the presence or absence of a given spliceosome component. Here, we provide a description of experimental considerations, approaches, and workflows for smFRET with an emphasis on applications for the splicing machinery. Copyright © 2017 Elsevier Inc. All rights reserved.
Compaction and condensation of DNA mediated by the C-terminal domain of Hfq
May 18, 2017   Nucleic Acids Research
Malabirade A, Jiang K, Kubiak K, Diaz-Mendoza A, Liu F, van Kan JA, Berret JF, Arluison V, van der Maarel JRC
Compaction and condensation of DNA mediated by the C-terminal domain of Hfq
May 18, 2017
Nucleic Acids Research
Hfq is a bacterial protein that is involved in several aspects of nucleic acids metabolism. It has been described as one of the nucleoid associated proteins shaping the bacterial chromosome, although it is better known to influence translation and turnover of cellular RNAs. Here, we explore the role of Escherichia coli Hfq's C-terminal domain in the compaction of double stranded DNA. Various experimental methodologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy, isothermal titration microcalorimetry and electrophoretic mobility assays have been used to follow the assembly of the C-terminal and N-terminal regions of Hfq on DNA. Results highlight the role of Hfq's C-terminal arms in DNA binding, change in mechanical properties of the double helix and compaction of DNA into a condensed form. The propensity for bridging and compaction of DNA by the C-terminal domain might be related to aggregation of bound protein and may have implications for protein binding related gene regulation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Human Myosin VIIa Is a Very Slow Processive Motor Protein on Various Cellular Actin Structures
May 16, 2017   The Journal Of Biological Chemistry
Sato O, Komatsu S, Sakai T, Tsukasaki Y, Tanaka R, Mizutani T, Watanabe TM, Ikebe R, Ikebe M
Human Myosin VIIa Is a Very Slow Processive Motor Protein on Various Cellular Actin Structures
May 16, 2017
The Journal Of Biological Chemistry
Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. While it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ≈0.3 s-1 for the mechanical turnover rate, hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm s-1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bi-directional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 on stereocilia actin bundles in inner-ear hair cells. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.
The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation
May 12, 2017   The Journal Of Biological Chemistry
Ragunathan P, Sielaff H, Sundararaman L, Biuković G, Subramanian Manimekalai MS, Singh D, Kundu S, Wohland T, Frasch W, Dick T, Grüber G
The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation
May 12, 2017
The Journal Of Biological Chemistry
The F1FO ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36 amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotation assays indicated that the introduction of this Mycobacterium-specific domain decreased the angular velocity of the power-stroke after ATP binding. Solution X-ray scattering data and NMR results revealed the solution shape of Mtα and the 3D structure of the subunit α C-terminal peptide 521PDEHVEALDEDKLAKEAVKV540 of M. tubercolosis (Mtα521-540), respectively. Together with crosslinking studies, the solution structural data lead to a model, in which Mtα521-540 comes in close proximity with subunit γ residues 104-109, whose interaction may influence the rotation of the camshaft-like subunit γ. Finally, we propose that the unique segment Mtα514-549, which is accessible at the C-terminus of mycobacterial subunit α, is a promising drug epitope. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.
Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase
May 12, 2017   Biophysical Journal
Scholl ZN, Yang W, Marszalek PE
Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase
May 12, 2017
Biophysical Journal
Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Rapid Microfluidic Dilution for Single-Molecule Spectroscopy of Low-Affinity Biomolecular Complexes
May 16, 2017   Angewandte Chemie (International Ed. In English)
Zijlstra N, Dingfelder F, Wunderlich B, Zosel F, Benke S, Nettels D, Schuler B
Rapid Microfluidic Dilution for Single-Molecule Spectroscopy of Low-Affinity Biomolecular Complexes
May 16, 2017
Angewandte Chemie (International Ed. In English)
To enable the investigation of low-affinity biomolecular complexes with confocal single-molecule spectroscopy, we have developed a microfluidic device that allows a concentrated sample to be diluted by up to five orders of magnitude within milliseconds, at the physical limit dictated by diffusion. We demonstrate the capabilities of the device by studying the dissociation kinetics and structural properties of low-affinity protein complexes using single-molecule two-color and three-color Förster resonance energy transfer (FRET). We show that the versatility of the device makes it suitable for studying complexes with dissociation constants from low nanomolar up to 10 μm, thus covering a wide range of biomolecular interactions. The design and precise fabrication of the devices ensure simple yet reliable operation and high reproducibility of the results. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Ferrocene- and Biferrocene-Containing Macrocycles towards Single-Molecule Electronics
May 12, 2017   Angewandte Chemie (International Ed. In English)
Wilson LE, Hassenrück C, Winter RF, White AJP, Albrecht T, Long NJ
Ferrocene- and Biferrocene-Containing Macrocycles towards Single-Molecule Electronics
May 12, 2017
Angewandte Chemie (International Ed. In English)
Cyclic multiredox centered systems are currently of great interest, with new compounds being reported and developments made in understanding their behavior. Efficient, elegant, and high-yielding (for macrocyclic species) synthetic routes to two novel alkynyl-conjugated multiple ferrocene- and biferrocene-containing cyclic compounds are presented. The electronic interactions between the individual ferrocene units have been investigated through electrochemistry, spectroelectrochemistry, density functional theory (DFT), and crystallography to understand the effect of cyclization on the electronic properties and structure. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Efficient modification of λ-DNA substrates for single-molecule studies
May 19, 2017   Scientific Reports
Kim Y, de la Torre A, Leal AA, Finkelstein IJ
Efficient modification of λ-DNA substrates for single-molecule studies
May 19, 2017
Scientific Reports
Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90-100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5'-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.
The molecular basis of thin filament activation: from single molecule to muscle
May 13, 2017   Scientific Reports
Longyear T, Walcott S, Debold EP
The molecular basis of thin filament activation: from single molecule to muscle
May 13, 2017
Scientific Reports
For muscles to effectively power locomotion, trillions of myosin molecules must rapidly attach and detach from the actin thin filament. This is accomplished by precise regulation of the availability of the myosin binding sites on actin (i.e. activation). Both calcium (Ca++) and myosin binding contribute to activation, but both mechanisms are simultaneously active during contraction, making their relative contributions difficult to determine. Further complicating the process, myosin binding accelerates the attachment rate of neighboring myosin molecules, adding a cooperative element to the activation process. To de-convolve these two effects, we directly determined the effect of Ca++ on the rate of attachment of a single myosin molecule to a single regulated actin thin filament, and separately determined the distance over which myosin binding increases the attachment rate of neighboring molecules. Ca++ alone increases myosin's attachment rate ~50-fold, while myosin binding accelerates attachment of neighboring molecules 400 nm along the actin thin filament.
Inkjet printing of NiO films and integration as hole transporting layers in polymer solar cells
May 12, 2017   Scientific Reports
Singh A, Gupta SK, Garg A
Inkjet printing of NiO films and integration as hole transporting layers in polymer solar cells
May 12, 2017
Scientific Reports
Stability concerns of organic solar cell devices have led to the development of alternative hole transporting layers such as NiO which lead to superior device life times over conventional Poly(3,4-ethylenedioxythiophene) Polystyrene sulfonate (PEDOT:PSS) buffered solar cells. From the printability of such devices, it is imperative to be able to print NiO layers in the organic solar cell devices with normal architecture which has so far remained unreported. In this manuscript, we report on the successful ink-jet printing of very thin NiO thin films with controlled thickness and morphology and their integration in organic solar cell devices. The parameters that were found to strongly affect the formation of a thin yet continuous NiO film were substrate surface treatment, drop spacing, and substrate temperature during printing. The effect of these parameters was investigated through detailed morphological characterization using optical and atomic force microscopy and the results suggested that one can achieve a transmittance of ~89% for a ~18 nm thin NiO film with uniform structure and morphology, fabricated using a drop spacing of 50 μm and a heat treatment temperature of 400 °C. The devices fabricated with printed NiO hole transporting layers exhibit power conversion efficiencies comparable to the devices with spin coated NiO films.
Isolation and genomic characterization of a Dehalococcoides strain suggests genomic rearrangement during culture
May 23, 2017   Scientific Reports
Yohda M, Ikegami K, Aita Y, Kitajima M, Takechi A,   . . . . . .   , Nakano K, Teruya K, Satou K, Hirano T, Yagi O
Isolation and genomic characterization of a Dehalococcoides strain suggests genomic rearrangement during culture
May 23, 2017
Scientific Reports
We have developed and characterized a bacterial consortium that reductively dechlorinates trichloroethene to ethene. Quantitative PCR analysis for the 16S rRNA and reductive dehalogenase genes showed that the consortium is highly enriched with Dehalococcoides spp. that have two vinyl chloride reductive dehalogenase genes, bvcA and vcrA, and a trichloroethene reductive dehalogenase gene, tceA. The metagenome analysis of the consortium by the next generation sequencer SOLiD 3 Plus suggests that a Dehalococcoides sp. that is highly homologous to D. mccartyi 195 and equipped with vcrA and tceA exists in the consortium. We isolated this Dehalococcoides sp. and designated it as D. mccartyi UCH-ATV1. As the growth of D. mccartyi UCH-ATV1 is too slow under isolated conditions, we constructed a consortium by mixing D. mccartyi UCH-ATV1 with several other bacteria and performed metagenomic sequencing using the single molecule DNA sequencer PacBio RS II. We successfully determined the complete genome sequence of D. mccartyi UCH-ATV1. The strain is equipped with vcrA and tceA, but lacks bvcA. Comparison with tag sequences of SOLiD 3 Plus from the original consortium shows a few differences between the sequences. This suggests that a genome rearrangement of Dehalococcoides sp. occurred during culture.
Discovery and biosynthesis of gladiolin: a Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis
May 22, 2017   Journal Of The American Chemical Society
Song L, Jenner M, Masschelein J, Jones C, Bull MJ,   . . . . . .   , Paisey C, Cole ST, Parkhill J, Mahenthiralingam E, Challis GL
Discovery and biosynthesis of gladiolin: a Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis
May 22, 2017
Journal Of The American Chemical Society
An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally-related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

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