1,720,968 research outputs found
Mass Action at the Single-Molecule Level
No full textWe developed a system to reversibly encapsulate small numbers of molecules in an array of nanofabricated "dimples". This system enables highly parallel, long-term, and attachment-free studies of molecular dynamics via single-molecule fluorescence. In studies of bimolecular reactions of small numbers of confined molecules, we see phenomena that, while expected from basic statistical mechanics, are not observed in bulk chemistry. Statistical fluctuations in the occupancy of sealed reaction chambers lead to steady-state fluctuations in reaction equilibria and rates. These phenomena are likely to be important whenever reactions happen in confined geometries.11Nsciescopu
Focused clamping of a single neuronal SNARE complex by complexin under high mechanical tension
No full textNeuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze synaptic vesicle fusion with presynaptic membranes through the formation of SNARE complexes. Complexin (Cpx) is the only presynaptic protein that tightly binds to SNAREs and regulates membrane fusion, but how it modulates the energy landscape of SNARE complex assembly, especially under mechanical tension on the complex, remains unclear. Here, using magnetic tweezers, we report how Cpx interacts with single SNARE complexes. The effects of Cpx manifest only under high mechanical tensions above 13 pN. Cpx stabilizes the central four-helix bundle of SNARE motifs and, at the same time, prevents the complete zippering of SNAREs by inhibiting linker-domain assembly. These results suggest that Cpx generates a focused clamp for the neuronal SNARE complex in a linker-open conformation. Our results provide a hint as to how Cpx cooperates with neuronal SNAREs to prime synaptic vesicles in preparation for synchronous neurotransmitter release.11Nsciescopu
Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley
No full textProteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a ‘DNA pulley’ for position-resolved nano-mechanical measurements of protein-DNA interactions. A molecule of λ DNA is tethered by one end to a glass surface, and by the other end to a magnetic bead. The DNA is stretched horizontally by a magnet, and a nanoscale knife made of silicon nitride is manipulated to contact, bend and scan along the DNA. The mechanical profile of the DNA at the contact with the knife is probed via nanometer-precision optical tracking of the magnetic bead. This system enables detection of protein bumps on the DNA and localization of their binding sites. We study theoretically the technical requirements to detect mechanical heterogeneities in the DNA itself.Version of Recor
Tension exerted on cells by magnetic nanoparticles regulates differentiation of human mesenchymal stem cells
No full textCells can ???sense??? physical cues in the surrounding microenvironment and ???react??? by changing their function. Previous studies have focused on regulating the physical properties of the matrix, such as stiffness and topog-raphy, thus changing the tension ???felt??? by the cell as a result. In this study, by directly applying a quantified magnetic force to the cell, a correlation between differentiation and tension was shown. The magnetic force, quantified by magnetic tweezers, was applied by incorporating magnetotactic bacteria-isolated magnetic nanoparticles (MNPs) in human mesenchymal stem cells. As the applied tension increased, the expression levels of osteogenic differentiation marker genes and proteins were proportionally upregulated. Additionally, the translocation of YAP and RUNX2, deformation of nucleus, and activation of the MAPK signaling pathway were observed in tension-based osteogenic differentiation. Our findings provide a platform for the quantitative control of tension, a key factor in stem cell differentiation, between cells and the matrix using MNPs. Furthermore, these findings improve the understanding of osteogenic differentiation by mechanotransduction.11Nsciescopu
Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes
No full textFueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.11Ysciescopu
Submicrometer elasticity of double-stranded DNA revealed by precision force-extension measurements with magnetic tweezers
Get PDFSubmicrometer elasticity of double-stranded DNA (dsDNA) governs nanoscale bending of DNA segments and their interactions with proteins. Single-molecule force spectroscopy, including magnetic tweezers (MTs), is an important tool for studying DNA mechanics. However, its application to short DNAs under 1 mm is limited. We developed an MT-based method for precise force-extension measurements in the 100-nm regime that enables in situ correction of the error in DNA extension measurement, and normalizes the force variability across beads by exploiting DNA hairpins. The method reduces the lower limit of tractable dsDNA length down to 198 base pairs (bp) (67 nm), an order-of-magnitude improvement compared to conventional tweezing experiments. Applying this method and the finite worm-like chain model we observed an essentially constant persistence length across the chain lengths studied (198 bp to 10 kbp), which steeply depended on GC content and methylation. This finding suggests a potential sequence-dependent mechanism for short-DNA elasticity.11Nsciescopu
Observing Extremely Weak Protein–Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy
No full textExtremely
weak protein–protein interactions (PPIs), signified
by micromolar or even millimolar dissociation constants, are one of
the keys to understanding the rapid responses of cellular systems.
Although single-molecule methods are particularly useful in determining
kinetics of biological processes, their application is largely limited
to rather strong interactions because of the diffraction-limited observation
volume. In this study, we report a single-molecule method that allows
the characterization of PPIs using a prey concentration 4 orders of
magnitude lower than the dissociation constant. Instead of increasing
the concentration of diffusing molecules, which is inevitably limited
by the optical diffraction limit, we employed an increased density
of surface bait protein. The low occupancy of the surface baits permitted
determination of the kinetics with single-molecule resolution. We
used this approach to study a PPI network consisting of Ras and its
downstream proteins including full-length Rafs and catalytic subunits
of phosphoinositide 3-kinase
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Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway
No full textTo understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the Escherichia coli rhomboid protease GlpG and the human ??2-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding
In Honor of W.E. Moerner: Confining Molecules for Single-Molecule Spectroscopy
No full textSingle-molecule spectroscopy provides a wealth of information on the dynamics and interactions of complex biological molecules. Yet these measurements are extremely challenging, partly because Brownian motion prevents molecules in free solution from remaining stationary. Here we describe several techniques that our lab has developed for confining single molecules for optical spectroscopy. These alternatives to surface immobilization provide confinement that is gentle enough to minimize perturbations to the molecule, but strong enough to allow long-time imaging of single fluorescent molecules, often in the presence of a high fluorescent background.Chemistry and Chemical BiologyPhysicsVersion of Recor
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