Polyelectrolyte brushes were formed at the interface between hydrophobized mica and water by adsorbing amphiphilic diblock copolymers consisting of a hydrophobic charged block. We measured force−distance profiles with surface force apparatus (SFA) for several polyelectrolyte different lengths as function aqueous 1:1 electrolyte concentration. Long-ranged electrostatic double-layer forces not observed in curves, our results suggest that more counterions than predicted Manning theory are...

The phosphorylation of nucleic acid catalyzed by polynucleotide kinase is an indispensible procedure involved in many vital cellular activities such as DNA recombination and repair. Herein, a novel strategy for the sensitive determination T4 (PNK) activity inhibition was proposed, which combined exonuclease enzyme reaction bimolecular beacons (bi-MBs)-based signal amplification. A hairpin probe (HP) with 5'-hydroxyl termini two different types molecular (MBs), MB1 MB2, designed. Taking...

Significance Synaptic vesicle fusion at synapses is the primary mechanism by which neurons communicate. A highly conserved membrane machine known as SNARE complex mediates this process. In addition, neuronal SNARE-binding regulatory proteins have evolved to control kinetics and speed of assembly synapses. One such protein, Complexin, has been found inhibit synaptic in absence an action potential activate SNARE-mediated release during stimulation. Here we examine molecular models for how...

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate fusion by pulling biological membranes together via a zippering mechanism. Recent biophysical studies have shown that t- and v-SNAREs can assemble in multiple stages from the N-termini toward C-termini. Here we show functionally, membrane requires sequential, two-step folding pathway assign specific distinct functions for each step. First, N-terminal domain (NTD) of v-SNARE docks to t-SNARE, which...

Significance Neurotransmission requires fusion of the synaptic vesicles that are closely apposed to presynaptic membrane. This apposition is achieved through initial association proteins known as soluble NSF attachment protein receptors (SNAREs). After a neurotransmission event, other must be rapidly prepared for next round fusion. Here we find kinetics formation SNARE complexes too slow spontaneously achieve this process because high activation energy barrier, but can accelerated by...

Previously, we showed that synaptotagmin1 (Syt1) forms Ca2+-sensitive ring-like oligomers on membranes containing acidic lipids and proposed a potential role in regulating neurotransmitter release (Zanetti et al., 2016). Here, report Syt1 assembles into similar solution when triggered by naturally occurring polyphosphates (PIP2 ATP) magnesium ions (Mg2+). These soluble rings were observed electron microscopy independently demonstrated quantified using fluorescence correlation spectroscopy....

Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as model system for cell membranes. However, the study proteo-membrane interactions in this is limited incorporation integral and lipid-anchored proteins into GUVs remains challenging. Here, we present simple generic method to incorporate GUVs. The basic principle break proteo-liposomes with an osmotic shock. They subsequently reseal larger which, if necessary, can endure same obtain even proteo-GUVs. This...

Recently, chemically synthesized short interfering RNA (siRNA) duplexes have been used with success for gene silencing. Chemical modification is desired therapeutic applications to improve biostability and pharmacokinetic properties; chemical may also provide insight into the mechanism of siRNA containing 2,4-difluorotoluyl ribonucleoside (rF) were evaluate effect noncanonical nucleoside mimetics on interference. 5′-Modification guide strand rF did not alter silencing relative unmodified...

Significance Intracellular membrane fusion is mediated by coupled folding and assembly of three or four soluble N -ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins into a four-helix bundle. A rate-limiting step the formation partial complex containing helixes called target (t)-SNARE on plasma membrane. The t-SNARE then serves as template to guide stepwise zippering fourth helix, process that further regulated other proteins. synaptic readily misfolds....

Synaptic vesicle fusion is mediated by SNARE proteins-VAMP2 on the and Syntaxin-1/SNAP25 presynaptic membrane. Chaperones Munc18-1 Munc13-1 cooperatively catalyze assembly via an intermediate 'template' complex containing Syntaxin-1 VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that recruits to initiate ternary direct binding, as judged bulk FRET spectroscopy single-molecule optical tweezer studies. Detailed structure-function analyses show binding MUN domain...

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle plasma membranes to neurotransmitters. Previous studies suggested sensor synaptotagmin (Syt) facilitator of docking both inhibitor fusion. On phospholipid monolayers, Syt C2AB domain spontaneously oligomerized into rings are disassembled Ca 2+ , suggesting may clamp fusion as membrane-separating “washers” until -mediated disassembly triggers [J. Wang et al., Proc....

2'-Deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic (ANA) paired to RNA are substrates of RNase H. The conformation the natural DNA/RNA hybrid appears be neither A-form nor B-form. Consistent with this, conformations FANA ANA were found intermediate between A- B-forms. However, opposite is preferred by H over ANA, affinity considerably exceeds that ANA. By investigating conformational boundaries residues in crystal structures B-form DNA duplexes at atomic resolution, we...

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required therapeutic applications in order improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements the efficacy oligoribonucleotides, chemical may also provide insight into mechanism mRNA downregulation mediated by RNA–protein effector complexes (RNA-induced silencing complex or RISC). We...

We propose a new way to determine weak repulsive forces operative between colloidal particles by measuring the rate of slow coagulation. The coagulation is directly related competition repulsion with thermal motion. Since are weak, measurements can lead precise information on potentials having magnitude just few kT. demonstrate this novel studying spherical polyelectrolyte brush (SPB) in aqueous solution containing trivalent La3+ counterions. consist monodisperse polystyrene core 121 nm...

We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable detect the clamping of and therefore questioned previous interpretation fluorescence resonance energy transfer isothermal titration calorimetry data which model originally based. Here we present new biochemical underscore validity our continued relevancy insertion for clamping.

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of entails an accurate in vitro reproduction the process. To date, supported bilayers have been commonly used to mimic Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce usually limited fluidity when embedded bilayers. This alters kinetics prevents correct reconstitution overall Also, observing content release across is hindered...

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming release in a geometry that enables detailed characterization fate docked vesicles both before after is triggered with Ca 2+ . Using this setup, identify new roles diacylglycerol (DAG) regulating -triggered involving SNARE assembly chaperone Munc13. We find low concentrations DAG profoundly accelerate rate...

Munc13-1 is a large banana-shaped soluble protein that involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest multiple copies form nano-assemblies active zones neurons. However, it not known whether such clustering correlated with multivalent binding to vesicles or specific plasma membrane domains at sites zone. The functional significance putative also unknown. Here, we report nano-clustering an inherent property indeed required for bilayers containing...

Significance Membrane fusion is the key step in cellular traffic, which induced by assembly of membrane protein, namely SNARE. How protein induces remains unknown. Answering this question requires knowledge intermediates, cannot be accessed conventional methods. We developed an instrument not only to freeze a continuous series intermediates SNARE but also monitor formation these domains. Here, we demonstrate that N-terminal initializing prior fusion.