Biology subjects

Vishvakarma, V.

Publications and source records attributed to Vishvakarma, V..

3 recordsLinked to original sources

bioRxiv2026

Munc18 reprograms the intrinsic neuronal SNARE complex assembly pathway

The SNARE proteins syntaxin/SNAP-25B (t-SNAREs) and synaptobrevin (v-SNARE) contain motifs that assemble into four-helix bundles to drive synaptic vesicle exocytosis; SNAP-25B contributes two helices, D1 and D2. The sequence in which these motifs interact remains unresolved. To address this, we used fluorescence anisotropy of SNARE motifs to conduct real-time order-of-addition experiments and found that the order in which components are mixed can determine whether on- or off-pathway complexes are formed. Beginning with soluble SNARE fragments alone, the first step in assembly is the binding of D1 to syntaxin, followed by the binding of synaptobrevin and D2, where the latter motif acts as a gatekeeper to control v-SNARE*t-SNARE interactions. We then examined the impact of two regulatory factors, Munc18 and the MUN domain of Munc13-1. Strikingly, in the presence of Munc18, all four isolated SNARE motifs must be present at the same time for assembly to occur, revealing a concerted mechanism, while the Munc13-1 fragment was without effect. We created C-SCORE, which reports assembly of the two SNARE motifs of SNAP-25B via FRET, and confirmed that in the presence of Munc18, SNARE assembly becomes concerted. Munc18 also disaggregated syntaxin, potentially contributing to its activation. Finally, our findings regarding SNARE motif folding were well-correlated with function using full-length SNAREs in in vitro lipid mixing assays. Hence, Munc18 acts as a molecular chaperone that directly promotes the concurrent assembly of SNARE proteins into functional fusion machines.

biochemistry↗

bioRxiv2024

Serotonin Promotes Vesicular Association and Fusion by Modifying Lipid Bilayers

The primary event in chemical neurotransmission involves the fusion of a membrane-limited vesicle at the plasma membrane and the subsequent release of its chemical neurotransmitter cargo. The cargo itself is not known to have any effect on the fusion event. However, amphiphilic monoamine neurotransmitters (e.g. serotonin and dopamine) are known to strongly interact with lipid bilayers and to affect their mechanical properties, which can in principle impact membrane-mediated processes. Here we probe whether serotonin can enhance the association and fusion of artificial lipid vesicles in vitro. We employ Fluorescence Correlation Spectroscopy and Total Internal Reflection Fluorescence microscopy to measure the attachment and fusion of vesicles whose lipid compositions mimic the major lipid components of synaptic vesicles. We find that association between vesicles and supported lipid bilayers are strongly enhanced in a serotonin dose-dependent manner, and this drives an increase in the rate of spontaneous fusion. Molecular dynamics simulations and fluorescence spectroscopy data show that serotonin insertion increases the water content of the hydrophobic part of the bilayer. This suggests that the enhanced membrane association is likely driven by an energetically favourable drying transition. Other monoamines such as dopamine and norepinephrine, but not other related species such as tryptophan, show similar effects on membrane association. Our results reveal a lipid bilayer-mediated mechanism by which monoamines can themselves modulate vesicle fusion, potentially adding to the control toolbox for the tightly regulated process of neurotransmission in vivo. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/576155v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@2717c5org.highwire.dtl.DTLVardef@899c21org.highwire.dtl.DTLVardef@697232org.highwire.dtl.DTLVardef@c850a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Rational design of protein-specific folding modifiers

Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a xenonucleus, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Forster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 {+/-} 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

biophysics↗