Data from: Munc18 reprograms the intrinsic neuronal SNARE complex assembly pathway

Vishvakarma, Vicky 1 ; Chapman, Edwin 1

Published Jul 27, 2026; Updated Jul 31, 2026 on Dryad. https://doi.org/10.5061/dryad.z34tmpgw9

Data files

Jul 27, 2026 version files 13.03 MB

PNAS-Dryad-VV-ERC.zip

13.03 MB
README.md

4.97 KB

Jul 31, 2026 version files 13.11 MB

README.md

4.99 KB
SNARE-assembly-manuscript-data-Dryad.zip

13.11 MB

Abstract

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.

Dataset DOI: 10.5061/dryad.z34tmpgw9

Description of the data and file structure

These data were collected to understand the intrinsic interactions of SNARE proteins and how they are regulated. There are four different types of experiments performed.

1) Fluorescence anisotropy

2) Fluorescence correlation spectroscopy

3) Forster resonance energy transfer

4) Fluorescence donor-acceptor lipid-based lipid-mixing assay

A brief description of the steps to curate these data is as follows:

1) Fluorescence anisotropy: The experiments were performed in a spectrofluorometer in T-format, which utilizes two detector channels to collect the fluorescence signal in two orthogonal directions (parallel and perpendicular to the excitation). We calculated the fluorescence anisotropy from these two data sets by correcting for the instrument response factor (g-factor) and then taking the difference. For our study, a 'g-factor' value of "0.285023" was used as a correction factor.

2) Fluorescence correlation spectroscopy (FCS): This method measures the diffusion of fluorescently labeled proteins and helps to understand their interaction with binding proteins quantitatively. We used this to study protein-protein interactions in SNARE proteins. We extracted diffusion time from the FCS autocorrelation traces via non-linear regression analysis using a well-known 3D-diffusion model.

3) Forster resonance energy transfer (FRET): It is tool that measure atomic distances on a scale of 2-10 nm. We used it to measure the folding of SNARE-protein (SNAP-25B) with syntaxin and synaptobrevin. FRET pair were engineered within the protein, and spectra were acquired using spectrofluorometer. An increase in the fluorescence ratio of the acceptor/donor serves as an indicator of the SNARE protein folding.

4) Fluorescence donor-acceptor based lipid-mixing assay: SNARE proteins catalyze membrane fusion by folding into a four alpha-helix bundle. We mimic this action in in vitro using reconstitution fusion assay which utilized dequenching of donor-acceptor lipids upon lipid mixing.

Files and variables

File: SNARE-assembly-manuscript-data-Dryad.zip

Description: Different folders of this zip file contain data obtained using the methodology described in the data description section. Below is a brief segregation.

Fig-1 to Fig-5: Fluorescence anisotropy; Each file contains the data value arranged in two columns. First column describes the time (in seconds), and second column describes the fluorescence value (arbitrary unit).

Fig-6A-B: Fluorescence correlation spectroscopy; Each file contains the data value arranged in two columns. First column describes the time (in milliseconds), and second column describes the value of the autocorrelation amplitude.

Fig-6D-E: Forster resonance energy transfer; Each file contains the data value arranged in two columns. First column describes the wavelength (in nanometer), and second column describes the value of the fluorescence value (arbitrary unit).

Fig-7: Fluorescence donor-acceptor based lipid-mixing assay; Each file contains the data value arranged in two columns. First column describes the time (in minutes), and second column describes the value of the fluorescence value (arbitrary unit).

Fig-S1-S7: Fluorescence anisotropy; Each file contains the data value arranged in two columns. First column describes the time (in seconds), and second column describes the fluorescence value (arbitrary unit).

Code/software

A) Code for calculation of fluorescence anisotropy (r):

Anisotropy (r) = (I_VV - GI_VH)/(I_VV + 2GI_VH)

The fluorescence anisotropy was calculated using the equation shown above, where ‘I’ is the fluorescence intensity, and H and V indicate horizontally and vertically polarized light, respectively. The first letter (subscript) defines the direction of excitation light, while the second letter indicates the direction of light detection. The anisotropy values were corrected for the instrument correction factor (G-factor, G), which was calculated as I_HV/I_HH.

B) Code for analyzing FCS autocorrelation traces:

G(τ) = ((1-f_T+f_Texp(-τ/τ_T))/(1-f_T))(g₁/((1+τ/τ_D1)(1+a²τ/τ_D1)^0.5) + g₂/((1+τ/τ_D2)(1+a²τ/τ_D2)^0.5)) + bl

We analyzed the FCS data using the above equation, where G(τ) represents the autocorrelation function and τ is the lag time. The analysis was performed using Origin Pro software (Northampton, MA, USA) to measure the following parameters; f_T : triplet-state fraction; τ_T : triplet-state relaxation time, τ_D : diffusion time of the fluorescent molecule, and g: amplitude of diffusion component. In the equation, ‘a’ serves as the structure factor and ‘bl’ as the background.