# Single-molecule manipulation of proteins involved in membrane fusion, lipid exchange, and mechanosensation

> **NIH NIH R35** · YALE UNIVERSITY · 2021 · $601,113

## Abstract

Abstract
SNARE proteins and Sec1p/Munc18-family (SM) proteins constitute the core molecular machines that
mediate nearly all intracellular membrane fusion. Other proteins regulate the core machinery to
enable fusion at the right time and location. Dysfunction of these proteins has been linked to
neurological and immunological disorders, cancers, diabetes, and other diseases. Decades of
research have established that SNAREs couple their ordered folding and assembly to membrane
fusion in a SM protein-dependent manner. However, it remains unclear what mechanistic role SM
proteins plays in SNARE assembly and how SNARE assembly is coupled to membrane fusion. Using
high-resolution optical tweezers, we recently found that neuronal SM protein Munc18-1 catalyzes
step-wise assembly of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix
bundle, a process essential for neurotransmission and insulin secretion. Importantly, Munc18-1
serves as a template to guide directional SNARE assembly along a new pathway. In this application,
we plan to first establish that the template complex is a conserved intermediate for SNARE-SM fusion
machineries and a key target for other proteins to regulate SNARE assembly and membrane fusion.
We will examine effects of key regulators involved in calcium-triggered exocytosis (Munc13-1,
complexin, synaptotagmin, NSF, and alpha-SNAP) and phosphorylation of SNARE and SM proteins
on SNARE assembly and disassembly. Then, we will develop new assays to simultaneous detect
step-wise SNARE assembly and state-wise membrane fusion using large nanodiscs and trapped
GUVs. A major goal is to reconstitute the calcium-triggered membrane fusion under controlled
experimental conditions and to understand their working mechanism. Finally, we will extend our
methodologies to pinpoint the molecular mechanisms of membrane binding and lipid exchange by
extended synaptotagmins (E-Syts) and of mechanosensation by ion channel NOMPC. Our long-term
goal is to develop a general approach to elucidate stability, folding, and dynamics of membrane
proteins.

## Key facts

- **NIH application ID:** 10152615
- **Project number:** 5R35GM131714-03
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Yongli Zhang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $601,113
- **Award type:** 5
- **Project period:** 2019-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10152615

## Citation

> US National Institutes of Health, RePORTER application 10152615, Single-molecule manipulation of proteins involved in membrane fusion, lipid exchange, and mechanosensation (5R35GM131714-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10152615. Licensed CC0.

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