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

> **NIH NIH R35** · YALE UNIVERSITY · 2023 · $78,941

## 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:** 10799300
- **Project number:** 3R35GM131714-05S1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Yongli Zhang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $78,941
- **Award type:** 3
- **Project period:** 2019-05-01 → 2024-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10799300

## Citation

> US National Institutes of Health, RePORTER application 10799300, Single-molecule manipulation of proteins involved in membrane fusion, lipid exchange, and mechanosensation (3R35GM131714-05S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10799300. Licensed CC0.

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