# Unravelling Membrane Protein-Lipid Interactions using Nanodiscs and Mass Spectrometry

> **NIH NIH R35** · UNIVERSITY OF ARIZONA · 2022 · $365,208

## Abstract

PROJECT SUMMARY/ABSTRACT
 Membrane proteins are involved in many cellular processes and thus are critical drug targets for a wide range
of diseases. However, there is a fundamental gap in understanding how the lipid environment affects membrane
protein structure and function. Mounting evidence indicates that lipids can be essential for membrane protein
function, but it is challenging to determine the molecular mechanisms underlying the importance of protein-lipid
interactions. The primary challenge is that conventional structural biology tools and binding assays are poorly
suited to characterizing transient and heterogeneous protein-lipid interactions.
 To understand how lipids modulate membrane protein structure and function, my research program is
developing new tools to study protein-lipid interactions by combining lipoprotein nanodiscs with mass
spectrometry (MS). These new technologies will be developed using well-characterized bacterial membrane
protein complexes before being applied to more complex mammalian proteins such as rhodopsin and uncoupling
protein 2. Our goal is to answer four questions for a given membrane protein target. 1) What lipids interact with
the target? To identify the endogenous lipids that surround the target, we are developing a hybrid
lipopeptide/lipoprotein approach to solubilize membranes surrounded by their natural lipids into nanodiscs
without the need for detergent. Following purification of the target, we will extract and identify the lipids that are
naturally associated with the target. 2) How strongly do lipids bind to the target? To distinguish tightly bound
lipids from weakly associated lipids, we will assemble lipoprotein nanodiscs with different mixtures of lipids and
use native MS to ionize the intact nanodisc assembly. Using collisional activation, we will gradually dissociate
the nanodisc to measure the composition of the lipid annular belt and tightly associated structural lipids.
Furthermore, we will use lipid exchange between nanodiscs to measure lipid binding constants in an experiment
analogous to equilibrium dialysis. 3) Where do lipids interact with the protein structure? After predicting lipid-
binding sites using molecular dynamics (MD), we will test the predictions by mutating interacting residues and
using native MS to detect the disruption of the binding sites. 4) Why are specific protein-lipid interactions
important for function? By pairing MD, mutagenesis, and native MS with functional studies, we will connect
lipid-dependent effects on protein function with specific lipid binding sites.
 Our overarching goal is to develop a toolbox for unravelling the molecular mechanisms of protein-lipid
interactions. This will impact biomedical research by identifying lipids important for maintaining protein activity
and aiding in elucidating the physiological mechanisms of membrane proteins inside natural bilayers. Ultimately,
an improved understanding of protein-lipid interactions holds the potentia...

## Key facts

- **NIH application ID:** 10475805
- **Project number:** 5R35GM128624-05
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** Michael T Marty
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $365,208
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10475805

## Citation

> US National Institutes of Health, RePORTER application 10475805, Unravelling Membrane Protein-Lipid Interactions using Nanodiscs and Mass Spectrometry (5R35GM128624-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10475805. Licensed CC0.

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