Abstract
Membrane proteins (MPs) influence all aspects of life, such as tumorigenesis, immune response, and neural transmission. However, characterization of MPs is challenging, as it often needs highly specialized techniques inaccessible to many labs. We herein introduce nanodisc-ID that enables quantitative analysis of membrane proteins using a gel electrophoresis readout. By leveraging the power of nanodiscs and proximity labeling, nanodisc-ID serves both as scaffolds for encasing biochemical reactions and as sensitive reagents for detecting membrane protein-lipid and protein-protein interactions. We demonstrate this label-free and low-cost tool by characterizing a wide range of integral and peripheral membrane proteins from prokaryotes and eukaryotes.
Highlights
Membrane proteins (MPs) influence all aspects of life, such as tumorigenesis, immune response, and neural transmission
We screened a panel of proximity labeling (PL) enzymes to test their compatibility with NDs for detecting the interaction between peripheral membrane proteins and lipids
We tested three classes of proximity labeling enzymes: (1) APEX2 derived from peroxidase[12,13]; (2) BioID and TurboID derived from biotin ligase[14,15]; (3) PafA, a bacterial ubiquitin-like protein ligase[16]
Summary
Membrane proteins (MPs) influence all aspects of life, such as tumorigenesis, immune response, and neural transmission. By leveraging the power of nanodiscs and proximity labeling, nanodisc-ID serves both as scaffolds for encasing biochemical reactions and as sensitive reagents for detecting membrane protein-lipid and protein-protein interactions. We demonstrate this label-free and low-cost tool by characterizing a wide range of integral and peripheral membrane proteins from prokaryotes and eukaryotes. A pressing need exists to develop simple and straightforward methods that can rapidly interrogate these interactions To tackle this challenge, we set out to marry nanodiscs with proximity labeling (PL) for facile and sensitive characterizations of MPs. Over the past few years, PL has emerged as a powerful approach for mapping protein–protein, protein–RNA, and protein–DNA interactomes[4]. Nanodisc-ID could serve as a powerful and versatile approach for biochemical dissections of MPs
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