Investigating protein-membrane interactions using native reverse micelles constructed from naturally sourced lipids.
Investigating protein-membrane interactions using native reverse micelles constructed from naturally sourced lipids.
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DOI：
10.1002/pro.4786
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发表时间：
2023-11
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影响因子：
--
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推进膜相关蛋白及其相互作用的研究取决于准确的膜模型。虽然存在多种用于高分辨率膜蛋白研究的膜模型，但大多数模型并不能反映生物膜内发现的脂质的多样性。在这项工作中，我们开发了由多种真核来源的脂质配制而成的天然反胶束（nRM），它封装蛋白质并使它们能够像与生物膜一样相互作用。使用大豆卵磷脂、猪脑脂质或牛心脂质与正十二烷基磷酸胆碱结合开发了不同的 nRM 配方，并通过动态光散射和 31P-NMR 进行了表征。为了优化蛋白质封装，使用泛素作为标准品，蛋白质 NMR 验证了其结构的最小变化。可逆地与膜结合的外周膜蛋白被封装，包括谷胱甘肽过氧化物酶 4 (GPx4)、磷脂酰乙醇胺结合蛋白 1 (PEBP1) 和脂肪酸结合蛋白 4 (FABP4)。通过蛋白质 NMR 评估，所有三种蛋白质都显示出与 nRM 的膜状内表面的预期相互作用。与其他非天然膜模型相比，这里开发的 nRM 配方可以在更具生物学相关性的背景下，对高达或超过 ~21kDa 的膜相互作用蛋白进行高效、高分辨率的研究。这里概述的方法可以应用于广泛的脂质提取物，从而可以在其特定的生物学背景下研究各种膜相关蛋白。
Advancing the study of membrane associated proteins and their interactions is dependent on accurate membrane models. While a variety of membrane models for high‐resolution membrane protein study exist, most do not reflect the diversity of lipids found within biological membranes. In this work, we have developed native reverse micelles (nRMs) formulated with lipids from multiple eukaryotic sources, which encapsulate proteins and enable them to interact as they would with a biological membrane. Diverse formulations of nRMs using soy lecithin, porcine brain lipids, or bovine heart lipids combined with n‐dodecylphosphocholine were developed and characterized by dynamic light scattering and 31P‐NMR. To optimize protein encapsulation, ubiquitin was used as a standard and protein NMR verified minimal changes to its structure. Peripheral membrane proteins, which bind reversibly to membranes, were encapsulated and include glutathione peroxidase 4 (GPx4), phosphatidylethanolamine‐binding protein 1 (PEBP1), and fatty acid binding protein 4 (FABP4). All three proteins showed anticipated interactions with the membrane‐like inner surface of the nRMs as assessed by protein NMR. The nRM formulations developed here allow for efficient, high‐resolution study of membrane interacting proteins up to and beyond ~21 kDa, in a more biologically relevant context compared to other non‐native membrane models. The approach outlined here may be applied to a wide range of lipid extracts, allowing study of a variety of membrane associated proteins in their specific biological context.
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影响因子：
62.1
作者：
Chipot C;Dehez F;Schnell JR;Zitzmann N;Pebay-Peyroula E;Catoire LJ;Miroux B;Kunji ERS;Veglia G;Cross TA;Schanda P
通讯作者：
Schanda P
影响因子：
6.8
作者：
Brown, Chanelle J.;Trieber, Catharine;Overduin, Michael
通讯作者：
Overduin, Michael
影响因子：
2.9
作者：
Constantine, KL;Friedrichs, MS;Farmer, BT
通讯作者：
Farmer, BT
影响因子：
1.5
作者：
Kato, Tsuyoshi;Nishimiya, Mami;Inagaki, Minoru
通讯作者：
Inagaki, Minoru
DOI：
10.1021/je00017a014
发表时间：
1995-01-01
期刊：
JOURNAL OF CHEMICAL AND ENGINEERING DATA
影响因子：
--
作者：
FRANJO, C;JIMENEZ, E;ANDRADE, MIP
通讯作者：
ANDRADE, MIP