Membrane-Mimicking Reverse Micelles for High-Resolution Interfacial Study of Proteins and Membranes
Membrane-Mimicking Reverse Micelles for High-Resolution Interfacial Study of Proteins and Membranes
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DOI：
10.1021/acs.langmuir.1c03085
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发表时间：
2022-03-17
期刊：
影响因子：
3.9
通讯作者：
Fuglestad, Brian
中科院分区：
文献类型：
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作者：
Labrecque, Courtney L.;Nolan, Aubree L.;Fuglestad, Brian
尽管取得了重大进展，但蛋白质与膜相互作用的研究仍然是一个重大挑战。虽然整合膜蛋白一直是最近的努力的主要焦点，外周膜蛋白（PMP）和它们与膜和脂质的相互作用有少得多的高分辨率信息可用。它们的小尺寸和它们相互作用的动态性质阻碍了使用结构方法如cryo-EM和X射线晶体学进行详细的界面研究。PMP相互作用的结构分析的一个主要障碍是在膜模型研究膜招募状态的局限性。常用的膜模拟物，如脂质体，bicelles，nanodisces和胶束是非常大的或由非生物去污剂，限制了它们的实用性的NMR研究的PMP。虽然先前已经成功地使用了整合膜蛋白和外周膜蛋白，但目前采用的反胶束（RM）组合物针对其与蛋白质的惰性而不是其模拟膜的能力进行了优化。应用更多的天然膜状脂质和表面活性剂有望成为蛋白质和膜之间界面相互作用研究的一个有价值的进展。在这里，我们描述了基于磷酸胆碱的RM系统，模仿生物膜，并与高分辨率蛋白质NMR兼容的发展。我们展示了新的配方，能够封装模型的可溶性蛋白质，泛素，蛋白质结构的干扰最小。此外，一个公式，DLPC：DPC，允许封装的PMP谷胱甘肽过氧化物酶4（GPx 4）和磷脂酰乙醇胺结合蛋白1（PEBP 1），使这些蛋白质的嵌入，匹配预期的相互作用与生物膜。RM的动态光散射和小角度X射线散射表征揭示了小的、近似球形的和非聚集的颗粒，这是蛋白质NMR和其他研究途径的先决条件。这里提出的配方代表了一种新的工具，用于研究难以捉摸的PMP相互作用和其他膜界面的调查。
Despite substantial advances, the study of proteins interacting with membranes remains a significant challenge. While integral membrane proteins have been a major focus of recent efforts, peripheral membrane proteins (PMPs) and their interactions with membranes and lipids have far less high-resolution information available. Their small size and the dynamic nature of their interactions have stalled detailed interfacial study using structural methods like cryo-EM and X-ray crystallography. A major roadblock for the structural analysis of PMP interactions is limitations in membrane models to study the membrane recruited state. Commonly used membrane mimics such as liposomes, bicelles, nanodiscs, and micelles are either very large or composed of non-biological detergents, limiting their utility for the NMR study of PMPs. While there have been previous successes with integral and peripheral membrane proteins, currently employed reverse micelle (RM) compositions are optimized for their inertness with proteins rather than their ability to mimic membranes. Applying more native, membrane-like lipids and surfactants promises to be a valuable advancement for the study of interfacial interactions between proteins and membranes. Here, we describe the development of phosphocholine-based RM systems that mimic biological membranes and are compatible with high-resolution protein NMR. We demonstrate new formulations that are able to encapsulate the model soluble protein, ubiquitin, with minimal perturbations of the protein structure. Furthermore, one formula, DLPC:DPC, allowed the encapsulation of the PMPs glutathione peroxidase 4 (GPx4) and phosphatidylethanolamine-binding protein 1 (PEBP1) and enabled the embedment of these proteins, matching the expected interactions with biological membranes. Dynamic light scattering and small-angle X-ray scattering characterization of the RMs reveals small, approximately spherical, and non-aggregated particles, a prerequisite for protein NMR and other avenues of study. The formulations presented here represent a new tool for the study of elusive PMP interactions and other membrane interfacial investigations.