New Polymer Belts for Lipid-Bilayer Nanodiscs to Study the Molecular Basis of GPCR Signalling

膜蛋白在细胞通讯和物质转移中起着重要作用，约占所有药物靶点的一半。然而，它们的提取、纯化和体外研究都非常具有挑战性。目前用于分离膜蛋白的方法往往依赖于侵略性化学反应，导致蛋白质变性。对于许多药理学靶标蛋白来说也是如此，它们从天然膜环境中去除后稳定性差，严重损害了药物的发现。在新的化学启发策略中，由脂质和苯乙烯/马来酸（SMA）共聚物组装的纳米圆盘受到了广泛的关注。这些聚合物可以直接从膜上吸收膜蛋白和脂质，形成纳米级的脂质双层斑块。然而，这种有前途的技术目前受到嵌入膜蛋白和脂质的构象动力学改变的限制。进一步的限制是存在的紫外线吸收芳香环和高电荷密度由于聚合物的羧基。NanoBelt将开发新的聚合物，这些聚合物结合了良好的增溶率、低紫外线吸收、可忽略的电荷密度，以及维持蛋白质的功能和构象动力学。聚合物由合作伙伴1 （ibmm -阿维尼翁大学）通过现有聚合物的后功能化或从功能化单体重新合成制备。合作伙伴2 （TU Kaiserslautern）确定了新聚合物形成纳米圆盘和提取膜蛋白的效力，以选择敏感G蛋白偶联受体（gpcr）的最佳聚合物。ghrelin受体（GHSR）被Partner 3 （IBMM-CNRS）用作a类GPCR的原型。为此，重组受体被组装到新的纳米圆盘中，以确定其药理学性质。Partner 4（莱比锡大学）使用促肾上腺皮质激素释放因子受体（CRF1R）作为B类gpcr的模型。使用新的FRET传感器，这些具有挑战性的药物靶点的构象动力学将在新的聚合物基纳米圆盘的天然样但受控的环境中被剖析。由于基于伙伴2、3和4的互补技能的系统验证方法，nanobbelt将提供结构-活性关系，从而能够合理设计由伙伴1合成的改进聚合物。最终目标是确定和建立新的聚合物，以温和而有效的方式直接从细胞膜上提取膜蛋白及其周围的脂质，形成聚合物包裹的脂质双层纳米盘，保留提取蛋白质的天然结构和功能。

Membrane proteins play fundamental roles in cellular communication and material transfer and account for about half of all drug targets. However, their extraction, purification, and in vitro investigation are very challenging. Current methods used for isolating membrane proteins often depend on aggressive chemistry and result in protein denaturation. This is also true for many pharmacological target proteins, whose poor stability upon removal from the native membrane environment seriously impairs drug discovery. Among new chemically inspired strategies, nano-discs assembled from lipids and styrene/maleic acid (SMA) copolymers have received great attention. These polymers can recruit membrane proteins and lipids directly from membranes into nanoscale lipid-bilayer patches. Yet, this promising technology is currently limited by alterations in the conformational dynamics of embedded membrane proteins and lipids. Further limitations are the presence of UV-absorbing aromatic rings and the high charge density due to the polymer’s carboxyl groups.NanoBelt will develop new polymers that combine good solubilisation yields with low UV absorption, negligible charge density, and maintenance of the function and conformational dynamics of proteins. Polymers are prepared by Partner 1 (IBMM—Avignon University) by post-functionalisation of existing polymers or de novo synthesis from functionalised monomers. Partner 2 (TU Kaiserslautern) determines the potency of the new polymers to form nanodiscs and to extract membrane proteins to select optimal polymers for sensitive G protein-coupled receptors (GPCRs). The ghrelin receptor (GHSR) is used as a prototypical class A GPCR by Partner 3 (IBMM—CNRS). To this end, the recombinant receptor is assembled into the new nanodiscs for determination of its pharmacological properties. Partner 4 (Leipzig University) uses the corticotropin releasing factor receptor (CRF1R) as a model of class B GPCRs. Using new FRET sensors, the conformational dynamics of these challenging drug targets will be dissected in the native-like but controlled environment of new polymer-based nanodiscs. Owing to a systematic approach of validation based on the complementary skills of Partners 2, 3, and 4, NanoBelt will provide structure–activity relationships to enable the rational design of improved polymers synthesised by Partner 1.The ultimate goal consists in identifying and establishing new polymers that extract membrane proteins with their surrounding lipids directly from cellular membranes in a mild yet efficient manner to form polymer-encapsulated lipid-bilayer nanodiscs that retain the native structures and functions of the extracted proteins.