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

用于脂质双层纳米圆盘的新型聚合物带，用于研究 GPCR 信号转导的分子基础

• 批准号: 445989742
• 负责人: Professorin Dr. Irene Coin
• 金额: --
• 依托单位: Institut für Molekulare Biowissenschaften
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/445989742?language=en
• 关键词: New; Polymer; Belts; Lipid; Bilayer

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.

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、纯化和体外研究非常具有挑战性。目前用于分离膜蛋白的方法通常依赖于侵蚀性化学并且导致蛋白质变性。对于许多药理学靶蛋白也是如此，其在从天然膜环境中去除时的差的稳定性严重损害药物发现。在新的化学启发的策略，纳米盘组装从脂质和苯乙烯/马来酸（SMA）共聚物已受到极大的关注。这些聚合物可以将膜蛋白和脂质直接从膜招募到纳米级脂质双层贴片中。然而，这种有前途的技术目前受到嵌入的膜蛋白和脂质的构象动力学改变的限制。进一步的限制是紫外线吸收芳香环的存在和高电荷密度由于聚合物的羧基。NanoBelt将开发新的聚合物，结合联合收割机良好的增溶率与低紫外线吸收，可忽略的电荷密度，并保持蛋白质的功能和构象动力学。聚合物由Partner 1（IBMM-Avignon University）通过现有聚合物的后官能化或从官能化单体重新合成来制备。Partner 2（TU Kaiserelistern）确定新聚合物形成纳米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。生长激素释放肽受体（GHSR）被Partner 3（IBMM-CNRS）用作原型A类GPCR。为此，将重组受体组装到新的纳米盘中以测定其药理学性质。Partner 4（莱比锡大学）使用促肾上腺皮质激素释放因子受体（CRF 1 R）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标的构象动力学将在新的基于聚合物的纳米盘的天然但受控的环境中进行解剖。由于采用了以伙伴2、3和4的互补技能为基础的系统验证方法，NanoBelt将提供结构-活性关系，使Partner 1合成的改进聚合物的合理设计成为可能。最终目标包括鉴定和建立新的聚合物，这些聚合物以温和而有效的方式直接从细胞膜中提取膜蛋白及其周围的脂质，以形成聚合物。包封的脂质双层纳米盘，其保留了提取的蛋白质的天然结构和功能。