Mechanism of Formation and Functional Properties of Lipoprotein Discs Stabilized by Amphiphilic Maleic Acid-Containing Alternating Copolymers

两亲性含马来酸的交替共聚物（MACPs），马来酸单元与（可变）碳氢化合物单元交替，是最近膜蛋白研究中一个重要的方法突破。一些macp被发现可以溶解来自人工和天然脂质双层的膜蛋白，产生直径在10-30 nm范围内均匀的盘状macp包裹的脂质/脂蛋白颗粒。酶似乎在macp颗粒内保持功能；因此，这些颗粒甚至可以使那些在洗涤剂存在下不稳定的膜蛋白，在需要均质水溶性（单）颗粒的技术中变得容易。然而，目前尚不清楚的是，在没有洗涤剂的情况下，macp是如何提取膜蛋白的，macp脂质/脂蛋白颗粒的不同组分是如何相互作用的，以及这些颗粒中受限的脂质环境是如何干扰被包裹膜蛋白的大规模构象动力学的。在与莫斯科国立大学（俄罗斯）的Konstantin Shaitan教授的合作中，我们打算通过实验和理论相结合的方法来仔细研究macp粒子的性质。使用不同的macp，我们将通过尺寸排除色谱，动态光散射，原子力显微镜，透射电子显微镜和EPR光谱来表征macp -脂质/脂蛋白颗粒。作为一种功能探测器，光敏感古细菌视紫红质及其同源换能器蛋白的复合物将被插入到macp颗粒中。利用时间分辨光学和EPR光谱学在不同的时间尺度上跟踪其蛋白质动力学，以了解MACPs的性质如何影响复合物内的光诱导信号转导。同时，我们的合作伙伴将模拟macp粒子，并通过分子动力学模拟来分析它们，以评估静电、疏水和堆叠相互作用如何稳定粒子。该项目的目标是绘制macp包裹的脂质/脂蛋白纳米颗粒形成和稳定的物理化学图，并开发优化的macp提取蛋白质的方案，在macp脂质/脂蛋白颗粒中产生类似天然环境的macp。

Amphiphilic maleic acid-containing alternating copolymers (MACPs), in which units of maleic acid alternate with (variable) hydrocarbon units, account for a recent major methodical breakthrough in the study of membrane proteins. Several MACPs were found to solubilize membrane proteins both from artificial and natural lipid bilayers yielding discoidal MACP-encased lipid/lipoprotein particles with uniform diameter in the range from 10-30 nm. Enzymes seem to remain functional within MACP-particles; therefore, such particles make even those membrane proteins, which are unstable in the presence of detergent, accessible to techniques that require homogenous water-soluble (single) particles. It remains, however, unclear how MACPs extract membrane proteins in the absence of detergents, how the different components of the MACP-lipid/lipoprotein particle interact, and how the restricted lipid environment in these particles interferes with large scale conformational dynamics of the encased membrane protein. In a collaboration with Prof. Konstantin Shaitan, Lomonosov Moscow State University (Russia), we intend to scrutinize the properties of MACP-particles by combining experimental and theoretical approaches. Using different MACPs, we will characterize MACP-lipid/lipoprotein particles by size exclusion chromatography, dynamic light scattering, atomic-force microscopy, transmission electron microscopy, and EPR spectroscopy. As a functional detector the complex of the light sensitive archaeal sensory rhodopsin and its cognate transducer protein will be inserted into MACP-particles. Its protein dynamics will be followed on different time scales using time resolved optical and EPR spectroscopy to understand, how the nature of MACPs affects the light-induced signal transduction within the complex. Concurrently, our collaboration partner will model MACP-particles and analyze them by molecular dynamic simulations to assess how electrostatic, hydrophobic and stacking interactions stabilize the particles. The goals of the project are to draw a physico-chemical picture of formation and stabilization of MACP-encased lipid/lipoprotein nanoparticles and to develop optimized protocols of protein extraction by MACPs yielding native-like environments within MACP-lipid/lipoprotein particles.