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

含有马来酸的两亲性交替共聚物(MACPs)是近年来膜蛋白研究中的一项重大方法学突破。发现几种MACP可以溶解人工和天然脂双层中的膜蛋白，得到直径在10-30 nm范围内均匀分布的盘状MACP包覆脂/脂蛋白颗粒。酶似乎在MACP颗粒中保持功能；因此，这种颗粒甚至使那些在洗涤剂存在下不稳定的膜蛋白，可以通过要求均一的水溶性(单一)颗粒的技术来获得。然而，目前还不清楚MACP在没有洗涤剂的情况下是如何提取膜蛋白的，MACP-脂/脂蛋白颗粒的不同成分是如何相互作用的，以及这些颗粒中受限的脂环境如何干扰被包裹的膜蛋白的大规模构象动力学。在与俄罗斯洛蒙诺索夫国立大学康斯坦丁·沙坦教授的合作下，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，我们将通过尺寸排斥层析、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，光敏感的古细菌感觉性视紫红质与其同源转导蛋白的复合体将被插入到MACP-颗粒中。它的蛋白质动力学将使用时间分辨光学和EPR光谱在不同的时间尺度上进行跟踪，以了解MACP的性质如何影响复合体内的光诱导信号转导。同时，我们的合作伙伴将对MACP颗粒进行建模，并通过分子动力学模拟对其进行分析，以评估静电、疏水和堆积相互作用如何稳定颗粒。该项目的目标是绘制MACP包裹的脂/脂蛋白纳米颗粒的形成和稳定的物理化学图景，并开发通过在MACP-脂/脂蛋白颗粒内产生类似天然环境的MACP来提取蛋白质的优化方案。

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.