Novel Detergent Systems for Membrane Protein Studies

洗涤剂是对膜蛋白、脂类和其他疏水分子进行生物物理研究的重要工具。虽然传统洗涤剂仍在广泛使用，是必不可少的试剂，但没有一种洗涤剂具有适用于所有应用的理想性能。因此，几种新的替代增溶系统正在开辟新的研究途径。拟议的方案提议扩大私人实验室正在积极开发的三项新技术。 1)PicoDisk。我们已经发现，人类小蛋白皂苷A能够与各种脂质形成稳定的、明确的、低分子质量的盘状复合体。我们将这些组件称为“微微盘”，以强调与广泛使用的“纳米盘”的相似之处。纳米盘作为膜蛋白稳定剂的替代品已经引起了人们的兴奋，然而，由于其尺寸的原因，纳米盘具有相对较高的多分散性。相比之下，皮盘要小得多，因此具有更明确的化学计量标准。因此，它们只能容纳较小的两亲性分子，如特定的脂类或单一的跨膜α螺旋。因此，微微盘具有独特的性质，是对目前纳米盘可行的补充，我们将开发微微盘作为一种独特的增溶系统。 2)脂肽洗涤剂(LPD)。我们设计了20-25个氨基酸多肽共价偶联到脂肪酰链上，作为一类新的增溶两亲分子。LPD可以自组装成相对刚性的低聚集数胶束，并通过将这些胶束容纳在圆柱形LPD胶束内部来稳定膜蛋白。我们设计并合成了几个系列的液晶显示器，每个系列都有自己的特点。LPD很容易结晶，我们已经确定了LPDS胶束的高分辨晶体结构，这些胶束包含8、9和12个链排列成具有脂核的圆柱形胶束。我们建议改进和多样化具有新性质的脂肽的设计，并将这些新型两亲性化合物的潜力扩展到新的应用领域。 3)SDS/MPD。我们发现，一些小分子醇，如2，4-甲基戊二醇(MPD)，对离子洗涤剂的性能有很大的影响。例如，十二烷基硫酸钠通常是一种强烈的蛋白质变性剂。而十二烷基硫酸钠和甲氧基苯二胺的混合物可以促进膜蛋白的折叠，甚至可以用于膜蛋白的结晶。我们将研究MPD如何调节十二烷基硫酸钠的性质的机制，并利用这些信息来扩大这项技术的应用。 总体而言，拟议的研究计划描述了我们在这些新方法上扩展的计划。重点是开发和表征这些系统，以便在分子水平上了解它们，以及探索如何将它们应用于现有的和新的应用。

Detergents are essential tools for the biophysical study of membrane proteins, lipids, and other hydrophobic molecules. While traditional detergents remain in wide use and are essential reagents, no single detergent has ideal properties for all applications. As a result, several new alternative solubilization systems are opening up new avenues of research. The proposed program proposes to expand on three novel technologies that are being actively developed in the Privé lab. 1) Picodiscs. We have discovered that the small human protein Saposin A is able to form stable, defined, low-molecular weight discoidal complexes with various lipids. We have called these assemblies “picodiscs” in order to emphasize the similarities with the widely used “nanodiscs”. Nanodiscs have generated excitement as alternatives to detergents or bicelles for the stabilization of membrane proteins, however, nanodiscs suffer from a relatively high degree of polydispersity due to their size. Picodiscs, in contrast, are significantly smaller and thus have more defined stoicheometries. As a result, they can only accommodate smaller amphiphilic molecules such as specific lipids or single transmembrane alpha helices. Thus, picodiscs have unique properties and complement what is currently feasible with nanodiscs, and we will develop picodiscs as a unique solubilization system. 2) Lipopeptide detergents (LPDs). We have designed 20-25 amino acid polypeptides covalently coupled to fatty acyl chains as a new class of solubilizing amphiphile. LPDs can self-assemble into relatively rigid micelles with low aggregation numbers and can stabilize membrane proteins by accommodating these in the interior of cylindrical LPD micelles. We have designed and synthesized several series of LPDs, each with specific characteristics. LPDs readily crystallize, and we have determined high-resolution crystal structures of LPDs micelles containing 8, 9 and 12 chains arranged into cylindrical micelles with lipidic cores. We propose to refine and diversify the design of lipopeptides with novel properties, and to expand the potential of these novel amphiphiles into new applications. 3) SDS/MPD. We have found that some small alcohols, such as 2,4-methyl-pentanediol (MPD), have profound effects on the properties of ionic detergents. For example, SDS is normally a strong protein denaturant. However, mixtures of SDS and MPD can promote membrane protein folding and can even be used in membrane protein crystallization. We will investigate the mechanism of how MPD modulates the properties of SDS, and use this information to expand the applications of this technology. Overall, the proposed research program describes our plans to expand on these new methodologies. The focus is to develop and characterize these systems in order to understand them at the molecular level, as well as to explore how they can be applied to existing and novel applications.