Novel Detergent Systems for Membrane Protein Studies

洗涤剂是生物物理研究膜蛋白、脂质和其他疏水分子的基本工具。虽然传统的洗涤剂仍然被广泛使用，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，一些新的替代增溶系统正在开辟新的研究途径。该计划建议扩展privprive实验室正在积极开发的三种新技术。* * * * * * 1) Picodiscs。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、明确、低分子量的盘状复合物。为了强调与广泛使用的“纳米光盘”的相似性，我们将这些组件称为“微光盘”。纳米圆盘作为稳定膜蛋白的洗涤剂或单胞体的替代品已经引起了人们的兴奋，然而，纳米圆盘由于其大小而具有相对较高的多分散性。相比之下，微盘要小得多，因此具有更明确的化学计量学。因此，它们只能容纳较小的两亲性分子，如特定的脂质或单个跨膜α螺旋。因此，皮盘具有独特的性能，并补充了目前纳米盘的可行性，我们将开发皮盘作为一种独特的增溶体系。*********2)脂肽洗涤剂（lpd）。我们设计了20-25个氨基酸多肽与脂肪酸酰基链共价偶联，作为一类新的增溶性两亲体。LPD可以自组装成相对刚性的胶束，聚集数低，并通过将膜蛋白安置在圆柱形LPD胶束内部来稳定膜蛋白。我们设计并合成了几个系列的lpd，每个系列都具有特定的特性。lpd容易结晶，我们已经确定了含有8、9和12链的lpd胶束的高分辨率晶体结构，这些胶束排列成具有脂质核的圆柱形胶束。我们建议改进和多样化具有新性质的脂肽的设计，并扩大这些新型两亲分子的应用潜力。* * * * * * * * * 3) SDS / MPD。我们发现一些小醇，如2,4-甲基戊二醇（MPD），对离子洗涤剂的性能有深远的影响。例如，SDS通常是一种强蛋白质变性剂。然而，SDS和MPD的混合物可以促进膜蛋白折叠，甚至可以用于膜蛋白结晶。我们将研究MPD如何调节SDS性质的机制，并利用这些信息来扩展该技术的应用。*********总的来说，拟议的研究计划描述了我们扩展这些新方法的计划。重点是开发和表征这些系统，以便在分子水平上理解它们，以及探索如何将它们应用于现有和新的应用

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.**