Novel Detergent Systems for Membrane Protein Studies

去污剂是膜蛋白、脂质和其他疏水分子的生物物理研究的基本工具。虽然传统的洗涤剂仍在广泛使用，并且是必不可少的试剂，但没有一种洗涤剂具有适用于所有应用的理想特性。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Privé实验室正在积极开发的三项新技术。 1)Picodisks。我们已经发现小的人类蛋白Saposin A能够与各种脂质形成稳定的、确定的、低分子量盘状复合物。我们将这些组件称为“picodisks”，以强调与广泛使用的“nanodisks”的相似性。纳米盘作为去污剂或双胞的替代物用于稳定膜蛋白已经产生了令人兴奋的结果，然而，纳米盘由于其尺寸而遭受相对高程度的多分散性。相比之下，微微圆盘明显更小，因此具有更明确的化学计量。因此，它们只能容纳较小的两亲分子，如特定的脂质或单跨膜α螺旋。因此，picodisks具有独特的性质，并补充了目前纳米盘的可行性，我们将开发picodisks作为一种独特的增溶系统。 2)脂肽洗涤剂（LPD）。我们已经设计了20-25个氨基酸的多肽共价偶联到脂肪酰基链作为一类新的增溶两亲物。LPD可以自组装成具有低聚集数的相对刚性的胶束，并且可以通过将膜蛋白容纳在圆柱形LPD胶束的内部来稳定膜蛋白。我们已经设计和合成了几个系列的LPD，每一个都有自己的特点。LPDs很容易结晶，我们已经确定了高分辨率的晶体结构的LPDs胶束含有8，9和12个链排列成圆柱形胶束与adhesidic核心。我们建议完善和多样化的脂肽的设计与新的性能，并扩大这些新的两亲物的潜力到新的应用。 3)SDS/MPD。我们发现，一些小分子醇，如2，4-甲基戊二醇（MPD），对离子型洗涤剂的性能有深远的影响。例如，SDS通常是强蛋白质变性剂。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，以便在分子水平上了解它们，并探索它们如何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.