Synthesis and Evaluation of Novel Surfactants for Membrane Protein Structural Bio

在分析膜蛋白结构和功能的过程中，蛋白质生产一直被认为是主要的瓶颈。目前的结构基因组学工作几乎忽略了这类重要的蛋白质，因为很难生产出足够数量的功能形式的纯化样品，这是结晶实验所需的。在膜蛋白样品生产中的许多具有挑战性的步骤中，增溶过程？将疏水性膜蛋白从天然脂双层中去除，并通过使用洗涤剂胶束人工使其与水相兼容--通常是最大的问题。众所周知，洗涤剂的选择是一种反复试验的经验努力，已被证明是非常特定于蛋白质的。目前，这一领域受到表面活性剂的极大限制。 设置仅显示很小范围的化学和物理属性。只有少数几类表面活性剂被成功地用于纯化和结晶大约60种独特的膜蛋白，这些蛋白的结构已经存储在蛋白质数据库中。我们寻求开发有效的方法来筛选表面活性剂对膜蛋白结构生物学的适用性。我们最初将使用表面活性剂测试这些方案，这些表面活性剂在增溶和维持膜蛋白的结构和功能完整性方面具有已被证明的效用。然后，我们将修改这些协议以进行高通量筛选，以评估我们团队可从商业实体获得的表面活性剂文库。这些表面活性剂是为其他目的而开发的，但其中一些可能适用于这一新兴领域。同时，我们将探索新的合成分子， 旨在取代传统上用于膜蛋白增溶的洗涤剂。后一类分子的成员是专门设计用于膜蛋白增溶和结晶的。

Protein production has been identified as the major bottleneck in the process of analyzing structure and function of membrane proteins. Current structural genomics efforts all but ignore this important class of proteins due to the difficulty in producing sufficient quantities of purified samples in functional form, as required for crystallization experiments. Of the many challenging steps in the production of membrane protein samples, the process of solubilization ? removing the hydrophobic membrane proteins from their native lipid bilayer and artificially rendering them compatible with aqueous phases by the use of detergent micelles - is typically the most problematic. Detergent selection is known to be a trial-and-error, empirical endeavor that has proven to be very protein-specific. Currently, the field is extremely limited by surfactant sets that display only a narrow range of chemical and physical properties. Only a few classes of surfactants have been used successfully in the purification and crystallization of the approximately 60 unique membrane proteins for which structures have been deposited in the Protein Data Bank. We seek to develop efficient methods by which surfactants can be screened for their applicability to membrane protein structural biology. We will initially test these protocols using surfactants with proven utility in solubilization and maintenance of the structural and functional integrity of membrane proteins. We will then adapt these protocols for high-throughput screening in order to assess libraries of surfactants available to our team from commercial entities. These surfactants were developed for other purposes, but some of them might be applicable to this emerging field. In parallel, we will explore novel synthetic molecules that are intended to substitute for the detergents traditionally used for the solubilization of membrane proteins. Members of this latter class of molecules were specifically designed for use in membrane protein solubilization and crystallization.