Antibody Fragments, Chaperones, and Membrane Protein Crystals

很难纯化大量的膜蛋白，并且大多数可以纯化的膜蛋白已被证明是难结晶的。这个过程中每一步固有的困难导致了膜蛋白晶体结构的缺乏，同时对药物发现和设计产生了抑制作用。如果有这样的结构，它们将加速新药物的生产，并允许生产更有效和更具体的药物。对于可以大量纯化的蛋白质，结构测定的一种创新方法是尝试不仅使膜蛋白结晶，而且使膜蛋白与抗体片段形成复合物。不幸的是，很少有小组能够复制这种方法，部分原因是难以在 获得抗体片段。我们相信，噬菌体展示抗体片段和工程支架库将被证明是一个很好的来源，亲和试剂的纯化，稳定和结晶膜蛋白。我们建议的工作包括四个具体目标。首先，我们将通过亲和选择筛选我们的文库，并分离出一组抗体片段，11位合作者将在结晶试验中进行测试。其次，由于目前还不清楚抗体是否是用于膜蛋白结晶的最佳亲和试剂类型，我们将开发和测试基于β螺旋桨和纤连蛋白重复序列的替代支架。第三，我们将表征这些亲和试剂与其膜蛋白靶点的结合。最后，为了能够容易地使用这些抗体片段进行纯化和纯化， 为了提高稳定性，我们将设计它们的变体，使得它们与靶标的结合在二价阳离子的控制下。提高科学界纯化、稳定、表征和结晶膜蛋白的能力有可能在人类疾病的治疗中发挥巨大作用。例如，目前市场上超过一半的药物被认为是针对一类膜蛋白，即G蛋白偶联受体。然而，科学界一直无法确定晶体 任何人类G蛋白偶联受体的结构。

It is difficult to purify significant amounts of membrane proteins, and the majority of those which can be purified have proven refractory to crystallization. The difficulties inherent to every step in this process have led to a paucity of membrane protein crystal structures, with a concurrent dampening effect on drug discovery and design. If such structures were available, they would accelerate the production of new medications and allow the production of more potent and more specific drugs. For proteins which can be purified in significant amounts, one innovative approach to structure determination has been to attempt the crystallization of not just the membrane protein but the membrane protein in a complex with an antibody fragment. Unfortunately, few groups have been able to copy this approach, due in part to the difficulty in acquiring antibody fragments. We believe that phage-displayed libraries of antibody fragments and engineered scaffolds will prove to be an excellent source of affinity reagents for purifying, stabilizing, and crystallizing membrane proteins. Our proposed work contains four specific aims. First, we will screen our libraries by affinity selection and isolate a panel of antibody fragments which eleven collaborators will test in crystallization trials. Second, since it is not clear whether antibodies are the best type of affinity reagent for use in membrane protein crystallization, we will develop and test alternative scaffolds based on beta propellers and fibronectin repeats. Third, we will characterize the binding of these affinity reagents to their membrane protein targets. Finally, to enable the facile use of these antibody fragments for purification and stabilization, we will engineer variants of them such that their binding to their targets is under the control of divalent cations. Improving the ability of the scientific community to purify, stabilize, characterize, and crystallize membrane proteins has the potential to be of great use in the treatment of human diseases. For example, more than half of the medications currently on the market are thought to target a single class of membrane proteins, the G-protein coupled receptors. However, the scientific community has been unable to determine the crystal structure of any human G-protein coupled receptor.