Antibody Fragments, Chaperones, and Membrane Protein Crystals

很难提纯大量的膜蛋白，而且大多数可以提纯的膜蛋白被证明是难于结晶的。这一过程中每一步固有的困难导致了膜蛋白晶体结构的缺乏，同时也抑制了药物发现和设计的效果。如果有这样的结构，它们将加速新药的生产，并允许生产更有效、更具体的药物。对于可以大量纯化的蛋白质，一种确定结构的创新方法是尝试不仅使膜蛋白结晶，而且尝试与抗体片段形成复合体中的膜蛋白结晶。不幸的是，很少有组织能够复制这种方法，部分原因是 获取抗体片段。我们相信，噬菌体展示的抗体片段文库和工程支架将被证明是纯化、稳定和结晶膜蛋白的极好的亲和剂来源。我们提出的工作包含四个具体目标。首先，我们将通过亲和力选择来筛选我们的文库，并分离一组抗体片段，11名合作者将在结晶试验中测试这些抗体片段。其次，由于目前尚不清楚抗体是否是用于膜蛋白结晶的最佳亲和剂类型，我们将开发和测试基于β推进器和纤维连接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标的结合。最后，为了能够方便地使用这些抗体片段进行纯化和 为了稳定，我们将设计它们的变体，使它们与目标的结合在二价阳离子的控制下。提高科学界提纯、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单一类别的膜蛋白，即G蛋白偶联受体。然而，科学界一直无法确定这颗水晶 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.