Core D3: Synthetic Antigen Binder Generation & Crystallography

合成抗原结合剂"Sab Core"的总体目标是提供广泛的强大方法和新型试剂，以支持该联盟的研究目标。基于在NIH蛋白质结构倡议（PSI）的赞助下开发的专业知识和资源，Sab Core具有独特的能力，以高通量的方式产生针对广泛范围的靶标的合成抗原结合剂，所述靶标包括可溶性蛋白质、蛋白质复合物、膜蛋白和功能性蛋白质。 核糖核酸因此，具有有效产生高质量亲和试剂的广泛能力的基础设施将大大加速MPSD联盟以及整个膜蛋白研究社区内进行的膜蛋白研究。 我们之前的努力集中在产生用作"结晶伴侣"的Sabs（1）。这一努力导致了几个高挂水果系统的结晶和结构测定。 重要的是，Sab技术具有许多额外的属性，我们的团队计划利用这些属性以独特的方式研究膜蛋白的结构-动力学-功能关系。膜蛋白是动态机器，需要改变它们的形状来执行它们的功能。因此，在功能和结构上表征主要构象状态并确定如何 它们的种群在行动过程中受到调节。 我们的sab通常是精致的构象特异性，使他们 用于研究蛋白质构象动力学的强有力的探针。它们可用于 “锁定”蛋白质在特定的构象状态，这允许明确的 功能状态注释（图D3.1）。sabs可以稳定特定的 构象状态，以促进结构确定。它们也可以 也被设计用作亲和试剂以帮助膜蛋白纯化 以稳定膜蛋白靶用于储存。最后的重要性 属性不能被夸大。膜蛋白质本身就很脆弱。以来 MPSD联盟中的拟议项目通常涉及样品的运输 在不同的地点之间，必须将它们交付和储存在 原住民国家此外，可以使用Sabs将光谱探针连接到微阵列。 目标内的特定位置，对目标进行最小的修改。 Sab Core产生三种不同类型的Sabs，以抗体的抗原结合片段（Fab）和其他设计蛋白的形式，共同满足膜蛋白研究的不同需求。这些Sabs是从高性能噬菌体展示库中产生的， 蛋白质工程的革命性概念，而Sabs是在细菌中产生的。我们的论点是，在不久的将来。sabs将取代传统的单克隆抗体技术，这是缓慢和昂贵的。 Sab Core的目标是（i）使用最先进的技术为膜蛋白靶点提供高质量的合成亲和试剂，（ii）通过基于Sab-based分子伴侣辅助结晶学加速结构测定，以及（iii）开发Sabs的新应用，使Core用户能够显着提高膜蛋白功能的机制理解水平。生成的Sabs 在这个核心中，最终生产太阳能电池的技术将提供给更广泛的科学界。

The overarching goal of the Synthetic Antigen Binder "Sab Core" is to provide a broad range of powerful approaches and novel reagents to support the consortium's research objectives. The Sab Core is uniquely capable, based on the expertise and resources that have been developed under the auspices of the NIH Protein Structure Initiative (PSI), to generate in a high-throughput way synthetic antigen binders to an extensive range of targets including soluble proteins, protein complexes, membrane proteins and functional RNA. Thus, the infrastructure in place with extensive capability to efficiently generate high-quality affinity reagents will dramatically accelerate membrane protein research performed within the MPSD Consortium, as well as in the entire membrane protein research community. Our previous efforts had focused on generating Sabs for use as "crystallization chaperones" (1). This endeavor has led to the crystallization and structure determination of several high-hanging fruit systems. Importantly, the Sab technology has a number of additional attributes that our team plans to exploit to investigate structure-dynamics-function relationships of membrane proteins in unique ways. Membrane proteins are dynamic machines that need to change their shape to perform their function. Therefore, it is critically important to functionally and structurally characterize major conformational states and determine how their populations are modulated during the course of action. Our Sabs are often exquisitely conformation-specific, making them powerful probes for studying protein conformation dynamics. They can be used to "lock" a protein in a specific conformational state, which allows for unequivocal annotation of functional states (Fig. D3.1). Sabs can stabilize a specific conformational state to facilitate structural determination. They can also be engineered for use as affinity reagents to aid membrane protein purification as well as to stabilize membrane protein targets for storage. The importance of the last attribute cannot be overstated. Membrane proteins are inherently fragile. Since the proposed projects in the MPSD Consortium often involve shipment of samples between different locations, it is essential that they be delivered and stored in their native states. Further, Sabs can be used to attach spectroscopic probes to a specific location within a target with minimal modification to the target. The Sab Core produces three distinct classes of Sabs, in the forms of the antigen-binding fragment (Fab) of antibodies and other designer proteins that collectively fulfill diverse needs in membrane protein research. These Sabs are generated from high-performance phage-display libraries that are designed based on revolutionary concepts in protein engineering, and Sabs are produced in bacteria. It is our contention that, in the near future. Sabs will replace the traditional monoclonal antibody technology that is slow and expensive. The goals of the Sab Core are (i) to provide high-quality synthetic affinity reagents for membrane protein targets using state-of-the-art technologies, (ii) to accelerate structure determination by Sab-based chaperone-assisted crystallography and (iii) to develop novel applications of Sabs that will enable Core users to significantly elevate the level of mechanistic understanding of membrane protein functions. Sabs generated in this Core and ultimately the technology to produce Sabs will be made available to the broader scientific community.