Core D3: Synthetic Antigen Binder Generation & Crystallography

合成抗原粘合剂“SAB核心”的总体目标是提供广泛的强大方法和新试剂，以支持该联盟的研究目标。基于在NIH蛋白质结构倡议(PSI)的支持下开发的专业知识和资源，SAB核心具有独特的能力，能够以高通量的方式产生针对广泛目标的合成抗原结合物，包括可溶性蛋白质、蛋白质复合体、膜蛋白质和功能性蛋白质 核糖核酸。因此，具备高效生产高质量亲和试剂的广泛能力的基础设施将极大地加速在MPSD联盟内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制造可用作“结晶伴侣”的Sabb(1)。这一努力导致了几个高悬的水果系统的结晶和结构确定。 重要的是，SAB技术具有许多其他属性，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。膜蛋白是动态的机器，需要改变它们的形状来执行它们的功能。因此，从功能和结构上表征主要构象状态并确定它们是如何形成的是至关重要的 他们的人口在行动过程中受到调节。 我们的军刀通常是精致的构象特定的，使它们 研究蛋白质构象动力学的有力探针。他们可以用来 将蛋白质锁定在特定的构象状态，这允许明确的 功能状态的注释(图2)D3.1)。Sabs可以稳定特定的 构象状态，以便于结构确定。它们也可以是 设计用于亲和试剂，以帮助膜蛋白纯化 作为稳定膜蛋白储存的靶标。最后一项的重要性 属性不能被夸大。膜蛋白天生就是脆弱的。自.以来 MPSD财团中的拟议项目经常涉及样品运输 在不同的位置之间，它们必须被交付和存储在他们的 原住民的州。此外，Sabs可用于将光谱探测器连接到 目标内的特定位置，只需对目标进行最小修改。 SAB核心以抗体和其他设计蛋白的抗原结合片段(FAB)的形式产生三种不同类别的Sabs，共同满足膜蛋白研究的不同需求。这些Sabb是从高性能噬菌体展示库生成的，这些库是基于 在蛋白质工程的革命性概念上，Sabs是在细菌中生产的。我们的观点是，在不久的将来。Sabs将取代速度慢、成本高的传统单抗技术。 SAB Core的目标是(I)使用最先进的技术为膜蛋白靶标提供高质量的合成亲和试剂，(Ii)通过基于Sab的伴侣辅助结晶学加速结构确定，以及(Iii)开发Sabs的新应用，使Core用户能够显著提高对膜蛋白质功能的机械理解水平。生成的SAB 在这个核心中，最终生产Sabs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.