SYNTHETIC BINDING PROTEINS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 尽管有高通量的方法，许多重要的蛋白质仍然不适合结晶。最近，一种基于抗体的策略已经发展到抵消这一点。在该方法中，针对感兴趣的蛋白质开发的抗体抗原结合片段（Fab）被结合并且复合物共结晶。该策略稳定靶蛋白并提供非靶表面积以形成晶体接触，防止聚集并促进晶格形成。这种分子伴侣辅助的结晶学方法已被证明在膜蛋白的结晶中是成功的，并且通常对柠檬酸蛋白有很大的实用性。为此目的使用Fab的一些限制是高成本的低表达、氧化还原敏感的二硫键连接结构和大尺寸，由于靶结构扰动或包装的原因，大尺寸可能不适合所有情况。此外，尽管已经出现了用于从组合文库成功生产抗体的合成技术，但是这些文库的理论大小通常超过实际限制许多数量级，导致不完整的序列覆盖和获得给定靶标的结合物的潜在困难。我们的小组已经开发了一种更小的抗体模拟系统，使用纤连蛋白III型（FN 3）支架产生新的结合蛋白。特别是，我们的小组最近建立了新的工程技术，以产生高亲和力的结合蛋白（“单体”），采用严格限制的氨基酸多样性的目标识别区。小尺寸（约Fab的1/4）、不存在二硫键和易于生产使得单体成为Fab作为结晶分子伴侣的有吸引力的替代物。此外，小尺寸加上低化学多样性允许在我们的组合文库中完全覆盖序列。我们现在已经确定了4个单抗体-靶复合物的结构。在这里，我们建议使用APS资源，以进一步探索单抗体辅助晶体学的蛋白质结构测定的潜力。我们获得的数据将用于进一步优化我们的组合库设计，并为未来的结晶伴侣生产策略提供信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Despite high-throughput methods, many important proteins remain unamenable to crystallization. Recently, an antibody-based strategy has evolved to counteract this. In this method, antibody antigen-binding fragments (Fabs) developed against the protein of interest are bound and the complex co-crystallized. This strategy stabilizes the target protein and provides non-target surface area for formation of crystal contacts, preventing aggregation and promoting lattice formation. Such chaperone assisted crystallography methods have proven successful in the crystallization of membrane proteins, and promise great utility for recalcitrant proteins generally. Some limitations of Fab usage for this purpose are low expression at high cost, redox sensitive disulfide linked structure, and large size which for reasons of target structure perturbation or packing may not be suitable in all instances. Furthermore, although synthetic technologies have emerged for the successful production of antibodies from combinatorial libraries, the theoretical size of these libraries often exceeds practical limits by many orders of magnitude resulting in incomplete sequence coverage and potential difficulty in obtaining binders for a given target. Our group has developed a much smaller antibody-mimic system using the Fibronectin Type III (FN3) scaffold for the generation of novel binding proteins. In particular, our group has recently established novel engineering technology to generate high-affinity binding proteins ("monobodies") employing severely restricted amino acid diversity in the target recognition region. The small size (~1/4 that of Fabs), the absence of disulfide bonds, and ease of production make the monobodies an attractive alternative to Fabs as crystallzation chaperones. Furthermore, small size coupled with low chemical diversity allows for complete sequence coverage in our combinatorial libraries. We have now determined the structures of 4 monobody-target complexes. Here we propose the use of APS resources to further explore the potential of monobody-assisted crystallography for protein structure determination. The data we obtain will be used to further optimize our combinatorial library design and inform future strategies for crystallization chaperone production.