SYNTHETIC BINDING PROTEINS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 尽管有高通量的方法，但许多重要的蛋白质仍然不能结晶。最近，一种基于抗体的策略已经进化出来，以抵消这一点。在这种方法中，针对目标蛋白开发的抗体抗原结合片段(Fabs)被结合，并使复合体共结晶。这一策略稳定了靶蛋白，并为晶体接触的形成提供了非靶表面积，防止了聚集，促进了晶格的形成。这种伴侣辅助的结晶学方法已被证明在膜蛋白的结晶中取得了成功，并有望在顽固蛋白的结晶中发挥巨大的作用。用于此目的的FAB的一些限制是高成本的低表达、氧化还原敏感的二硫键结构、以及由于靶结构扰动或堆积的原因而不是在所有情况下都适合的大尺寸。此外，尽管已经出现了从组合文库中成功生产抗体的合成技术，但这些文库的理论大小往往超过实际限制数个数量级，导致不完整的序列覆盖，并可能难以获得给定靶标的结合。我们的团队已经开发了一个更小的抗体模拟系统，使用纤维连接蛋白III型(FN3)支架来产生新的结合蛋白。特别是，我们的团队最近建立了新的工程技术，利用目标识别区域严格受限的氨基酸多样性来产生高亲和力结合蛋白(单体)。这种单体的尺寸较小(约为Fabs的1/4)，没有二硫键，且易于生产，这使得它成为一种有吸引力的替代Fabs的结晶伴侣。此外，小尺寸加上低化学多样性允许我们的组合文库中完全覆盖序列。我们现在已经确定了4个单体靶配合物的结构。在这里，我们建议利用APS资源来进一步探索单体辅助结晶学在蛋白质结构确定方面的潜力。我们获得的数据将用于进一步优化我们的组合库设计，并为未来结晶伴侣的生产策略提供参考。

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.