Chaperone-Assisted RNA Crystallography-Equipment Supplement

分子伴侣辅助 RNA 晶体学设备补充品

• 批准号: 9895189
• 负责人: Joseph Anthony Piccirilli
• 金额: $ 8.6万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895189
• 关键词: Acute; Affinity; Amino Acids; Antibody Formation; Architecture; Award; Biological Process; Cellular biology; Chemistry; Complex; Crystallization; Crystallography; Development; Dimensions; Disease; Equipment; Gene Expression; Goals; Health; Human; Immunoglobulin Fragments; Knowledge; Libraries; Macromolecular Complexes; Mediating; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Evolution; Parents; Phage Display; Play; Population; Process; Property; Proteins; RNA; RNA Binding; RNA Folding; Reagent; Research; Resolution; Ribonucleoproteins; Role; Specificity; Structural Biologist; Structure; Surface; Techniques; X-Ray Crystallography; biological systems; improved; inorganic phosphate; macromolecule; portability; protein complex; synthetic antibodies; three dimensional structure; transcriptome

PROJECT SUMMARY FROM PARENT AWARD Biological systems possess a highly complex and dynamic cellular RNA population, collectively known as the transcriptome. Many RNAs fold into complex three-dimensional structures, both intrinsically and as ribonucleoprotein (RNP) complexes, and play fundamental roles in nearly every aspect of gene expression. Understanding cell biology, health, and disease requires knowledge of how RNA structure mediates biological function. X-ray crystallography provides a powerful method for structure determination, but RNA crystallization represents a major bottleneck in the process, reflecting in part the limited surface chemistry for mediating lattice interactions and repulsion among the phosphates. Considering the rapid pace of new RNA discovery, there remains an acute need to develop methods to facilitate RNA structure acquisition. For difficult protein targets, antibody fragments (Fab or scFv) have served as effective chaperones for crystallization, and we hypothesized that the large size, conformational properties and surface chemistry of Fabs will facilitate RNA crystallization as well. Using phage-display library selections we demonstrated that Fabs can bind RNA with high affinity and specificity, mediate the majority of lattice interactions in Fab-RNA co-crystals, and provide a molecular replacement model for solving the structures. The long-term goal of this project is to facilitate resolution of the RNA crystallization bottleneck through development of a high-throughput pipeline for antibody production against RNA. The objective of this application is to enable facile access to RNA- binding Fabs and pursue them as reagents for RNA and RNP crystallization and structure determination. To attain this objective we will (a) improve Fab libraries using phage display and molecular evolution approaches to identify amino acid types that tailor complementary determining regions (CDRs) for RNA binding, (b) develop general use crystallization modules with surface and conformational properties adjusted to facilitate crystallization, and (c) use these techniques to create and use Fab complexes of RNA and RNP targets for crystallization and structure determination. Completion of the research will allow facile access to RNA binding Fabs, provide structural biologists with a suite of portable modules for generalized use in RNA/RNP crystallization, and provide important new structural knowledge for understanding biological function.

父母奖的项目摘要 生物系统具有高度复杂和动态的细胞RNA群体，统称为 转录组。许多RNA折叠成复杂的三维结构，包括本质和AS 核糖核蛋白（RNP）复合物，几乎在基因表达的各个方面起着基本作用。 了解细胞生物学，健康和疾病需要了解RNA结构如何介导生物学 功能。 X射线晶体学为结构确定提供了强大的方法，但是RNA 结晶代表了此过程中的主要瓶颈，部分反映了有限的表面化学。 介导磷酸盐之间的晶格相互作用和排斥。考虑新RNA的快速速度 发现，仍需要急需开发促进RNA结构采集的方法。很难 蛋白质靶标，抗体片段（FAB或SCFV）已充当有效的结晶伴侣，并且 我们假设晶圆厂的大尺寸，构象性能和表面化学将有助于 RNA也结晶。使用噬菌体播放库的选择，我们证明了Fab可以结合RNA 具有高亲和力和特异性，介导Fab-RNA共晶中的大多数晶格相互作用，并介导 提供一个分子替代模型来解决结构。该项目的长期目标是 通过开发高通量管道来促进RNA结晶瓶颈的分辨率 用于针对RNA的抗体产生。该应用的目的是使可轻松访问RNA- 结合Fabs并将其作为RNA和RNP结晶和结构测定的试剂。到 实现这一目标，我们将（a）使用噬菌体显示和分子进化方法改善晶圆厂库 识别氨基酸类型，以量身定制互补确定区域（CDRS）以进行RNA结合，（b） 开发具有表面和构象特性的一般使用结晶模块以促进 结晶，（c）使用这些技术来创建和使用RNA和RNP目标的Fab复合物 结晶和结构确定。研究的完成将允许轻松访问RNA结合 Fabs，为结构生物学家提供一套便携式模块，用于RNA/RNP的广义使用 结晶，并提供重要的新结构知识来理解生物学功能。