PULSE DIPOLAR ESR OF PROTEIN-PROTEIN INTERACTIONS IN HUMAN COMPLEMENT SYSTEM

人体补体系统中蛋白质-蛋白质相互作用的脉冲偶极 ESR

• 批准号: 8364094
• 负责人: Gregory Brion Timmel
• 金额: $ 0.08万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364094
• 关键词: Bacterial Adhesins; Binding Sites; Buffers; CD97 gene; Chemicals; Collaborations; Complement; Complex; Data; Docking; Engineering; Escherichia coli; Funding; Grant; Human; Laboratories; Ligands; Maps; Methodology; Mutation; National Center for Research Resources; Physiologic pulse; Principal Investigator; Proteins; Research; Research Infrastructure; Resolution; Resources; Roentgen Rays; Solutions; Source; Spin Labels; Structure; Surface; T-Lymphocyte; Techniques; Technology; United States National Institutes of Health; complement system; cost; disulfide bond; improved; protein folding; protein protein interaction; research study

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. In collaboration with ACERT we wish to study the interactions between a key human complement regulator (DAF; CD55) and (i) bacterial adhesins e.g. the DraE of enteropathogenic Eschericia coli or (ii) the human T-cell co-stimulatory molecule, CD97 using DEER and DQC. For all three components we have high resolution X-ray crystallographic structures and also have chemical shift mapping data for the CD55 binding site on both ligands. We now seek to derive constraints from DEER and DQC (ACERT) that will allow us to dock the complexes together. Our CD55 construct is already engineered with a free Cys at the C-terminus and we have successfully used this to investigate another CD55-ligand pair using DEER. However, the combined application of our DEER technology with the DQC methodologies available at ACERT allows us to determine distance distributions for a larger range of distances at a much improved S/N, hence, providing superior data for protein docking. All three proteins are expressed in E. coli. The CD55 and CD97 are refolded post-expression to allow correct formation of their many disulphide bonds. The DraE is not refolded but also contains a single disulphide bond. It is the presence of natural disulphide bonds that makes this application adventurous as it is difficult to predict whether it will be possible to generate functional, correctly folded proteins, with additional Cys inserted. Our strategy will be to use the atomic structures to identify surface exposed residues (e.g. Ser) where mutation to Cys is unlikely to disrupt the structure (once folded). Once proteins are characterized by DEER and other cw and pulsed EPR technique here in Oxford they will be investigated further by the DQC methodology developed in the Freed laboratory yielding distances between the two spin-labeled proteins from as short as 10 ¿ and to as long as 90 ¿. In all experiments, we plan the use of deuterated spin labels as well as fully deuterated buffer solutions.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们希望与ACERT合作研究人类关键的人类补体调节剂（DAF； CD55）和（i）细菌粘附物之间的相互作用，例如使用鹿和DQC的人类T细胞共刺激分子CD97的肠病大肠杆菌或（ii）的DRAE。对于所有三个组件，我们都有高分辨率的X射线晶体学结构，并且还具有两个配体上CD55结合位点的化学移位图。现在，我们寻求从鹿和DQC（ACERT）中获得约束，这将使我们能够将复合物隔离在一起。我们的CD55构建体已经在C端的免费CYS进行了设计，我们已经成功地使用了此构建体来调查使用Deer的另一个CD55-crigand对。但是，我们的鹿技术与ACERT可用的DQC方法的合并应用使我们能够确定更大范围的S/N距离范围的距离分布，因此为蛋白质对接提供了出色的数据。所有三种蛋白质均以大肠杆菌表示。 CD55和CD97在表达后进行了重新折叠，以正确形成其许多二硫键。 DRAE不会重新折叠，但也包含单个二硫键。正是天然二硫键的存在使这种应用具有冒险精神，因为很难预测是否可能会产生功能，正确折叠的蛋白质，并插入其他CYS。我们的策略是使用原子结构来识别表面暴露的残差（例如SER），其中突变到CYS不太可能破坏结构（一旦折叠）。一旦蛋白质以鹿和其他CW和脉冲EPR技术为特征，在牛津的蛋白质中，将通过在自由实验室中开发的DQC方法进一步研究它们，从而在短达10€且长达90�之间产生了两个自旋标记的蛋白质之间的距离。在所有实验中，我们都计划使用氘代自旋标签以及完全氘化的缓冲溶液。