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.

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。子项目的主要支持 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。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 与ACERT合作，我们希望使用DEER和DQC研究关键的人补体调节因子（CD 55）与（i）细菌粘附素（例如肠道致病性大肠杆菌的DraE）或（ii）人T细胞共刺激分子（CD 97）之间的相互作用。对于所有三种成分，我们有高分辨率的X射线晶体结构，也有两个配体上的CD 55结合位点的化学位移映射数据。我们现在试图从DEER和DQC（ACERT）中获得约束，这将使我们能够将复合体对接在一起。我们的CD 55构建体已经在C-末端用游离Cys进行了工程化，并且我们已经成功地使用该构建体使用DEER研究了另一种CD 55-配体对。然而，我们的DEER技术与ACERT提供的DQC方法相结合的应用，使我们能够确定更大范围的距离在一个大大改善的S/N的距离分布，因此，提供上级数据的蛋白质对接。这三种蛋白质均在大肠杆菌中表达。杆菌CD 55和CD 97在表达后重折叠，以允许正确形成它们的许多二硫键。DraE不重折叠，但也含有单个二硫键。天然二硫键的存在使得这种应用具有冒险性，因为很难预测是否有可能产生功能性的、正确折叠的蛋白质，并插入额外的Cys。我们的策略将是使用原子结构来鉴定表面暴露的残基（例如Ser），其中突变为Cys不太可能破坏结构（一旦折叠）。 一旦蛋白质在牛津通过DEER和其他cw和脉冲EPR技术表征，它们将通过Freed实验室开发的DQC方法进一步研究，产生两种自旋标记蛋白质之间的距离，从短至10 º到长至90 º。在所有实验中，我们计划使用氘化自旋标签以及全氘化缓冲溶液。