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

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。 在与Acert的合作中，我们希望研究关键的人类补体调节因子(DAF；CD55)与(I)细菌粘附素之间的相互作用，例如肠道致病性大肠杆菌的DRE或(Ii)使用鹿和DQC的人类T细胞共刺激分子CD97。对于这三个组分，我们都有高分辨率的X射线晶体结构，也有两个配体上CD55结合位点的化学位移映射数据。我们现在寻求从Deer和DQC(Acert)获得约束，使我们能够将综合体停靠在一起。我们的CD55结构已经被设计成在C末端带有一个游离的半胱氨酸，我们已经成功地利用这一点来研究另一对使用鹿的CD55配体对。然而，将我们的DER技术与ACERT提供的DQC方法相结合，使我们能够以大大改善的S/N确定更大范围距离的距离分布，从而为蛋白质对接提供卓越的数据。这三种蛋白质都在大肠杆菌中表达。CD55和CD97在表达后折叠，以便正确形成它们的许多二硫键。DRE没有折叠，但也含有一个二硫键。正是天然二硫键的存在使这一应用变得冒险，因为很难预测是否有可能产生具有功能的、正确折叠的蛋白质，并插入额外的半胱氨酸。我们的策略将是使用原子结构来识别表面暴露的残基(例如Ser)，其中Cys的突变不太可能破坏结构(一旦折叠)。一旦牛津大学的Deer和其他CW和脉冲EPR技术对蛋白质进行了表征，它们将通过Freed实验室开发的DQC方法进行进一步研究，两种自旋标记蛋白质之间的距离从短到长从10？到90？在所有的实验中，我们都计划使用氢化自旋标记以及全氢化缓冲溶液。

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.