NEW APPROACHES TO STUDY TERNARY COMPLEXES OF OLIGOMERIC PROTEINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们概述了使用PDS约束通过三角法和刚体精化来阐明蛋白质和蛋白质复合体的三级和三级结构细节的方法[1]。有人强调，寡聚蛋白质的情况是一个困难的情况。讨论了两种方法：(1)使用明智的标记，使感兴趣的距离超出未解析的“不想要的”距离池的范围；(2)使用异二聚体或类似物。 这两种方法都被应用于解决maritima的CHEA/CHEW/MCP超分子复合体的结构。CHEA是一种二聚体，它结合了两个咀嚼和数量未知的二聚体MCP受体。原则上，整个结构可以由几个CHEA/CHEW复合体和一些受体组成。解决这一问题的方法首先包括使用未标记的组分来确定标记组分的构象变化，然后在每个组分上使用几个标记位点，最后使用异二聚体。后者减少了增加距离谱(即谱编辑)的不同对的数量，而标签组件的某些组合将产生不同的峰。此外，扫描自旋标记的位置将导致距离谱的逐渐变化。将整个P(R)‘S模型拟合到一个参数化的模型中，将收敛到定义该络合物的一组唯一的参数。络合物的化学计量可以通过结合使用标记和非标记组分的自旋计数来确定。然而，由于络合物的大小，似乎有必要进行蛋白质氢化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We outlined approaches to use PDS constraints to elucidate details of tertiary and ternary structure of proteins and protein complexes [1] by triangulation method and rigid-body refinement. It was stressed that the case of oligomeric proteins is a difficult one. Two approaches were discussed: (1) use judicious labeling to make distance of interest outside the range of unresolved pool of "unwanted" distances; (2) use heterodimers or like. Both approaches were applied to solve the structure of supramolecular complex of CheA/CheW/MCP from T. maritima. CheA is a dimer and it binds two CheW's and unknown number of dimeric MCP receptors. The whole construct could, in principle, consist of several CheA/CheW complexes and a number of receptors. The approach to the problem first includes using unlabeled components to determine conformational changes on labeled components, using several labeling sites on each component, and last using heterodimers. The latter reduces number of different pairs that add to distance spectrum (i.e. spectrum editing), whereas certain combinations of labeling components would yield distinct peaks. Furthermore, scanning the position of a spin label would lead to a gradual change of the distance spectrum. Fitting the whole set of P(r)'s to a parametrized model would converge to unique set of parameters defining the complex. The stoichiometry of complex could be determined by spin-counting using a combination of labeled and unlabeled components. However, protein deuteration seems to be necessary due to the size of complex.