NEW APPROACHES TO STUDY TERNARY COMPLEXES OF OLIGOMERIC PROTEINS

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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 CheWs 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.