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. Histidine kinases relay bacteria and plant receptor responses to the stimuli from their environments. CheA kinase is a dimer segregated into several subunits specialized for different tasks. The regulatory domain P5 binds the receptor-coupling protein CheW, whereas the P1 domain provides hystidine for autophosphorilation. A partial crystal structure of CheA (290-671) from Thermotoga maritima is known. This includes P3-P5 domains only. The exact locations and structures of the other two mobile domains, P1 and P2, are uncertain. The change in conformations of the subunits P4, P5 and their relative positions modulate kinase s activity in response to receptor activation. Our study is aimed at determining the locations of the P1 and P2 domains, structural changes upon ATP binding, and the structure of CheA/CheW complex. The method is based on measurement of the distance between nitroxide spin labels attached to cysteine residues, introduced at the sites of interest by site-directed mutagenesis. Due to the dimeric structure of CheA, there are two to four spin labels per labeled protein, with some of distances unwanted; even though they may provide some useful input. Our strategy was to introduce one or two pairs of spin labels and observe the difference in dipolar signals for two cases. Due to the large size of the protein it was possible in some cases to place nitroxides such that some of distances were very large, i.e. non measurable on the available time-scale. This simplified data interpretation. We have studied the following systems: CheA, S318C (26.3/28.7);CheA-Delta289, Q545C(~42Angstrom); Complex CheA- Delta289 Q545C/CheW, 15C(~55Angstom); CheA E387C/E12C(~53); CheA-ATP E387C/E12C,(~53); CheA-Delta289 E387C,(~53). We determined the distance to site 15C on CheW; the distances to CheW 80C, and CheW 72C are the subject of ongoing work. The location of the P1 subunit is a subject of future work.
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