Measuring distances by pulsed dipolar ESR spectroscopy: spin-labeled histidine kinases

Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has become a useful method that is aimed at resolving protein structure and functional mechanisms. The method of pulsed dipolar ESR spectroscopy (PDS) is outlined in the first half of the chapter, and it illustrates the simplicity and potential of this developing technology with applications to various biological systems. A more detailed description is presented of the implementation of PDS to reconstruct the ternary structure of a large dimeric protein complex from Thermotoga maritima, formed by the histidine kinase CheA and the coupling protein CheW. This protein complex is a building block of an extensive array composed of coupled supramolecular structures assembled from CheA/CheW proteins and transmembrane signaling chemoreceptors, which make up a sensor that is key to controlling the motility in bacterial chemotaxis. The reconstruction of the CheA/CheW complex has employed several techniques, including X-ray crystallography and pulsed ESR. Emphasis is on the role of PDS, which is part of a larger effort to reconstruct the entire signaling complex, including chemoreceptor, by means of PDS structural mapping. In order to precisely establish the mode of coupling of CheW to CheA and to globally map the complex, approximately 70 distances have already been determined and processed into molecular coordinates by readily available methods of distance geometry constraints.