Motile bacteria have a chemotaxis system that enables them to sense their environment and direct their swimming toward favorable conditions. Chemotaxis involves a signaling process in which ligand binding to the extracellular domain of the chemoreceptor alters the activity of the histidine kinase, CheA, bound ~300 Å away to the distal cytoplasmic tip of the receptor, to initiate a phosphorylation cascade that controls flagellar rotation. The cytoplasmic domain of the receptor is thought to propagate this signal via changes in dynamics and/or stability, but it is unclear how these changes modulate the kinase activity of CheA. The major objective of this study is to address this question by employing hydrogen deuterium exchange mass spectrometry to probe the structure and dynamics of CheA within functional signaling complexes of the Escherichia coli aspartate receptor cytoplasmic fragment, CheA, and CheW. Our results reveal that stabilization of the P4 catalytic domain of CheA correlates with kinase activation. Furthermore, differences in activation of the kinase that occur during sensory adaptation depend on receptor destabilization of the P3 dimerization domain of CheA. Our HDX-MS results of CheA in complexes led us to initiate two new directions to provide additional insights into kinase regulation of CheA. Since P1-P4 interactions at a non-productive and/or productive site may be influenced by P1 dimerization, we have designed single molecule FRET (smFRET) experiments based on our HDX-MS results to inveistigate these P1 interactions. These complementary experiments have the potential to provide details into P1 conformation and positioning that remain elusive to date. Lastly, we explored whether the mechanism of CheA P4 stabilization that was identified in this study is conserved between bacterial species. HDX-MS comparison between thermophilic T. maritima and E.coli CheA in solution reveals that stabilization may not be a requirement for T.maritima CheA activation. Therefore, it is unlikely that these CheAs work similarly as generalized within the chemotaxis field. In all, these studies have yielded novel approaches for investigating protein complexes, insights into long-range membrane signal transduction, and evidence for divergent mechanisms of related enzymes.