Single-domain protein superfamilies mediate essential signaling functions in both prokaryotes and eukaryotes. Signal transduction mediated by single-residue phosphorylation involves subtle secondary structure, side chain rotamer, and hydrogen-bond fluctuations for transitions between active and inactive conformational states, rather than large fold variations. Its study requires molecular dynamics simulations for adequate sampling of the conformational landscape. Discrimination between functional states and subfamilies is achieved by subsequent network analysis designed to couple variations at the local residue or fragment levels to functional collective motions. We describe an integrated protocol for quantitative measurement of local fluctuations and their coupling to functional motions with phosphorylation of the CheY bacterial chemotaxis signal protein as a case study.