Fast automated NMR spectroscopy of short-lived biological samples.

Faster than death: NMR techniques that make use of nonlinear sampling and hyperdimensional processing enable the recording of complete NMR data sets for the automated assignment of the backbone and side-chain resonances of short-lived protein samples of cell lysates.

Structural insights into Rcs phosphotransfer: the newly identified RcsD-ABL domain enhances interaction with the response regulator RcsB.

The Rcs-signaling system is one of the most remarkable phosphorelay pathways in Enterobacteriaceae, comprising several membrane-bound and soluble proteins. Within the complex phosphotransfer pathway, the histidine phosphotransferase (HPt) domain of the RcsD membrane-bound component serves as a crucial factor in modulating the phosphorylation state of the transcription factor RcsB. We have identified a new domain, RcsD-ABL, located N terminally to RcsD-HPt that interacts with RcsB as well. We have determined its structure, characterized its interaction interface with RcsB, and built a structural model of the complex of the RcsD-ABL domain with RcsB. Our results indicate that the effector domain of RcsB, which normally binds to DNA, is recognized by RcsD-ABL, whereas the HPt domain interacts with the phosphoreceiver domain of RcsB.

Modulation of the Rcs-mediated signal transfer by conformational flexibility.

The Rcs (regulator of capsule synthesis) signalling complex comprises the membrane-associated hybrid sensor kinases RcsC and RcsD, the transcriptional regulator RcsB and the two co-inducers RcsA and RcsF. Acting as a global regulatory network, the Rcs phosphorelay controls multiple cellular pathways including capsule synthesis, cell division, motility, biofilm formation and virulence mechanisms. Signal-dependent communication of the individual Rcs domains showing histidine kinase, phosphoreceiver, phosphoryl transfer and DNA-binding activities is characteristic and essential for the modulation of signal transfer. We have analysed the structures of core elements of the Rcs network including the RcsC-PR (phosphoreceiver domain of RcsC) and the RcsD-HPt (histidine phosphotransfer domain of RcsD), and we have started to characterize the dynamics and recognition mechanisms of the proteins. RcsC-PR represents a typical CheY-like alpha/beta/alpha sandwich fold and it shows a large conformational flexibility near the active-site residue Asp(875). NMR analysis revealed that RcsC-PR is able to adopt preferred conformations upon Mg(2+) co-ordination, BeF(3)(-) activation, phosphate binding and RcsD-HPt recognition. In contrast, the alpha-helical structure of RcsD-HPt is conformationally stable and contains a recognition area in close vicinity to the active-site His(842) residue. Our studies indicate the importance of protein dynamics and conformational exchange for the differential response to the variety of signals perceived by complex regulatory networks.