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Neuromedin C (NMC) is a peptide that regulates various processes in the central nervous system and gastrointestinal tract through its interaction with the bombesin receptor subtype-2 (BB2R). Hence, BB2R antagonists hold the potential to treat disorders that occur as a result of NMC dysfunction or misregulation. However, their efficient design requires a detailed understanding of the structural features of NMC, which hitherto are unknown. Herein, we describe the conformational ensembles of NMC in an aqueous solution, at five different TFE concentrations to decode its folding pathway and under its SDS micelle bound state. NMC displays a disordered but well defined backbone architecture that undergoes a progressive coil-helix transition with increasing TFE concentration, first at the C-terminus and then at the N-terminus. NMC also adopts a C-terminal alpha-helical conformation upon binding to SDS micelles. This micelle binding is directed by hydrophobic interactions that concur with the unfavourable deprotonation of His(8) and its further insertion into the micelle. Moreover, NMR relaxation data reveal that the acquisition of the micelle bound alpha-helical conformation constrains the NMC flexibility more than the confinement itself. This comprehensive study of the structural behaviour of NMC provides essential mechanistic information that could be useful for the development of new therapeutics to treat neurological, cancer-related or eating disorders.

14 páginas, 8 figuras, 1 tabla, PipX, an 89-residue protein, acts as a co-activator of the global nitrogen regulator NtcA in cyanobacteria. NtcA-PipX interactions are regulated by 2-oxoglutarate (2-OG), an inverse indicator of the ammonia abundance, and by PII, a protein that binds to PipX at low 2-OG concentrations. The structure of PipX, when bound to NtcA or PII, consists of an N-terminal, five-stranded -sheet (conforming a Tudor-like domain), and two long -helices. These helices adopt either a flexed conformation, where they are in close contact and in an antiparallel mutual orientation, also packing against the -sheet; or an open conformation (observed only in the PII-PipX complex) where the last -helix moves apart from the rest of the protein. The aim of this work was to study the structure and dynamics of isolated PipX in solution by NMR. The backbone chemical shifts, the hydrogen-exchange and the NOE patterns indicated that the isolated, monomeric PipX structure was formed by an N-terminal five-stranded -sheet and two C-terminal -helices. Furthermore, the observed NOEs between the two helices, and of -helix2 with -strand2 suggested that PipX adopted a flexed conformation. The -strands 1 and 5 were highly flexible, as shown by the lack of inter-strand backbone-backbone NOEs; in addition, the 15N-dynamics indicated that the C terminus of -strand4 and the following -turn (Phe42-Thr47), and the C-cap of -helix1 (Arg70-Asn71) were particularly mobile. These two regions could act as hinges, allowing PipX to interact with its partners, including PlmA in the newly recognized PII-PipX-PlmA ternary complex., This work was supported by the Spanish Ministerio de Economía y Competitividad (CTQ2013-4493-R to JLN; BFU2014-58229-P to VR) and by the Generalitat Valenciana (PrometeoII/2014/029 to VR).

Protein dynamics can be studied by NMR measurements of aqueous dilute liquid crystalline samples. However, the measured residual dipolar couplings are sensitive not only to internal fluctuations but to all changes in internuclear vectors relative to the laboratory frame. We show that side-chain fluctuations and bond librations in the ps-ns time scale perturb the molecular shape and charge distribution of a small globular protein sufficiently to cause a noticeable variation in the molecular alignment. The alignment variation disperses the bond vectors of a conformational ensemble even further from the dispersion already caused by internal fluctuations of a protein. Consequently RDC-probed order parameters are lower than those obtained by laboratory frame relaxation measurements.