| Description |
Nuclear magnetic resonance (NMR) spectroscopy was employed to characterize structural and dynamic properties of bacteriophage ƛN protein (ƛN). ƛN is an intrinsically disordered protein (IDP) that interacts with multiple partners to prevent termination in the phage ƛ-Escherichia coli transcription apparatus. Limited dispersion in the 1H dimension of the 1H-15N heteronuclear correlation spectra confirmed the extensively disordered nature of ƛN. Resonance assignments were made for the amide-15N, amide-1H, 13Cα and 13Cβ nuclei of more than 90% of the nonproline residues at pH 7 and 5.5, which were subsequently used to calculate secondary structure propensities. Residues 2-7 and 55-75 showed propensities to form α-helical structures, whereas the residues 34-47 and 95-107 showed propensities to form extended structures. Previous studies have shown that residues 1-22 of ƛN adopt a helical structure when bound to a site in the RNA transcript (boxB) and residues 34-47 form an extended structure to interact with E. coli host transcription factor NusA protein. We have discovered that the residues 55-75 of ƛN protein, hitherto uncharacterized, have propensities to form transient helical secondary structures. This putative transient helical region spanning residues 55-75 is amphipathic and may form coiled-coil structures, which further suggests a possible structural or functional role of this segment in the antitermination apparatus. To characterize the backbone dynamics of ƛN, 15N longitudinal relaxation rates (R1), transverse relaxation rates (R2), and steady-state 15N-1H nuclear Overhauser effects were measured. Significantly elevated transverse relaxation rates (R2) for the amide groups of residues 55-75 indicated slow conformational exchange in the ƛs-ms timescale, consistent with a transient secondary structure in this segment of ƛN. Faster amide-bond motions were analyzed by mapping reduced spectral density functions, derived from the 15N relaxation parameters, which further revealed backbone motions on two or more timescales, as expected for a nonglobular disordered protein. The results of this NMR study suggest the presence of previously unknown functional domains of ƛN protein, which may enhance our understanding of the phage ƛ-Escherichia coli antitermination apparatus and allow further investigations of the binding mechanisms of IDPs with their interacting partners. |
