The 179 amino acid long self-processing module (SPM) is present in a disordered form in the bacterial cell and folds only in calcium rich environment. The structural change induces autocatalytic cleavage of the Asp-Pro peptide bond. An almost complete NMR resonance assignment of the intrinsically disordered and folded SPM was obtained. Non-uniformly sampled multi-dimensional spectra were recorded in order to resolve poorly dispersed resonance frequencies of the disordered protein. The assignment was used to evaluate secondary structure propensities of the disordered form of SPM. The results show that disordered SPM exhibits a tendency to form helical structures in two regions.
The standard set of NMR experiments was used to obtain the resonance assignment of the ordered form of SPM except a region between alanine A15 and glycine G36, that exhibits different motional behavior causing a signal disappearing. The 1H - 1H distance restraints were obtained from NOE cross peaks observed and unambiguously assigned in 15N-edited and 13C-edited NOESY spectra by the program ARIA 2.1. The structure was calculated by running restrained molecular dynamics in the program CNS 1.2 using the standard protocol.
The amount of bound calcium ions to SPM was determined by titration with EDTA as a chelating agent. Concentration of free and calcium-bound EDTA was monitored by 1D proton NMR spectroscopy. The possible calcium-binding sites were predicted from SPM sequence.