NMR observation of selected segments in a larger protein: Central-segment isotope labeling through intein-mediated ligation

Peptide segments in a protein, which can include an active site of interest or be a series of parts constituting the entire structure, are now selecti vely observed by nuclear magnetic resonance (NMR) spectroscopy using sample s prepared by the intein-mediated ligation method. Two separate inteins wer e used to ligate NMR-transparent segments to both the ends of an NMR-visibl e segment, producing a partly visible intact protein molecule. The N-15-H-1 correlation spectrum of a 370-residue maltose binding protein labeled with N-15 at a continuous segment comprising residues Gly(101)-Ser(238) showed the essential elimination of signal overlapping, the signals being at the s ame positions as for the uniformly labeled sample. This method will allow s tructural analysis by NMR of over 50-kDa proteins in combination with conte mporary NMR techniques suppressing the signal decays of larger proteins.