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 selectively observed by nuclear magnetic resonance (NMR) spectroscopy using samples prepared by the intein-mediated ligation method. Two separate inteins were used to ligate NMR-transparent segments to both the ends of an NMR-visible segment, producing a partly visible intact protein molecule. The (15)N-(1)H correlation spectrum of a 370-residue maltose binding protein labeled with (15)N at a continuous segment comprising residues Gly(101)-Ser(238) showed the essential elimination of signal overlapping, the signals being at the same positions as for the uniformly labeled sample. This method will allow structural analysis by NMR of over 50-kDa proteins in combination with contemporary NMR techniques suppressing the signal decays of larger proteins.
Otomo
T
T
Institute for Protein Research, Osaka University, Suita, Japan.
Ito
N
N
Kyogoku
Y
Y
Yamazaki
T
T
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D016254:Mutagenesis, Insertional; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing / Q000235:genetics; D011522:Protons; D020027:Pyrococcus furiosus
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011498:Protein Precursors; D011522:Protons; C461599:maltose transport system, E coli