Investigation of the paramagnetic domain of putidaredoxin by nitrogen-15 NMR spectroscopy

✍ Scribed by Bruce Coxon; Nese Sari; Marcia J. Holden; Vincent L. Vilker

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 442 KB
• Volume: 35
• Category: Article
• ISSN: 0749-1581
• DOI: 10.1002/(sici)1097-458x(199711)35:11<743::aid-omr166>3.0.co;2-w

✦ Synopsis

The oxidized forms of the redox proteins putidaredoxin and putidaredoxin-15N were prepared by insertion of suitable plasmids into E. coli and the structures of the proteins were studied by homo-and heteronuclear 1H and 15N NMR methods, including identiÐcation of 15N resonance types by one-and two-dimensional DEPT spectrum editing and measurements of 15N chemical shifts, coupling constants, nuclear Overhauser e †ects and spin-lattice relaxation times. Direct detection of 15N NMR spectra revealed a number of 15N resonances that have not been found in previous 1H detected NMR studies. These resonances include a set of 16 paramagnetically broadened signals from 15N nuclei that are structurally close to the iron-sulfur cluster of the protein, proline backbone 15N resonances and 15N signals from the side-chains of glutamine and lysine residues. Reduced distances between certain paramagnetically a †ected 15N nuclei and the center of the iron-sulfur cluster were calculated from the 15N spin-lattice relaxation times, based on the assumption of a dominant electron-nuclear dipole-dipole relaxation mechanism. Ranked by size, these distances agree with those computed from coordinates of a published structure of putidaredoxin determined by 1H NMR and analogy with an Anabaena ferredoxin. 1997