ja037077i_si_001.pdf (29.04 kB)
Download file

Strategy for the Study of Paramagnetic Proteins with Slow Electronic Relaxation Rates by NMR Spectroscopy:  Application to Oxidized Human [2Fe-2S] Ferredoxin

Download (29.04 kB)
journal contribution
posted on 05.05.2004, 00:00 by Timothy E. Machonkin, William M. Westler, John L. Markley
NMR studies of paramagnetic proteins are hampered by the rapid relaxation of nuclei near the paramagnetic center, which prevents the application of conventional methods to investigations of the most interesting regions of such molecules. This problem is particularly acute in systems with slow electronic relaxation rates. We present a strategy that can be used with a protein with slow electronic relaxation to identify and assign resonances from nuclei near the paramagnetic center. Oxidized human [2Fe-2S] ferredoxin (adrenodoxin) was used to test the approach. The strategy involves six steps:  (1) NMR signals from 1H, 13C, and 15N nuclei unaffected or minimally affected by paramagnetic effects are assigned by standard multinuclear two- and three-dimensional (2D and 3D) spectroscopic methods with protein samples labeled uniformly with 13C and 15N. (2) The very broad, hyperfine-shifted signals from carbons in the residues that ligate the metal center are classified by amino acid and atom type by selective 13C labeling and one-dimensional (1D) 13C NMR spectroscopy. (3) Spin systems involving carbons near the paramagnetic center that are broadened but not hyperfine-shifted are elucidated by 13C{13C} constant time correlation spectroscopy (CT-COSY). (4) Signals from amide nitrogens affected by the paramagnetic center are assigned to amino acid type by selective 15N labeling and 1D 15N NMR spectroscopy. (5) Sequence-specific assignments of these carbon and nitrogen signals are determined by 1D 13C{15N} difference decoupling experiments. (6) Signals from 1H nuclei in these spin systems are assigned by paramagnetic-optimized 2D and 3D 1H{13C} experiments. For oxidized human ferredoxin, this strategy led to assignments (to amino acid and atom type) for 88% of the carbons in the [2Fe-2S] cluster-binding loops (residues 43−58 and 89−94). These included complete carbon spin-system assignments for eight of the 22 residues and partial assignments for each of the others. Sequence-specific assignments were determined for the backbone 15N signals from nine of the 22 residues and ambiguous assignments for five of the others.