NMR of Paramagnetic Proteins

One of the biggest difficulties facing NMR studies of paramagnetic enzymes such as ARD and P450 is the broadening of resonances in the vicinity of the metal cluster due to paramagnetism. The extent to which a resonance is broadened by paramagnetic relaxation is proportional to the gyromagnetic ratio of the nucleus being observed. For this reason, 15N and 13C NMR spectroscopy are useful structural probes in the vicinity of metal centers. Both 15N and 13C have lower gyromagnetic ratios and so are less affected by the paramagnetism of the metal center than 1H. We have developed 13C-detected 2D NMR methods that remove the need for selective labeling in order to accomplish sequential NMR assignments near paramagnetic centers.

Figure 1

2D CAN spectrum of 1 mM ²H,¹⁵N,¹³C-labeled ARD, pH 7.4 HEPES in 100% D₂O, 298 K, showing sequential ¹³Cα-¹⁵N resonance assignments in the ARD active site. Except for Gly 161 (upper right-hand corner of spectrum), all annotated peaks identify previously unassigned resonances relaxed by proximity to the Ni⁺² in the active site of the enzyme.

Figure 2

Structure of Ni-bound ARD (PDB entry 1ZRR) showing location of newly assigned residues with backbone atoms in red. Regions shown in blue were assigned by standard methods; those in white are still unassigned. The Ni⁺² ion is shown as a green sphere.

Publications

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Completing the circuit: direct-observe 13C,15N double-quantum spectroscopy permits sequential resonance assignments near a paramagnetic center in acireductone dioxygenase. S. S. Pochapsky; J. C. Sunshine; T. C. Pochapsky, Journal of the American Chemical Society 2008, 130 (7), 2156-2157.
M. Kostic; T. C. Pochapsky, Structure and Dynamics of Paramagnetic Proteins by NMR. In Paramagnetic Resonance of Metallobiomolecules, American Chemical Society: 2003; Vol. 858, pp 214-226.
Rapid Recycle 13C‘,15N and 13C,13C‘ Heteronuclear and Homonuclear Multiple Quantum Coherence Detection for Resonance Assignments in Paramagnetic Proteins: Example of Ni2+-Containing Acireductone Dioxygenase. M. Kostic; S. S. Pochapsky; T. C. Pochapsky, Journal of the American Chemical Society 2002, 124 (31), 9054-9055.
A New Assignment Strategy for the Hyperfine-Shifted13C and15N Resonances in Fe2S2Ferredoxins. N. U. Jain; T. C. Pochapsky, Biochemical and Biophysical Research Communications 1999, 258 (1), 54-59.

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