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

Abstract

NMR resonance assignments in the vicinity of paramagnetic metals in proteins are often difficult or impossible to make using conventional 1H detected 2-D and 3-D methods due to paramagnetic line broadening. The applicability of 13Calpha{13C'} and 13C'{15N} multiple quantum coherence methods for residue-specific assignments of resonances near paramagnetic centers is described, using the Ni2+-containing enzyme acireductone dioxygenase as an example.

Nickel in Acireductone Dioxygenase

“…Thus, direct 13 C detection 2D NMR methods offer the promise of sequential resonance assignments and structural insights into the active sites of ARD and related proteins. We have described the use of 2D [47], and other researchers have published experiments that offer the hope of direct structural methods for determining structures of metal binding sites in paramagnetic proteins [48][49][50][51].…”

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

“…[1][2][3] Progress in this area was initially stimulated by its application to the investigation of paramagnetic proteins, [4][5][6][7][8][9] where the presence of a paramagnetic center leads to relaxation-rate enhancements that depend, among other factors, on the square of the gyromagnetic ratio of the observed nucleus. Therefore, experiments based on 13 C direct detection that only rely on heteronuclei (protonless NMR) [1,10] proved particularly useful for characterization of paramagnetic proteins also in regions where 1 H resonances are broadened beyond detection.…”

A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

“…In the current refinement, we made use a greater number of dihedral restraints generated from chemical shift database correlations (TALOS) than in the previous calculations (Cornilescu, Delaglio et al, 1999). Improved 2D methods for sequential assignment of paramagnetically relaxed 13 C and 15 N resonances have permitted us to make some assignments in the region of the NiARD active site (Kostic et al, 2002). However, these assignments are not yet sufficiently complete to forgo modeling in the current structural refinement.…”