A heteronuclear zero quantum coherence Nz-exchange experiment that resolves resonance overlap and its application to measure the rates of heme binding to the IsdC protein.
Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei. To overcome the problem of (15)N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence N(z)-exchange experiment that resolves (15)N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow k(off) rate of heme from IsdC (<10 s(-1)), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to (13)C nuclei. We expect our heteronuclear zero-quantum coherence N(z)-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.
Robson
Scott A
SA
University of California, Los Angeles, Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics and the California NanoSystems Institute, UCLA, 611 Charles E. Young Drive, Los Angeles, California 90095, USA.
Peterson
Robert
R
Bouchard
Louis-S
LS
Villareal
Valerie A
VA
Clubb
Robert T
RT
D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D011789:Quantum Theory
D002352:Carrier Proteins; C488727:IsdC protein, Staphylococcus aureus; D006418:Heme