Cofactor optimization

Often an NMR sample can be improved by adding an appropriate cofactor such as a metal or a small organic molecule. 

Here we show two examples:

1. Addition of Zn to IscU
2. Addition of CoA to YhhK
   

Addition and removal of Zinc from Haemophilus influenzae IscU

As many as 30% of proteins are predicted to be metal binding proteins and many of these may require metal for proper folding. In fact, a recent analysis of the PDB revealed that 14% of non-redundant entries contain stoichiometric amounts of six transition metals: Mn, Fe, Co, Ni, Cu or Zn

Zincfinder by Passerini (2006)

In this example (NESG ID IscU)the effect of metal chelation on protein stability was probed by a 2D 1H–15N HSQC NMR experiment. A well dispersed spectra is indicative of global stability, collapsed spectra of instability . Due to the adverse effect of metal chelation on protein stability, and the high prevalence of zinc, it is likely that a subset of NESG targeted proteins with poorly resolved 2D 1H–15N HSQC spectra, may have inadvertently lost structural zinc during the protein sample preparation.

Insert IscU NHSQC figures here

Addition of acetyl CoA to E. coli YhhK

E.coli YhhK (NESG ID ET106) is a putative GCN5-related N-acetyltransferase (NAT) whose substrate is unknown (below, left). The protein purified with partial occupancy of the acetyl CoA binding site, thereby yielding a N-HSQC spectrum during screening with too many peaks (below, middle) because both apo- and holo-protein were present. After additional CoA was added the spectrum showed only one set of peaks (below, right), from ligand-bound protein. The observation of multiple peaks initially also indicates ligand binding in slow exchange on the NMR time scale. Additional CoA was added and the spectrum showed only one set of peaks, from ligand-bound proteins.