A Simple Protocol to Study Blue Copper Proteins by NMR

In the case of oxidized plastocyanin from Synechocystis sp. PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids. A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of nine of the above 14 residues. The proposed protocol differs from previous aproaches in that it does not involve the use of unconventional experiments designed specifically for paramagnetic systems, and does not exploit the occurrence of a corresponding diamagnetic species in chemical exchange with the blue copper form. This protocol is expected to extend the popularity of NMR in the structural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requiting a specific expertise in the field. The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis.