Abstract : Herein we present examples of the application of the 13C-13C Nuclear Overhauser Effect Spectroscopy (NOESY) experiment to the study of metalloproteins and we critically discuss the advantages and drawbacks of the method as a function of the molecular size of the investigated systems. The contribution is focused on a few case studies among the systems analyzed in the group of the corresponding author. The 13C-13C NOESY experiment represents the gold standard for the observation of NMR signals in the 480 kDa ferritin nanocage and for monitoring its interaction with iron. By decreasing the protein size, the experiment progressively loses its importance as a tool for the detection of the complete spin pattern of the amino acid side chains, as exemplified by nickel-dependent regulatory protein, NikR (molecular mass of the homo-tetramer ~80 kDa). In very small proteins, such as mitochondrial cytochrome c (12.3 kDa), we are only able to detect cross peaks between adjacent 13C nuclei; this feature turned out to be useful for the assignment of the 13C core resonances of the porphyrin in a uniformly enriched heme. © 2018 The Authors
