Abstract
Paramagnetic NMR spectroscopy and iron-sulfur (Fe-S) proteins have maintained a synergic relationship for decades. Indeed, the hyperfine shifts with their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues have been extensively used as a fingerprint of the type and of the oxidation state of the Fe-S cluster within the protein frame. The identification of NMR signals from residues surrounding the metal cofactor is crucial for understanding the structure-function relationship in Fe-S proteins, but it is generally impaired in standard NMR experiments by paramagnetic relaxation enhancement due to the presence of the paramagnetic cluster(s). On the other hand, the availability of systems of different sizes and stabilities has, over the years, stimulated NMR spectroscopists to exploit iron-sulfur proteins as paradigmatic cases to develop experiments, models, and protocols. Here, the cluster-binding properties of human mitoNEET have been investigated by 1D and 2D H diamagnetic and paramagnetic NMR, in its oxidized and reduced states. The NMR spectra of both oxidation states of mitoNEET appeared to be significantly different from those reported for previously investigated proteins. The protocol we have developed in this work conjugates spectroscopic information arising from "classical" paramagnetic NMR with an extended mapping of the signals of residues around the cluster which can be taken, even before the sequence-specific assignment is accomplished, as a fingerprint of the protein region constituting the functional site of the protein. We show how the combined use of 1D NOE experiments, direct-detected experiments, and double- and triple-resonance experiments tailored using R- and/or R-based filters significantly reduces the "blind" sphere of the protein around the paramagnetic cluster. This approach provided a detailed description of the unique electronic properties of mitoNEET, which are responsible for its biological function. Indeed, the NMR properties suggested that the specific electronic structure of the cluster possibly drives the functional properties of different proteins.
Highlights
After 40 years of a life-long relationship, iron–sulfur (Fe– S) proteins and paramagnetic NMR still maintain an active and fruitful “liaison”
Relaxation data of the protein regions not affected by the paramagnetic center (Appendix A, Fig. A4) account for a molecular reorientational correlation time of 11.6 ± 0.8 ns, which is consistent with the 19.4 kDa molecular weight of dimeric mitoNEET (Mori et al, 2008; Rossi et al, 2010)
The NMR characterization via 1D paramagnetic NMR experiments offers insights into the electronic properties of the clusters, revealing features previously unobserved and unexpected. It is another tempo of the tango relationship between the electronic structure of Fe–S clusters and the biological functions of Fe–S proteins
Summary
After 40 years of a life-long relationship, iron–sulfur (Fe– S) proteins and paramagnetic NMR still maintain an active and fruitful “liaison”. Combined with Mössbauer, EPR, and magnetic susceptibility data (Dunham et al, 1971), the chemical shift properties of the paramagnetically shifted signals and their temperature dependencies were used to propose, with alternate fortune, models for the type of the Fe– S clusters and of their electronic structure within these proteins. The first NMR spectra of non-heme metalloproteins showed everyone the huge potential of NMR spectroscopy, capable of combining on the one hand the information on the electronic structure of the paramagnetic center and, on the other hand, its unique ability to identify individual hydrogen atoms within the protein framework These features were extremely attractive for biochemists and biophysicists engaged in the understanding of Fe–S proteins.
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