Nuclear Magnetic Resonance of Paramagnetic Metalloproteins: Methodological and Practical Aspects

Metalloproteins containing paramagnetic metal clusters, particularly Iron-Sulfur (FeS) proteins, play crucial roles in rare and infectious dis- eases. Understanding their function and dynamics is essential for de- veloping new therapeutic strategies, and accurate structural data and spectroscopic probes are mandatory to achieve this. This PhD project aimed to advance the structural characterization of paramagnetic met- alloproteins using nuclear magnetic resonance (NMR) spectroscopy, with a focus on developing tailored pulse sequences and methodologi- cal approaches for studying potential therapeutic targets. Within this frame, 13C direct-detected NMR pulse sequences were designed and optimized for fast-relaxing nuclei and 13C paramagnetic relaxation enhancement (PRE)–based relaxation measurements were employed to refine 3D structures of paramagnetic systems. Biochemical stabil- ity and redox-dependent reactivity of FeS protein CISD3 were eval- uated under oxidative and nitrosative stress. Human oxidized FDX2 protein was investigated to characterize electron delocalization path- ways relevant to the rare disease MEOAL and to evaluate Optimal Control (OC) pulses for inversion over large spectral windows. Addi- tionally, in two collaborative studies, I conducted structural and dy- namic NMR analysis of the nuclear complex formed by BET (bromo (BD) domain- and extra-terminal (ET) domain-containing) protein BRD3 and the C-terminal domain (CTD) of the Murine Leukaemia Virus Integrases (MLV-IN) using a lanthanide-tagged ET mutant and contributed to the conformational characterization of a Trastuzumab- Remdesivir antibody-drug conjugate for antiviral therapeutics. This work advances the understanding of structure-function relationships in metalloproteins and provides novel NMR methodologies for study- ing paramagnetic systems. These contributions support future drug discovery efforts aimed at rare and infectious diseases.