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Subject of the Master's internship

Investigation of the peculiar nickel-binding site of the metalloprotein CooT by hyperpolarized solid-state NMR


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Published on 11 October 2019
Summary
With its tuneable chemical reactivity, cysteine plays crucial roles as Ni ligands in active sites of redox Ni enzymes. Instead, histidines are preferred to coordinate Ni(II) ions in non-redox proteins. Interestingly, cysteine exhibits extreme patterns of conservation, being either highly conserved or completely degenerated, with a strong tendency to form buried cysteine clusters. The case of the nickel chaperone CooT is intriguing as the binding site is formed by the dimerization of the protein that only contains a single strictly conserved and solvent-exposed cysteine. The solvent exposure of the Ni-binding site as well as its position at the dimer interface are two drawbacks for structural studies. The goal of this project is to use Dynamic Nuclear Polarization (DNP)-enhanced solid-state NMR to investigate precisely this original nickel-binding site, by enhancing significantly the sensitivity and selectivity.

Full description of the subject
Metalloenzymes often contain multi-metallic active sites whose biogenesis requires specific maturation pathways. Thus, the active site of the carbon monoxide dehydrogenase (CODH), a key enzyme of carbon metabolism that reversibly catalyzed the reduction of CO2 in CO, is composed of a NiFe4S4 center, unique in biology. Its still unknown detailed biosynthesis pathway requires three poorly characterized nickel proteins. One of them, CooT is a small protein of 66 residues, which contains a single cysteine (Cys2) essential for Ni-binding. Nickel-binding motifs in proteins are usually made up of multiple histidine and/or cysteine residues. CooT is unusual as the binding site is formed by the dimerization of the protein that only contains a single strictly conserved cysteine. Although a crystal structure has been obtained by LCBM, the precise nature of the ligands as well as the metal coordination mode in the protein are still unknown. The high flexibility of the N-terminal region as well as the protein propensity to form biologically irrelevant tetramers in concentrated solutions makes the structural study of CooT by standard NMR and X-ray diffraction difficult.
The originality of the project relies on the use of Dynamic Nuclear Polarization (DNP)-enhanced solid-state NMR, an innovative NMR technique whose sensitivity and selectivity for binding sites should be best suited for the study of nickel binding to CooT. For instance, the sensitivity gain brought by DNP should compensate for the low concentration required for the homodimer formation. The approach will be strongly multidisciplinary and conducted in close collaboration with the Laboratory of Chemistry and Biology of Metals (LCBM), specialized in biochemical and structural characterization of metalloproteins. Targeted competences are in biochemistry, structural biology and NMR.

Requested skills
This internship will be performed in strong collaboration between MEM and LCBM. Competences at the Chemistry-Biology interface are therefore required.

Keywords
Biomolecular solid-state NMR, Hyperpolarization, DNP, metalloprotein

Contacts
Sabine Hediger
Phone: 04 38 78 65 79
or
Christine Cavazza
Phone: 04 38 78 91 10

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