Nuclear Magnetic Resonance (NMR) is a highly versatile and powerful spectroscopic technique, and developing novel experimental approaches is essential to fully exploit its potential. At CERM/CIRMMP, advanced instrumentation provides a broad platform for method development and applications, spanning biomolecular structure, biomedical imaging, and pharmacology.

A suite of tailored experiments has been developed, with pulse sequences and reference datasets now available through Bruker releases. These tools enable sequence-specific protein assignments as well as detailed structural and dynamic analyses.

Solid-state NMR (SSNMR) is a powerful technique for studying the structure and dynamics of materials and biomolecules at the atomic level. In recent times, solid state NMR has witnessed a profound advancement in terms of sensitivity and resolution. Fast magic angle spinning (MAS, beyond 100 kHz) and magnetic fields now exceeding 28 T, have promoted biomolecular SSNMR by providing high resolution and permitting 1H detection, which results in a large sensitivity gain with respect to standard heteronuclear detection. In this context there is a need to define proper sample manipulation stategies, time-optimization of the experiments and dedicated processing approaches .

Another key area of research focuses on paramagnetic systems. NMR offers quantitative structural insights on these systems. The key observables are the hyperfine interaction, self-orientation effects, relaxation and cross-correlation phenomena. These can be used as long-range restraints for structural and dynamical studies in paramagnetic metalloproteins, complementing the local information that comes from traditional NMR experiments. Integration with quantum chemical calculations can also offer access to the details of the electronic structure, and, through these, the possibility of deriving magnetostructural correlations. Paramagnetic relaxation can be measured from low to ultra-high magnetic fields. These relaxometry studies have a broad range of applications, including direct applications in MR imaging, where chelates of manganese(II) and gadolinium(III) are used as contrast agents, i.e.: to accelerate proton relaxation, enhancing signal intensity and producing clearer tissue images. Using field cycling relaxometry, researchers explore how these complexes interact with water, move, and sometimes aggregate. This information can be used to understand contrast mechanisms and therefore to increase the efficiencyof the contrast agents.

Selected papers:

Bermel, W.; Bertini, I.; Felli, I. C.; Kümmerle, R.; Pierattelli, R. 13C Direct Detection Experiments on the Paramagnetic Oxidized Monomeric Copper, Zinc Superoxide Dismutase. J. Am. Chem. Soc. 2003, 125 (52). https://doi.org/10.1021/ja037676p
Arnesano, F.; Banci, L.; Bertini, I.; Felli, I. C.; Luchinat, C.; Thompsett, A. R. A Strategy for the NMR Characterization of Type II Copper(II) Proteins: The Case of the Copper Trafficking Protein CopC from Pseudomonas Syringae. J. Am. Chem. Soc. 2003, 125 (24). https://doi.org/10.1021/ja034112c
Bertini, I.; Felli, I. C.; Luchinat, C.; Parigi, G.; Pierattelli, R. Towards a Protocol for Solution Structure Determination of Copper(II) Proteins: The Case of CuIIZnII Superoxide Dismutase. ChemBioChem 2007, 8 (12), 1422–1429. https://doi.org/10.1002/cbic.200700006
Bermel, W.; Bertini, I.; Felli, I. C.; Piccioli, M.; Pierattelli, R. 13C-Detected Protonless NMR Spectroscopy of Proteins in Solution. Prog. Nucl. Magn. Reson. Spectrosc. 2006, 48 (1). https://doi.org/10.1016/j.pnmrs.2005.09.002
Turano, P.; Lalli, D.; Felli, I. C.; Theil, E. C.; Bertini, I. NMR Reveals Pathway for Ferric Mineral Precursors to the Central Cavity of Ferritin. Proc. Natl. Acad. Sci. 2010, 107 (2), 545–550. https://doi.org/10.1073/pnas.0908082106
Bertini, I.; Felli, I. C.; Kümmerle, R.; Moskau, D.; Pierattelli, R. 13C-13C NOESY: An Attractive Alternative for Studying Large Macromolecules. J. Am. Chem. Soc. 2004, 126 (2), 464–465. https://doi.org/10.1021/ja0357036
Matzapetakis, M.; Turano, P.; Theil, E. C.; Bertini, I. 13C-13C NOESY Spectra of a 480 KDa Protein: Solution NMR of Ferritin. J. Biomol. NMR 2007, 38 (3), 237–242. https://doi.org/10.1007/s10858-007-9163-9
Felli IC, Pierattelli R. J Magn Reson. Novel methods based on (13)C detection to study intrinsically disordered proteins. 2014 Apr;241:115-25. doi: 10.1016/j.jmr.2013.10.020. PMID: 24656084 Review
Bertini, I.; Emsley, L.; Lelli, M.; Luchinat, C.; Mao, J.; Pintacuda, G. Ultrafast MAS Solid-State NMR Permits Extensive 13C and 1H Detection in Paramagnetic Metalloproteins. J. Am. Chem. Soc. 2010 132 (16) 5558-5559. https://doi.org/10.1021/ja100398q
Knight, M.J.; Webber, A.L.; Pell, A.J.; Guerry, P.; Barbet-Massin, E.; Bertini, I.; Felli, I.C.; Gonnelli, L.; Pierattelli, R. Emsley, L.; Lesage, A.; Herrmann, T.; Pintacuda, G. Fast Resonance Assignment and Fold Determination of Human Superoxide Dismutase by High‐Resolution Proton‐Detected Solid‐State MAS NMR Spectroscopy. Angew. Chem. Int. Ed. 2011 50, 11697-11701. https://doi.org/10.1002/anie.201106340
Knight, M.J.; Felli, I.C.; Pierattelli, R.; Bertini, I.; Emsley, L.; Herrmann, T.; Pintacuda, G. Rapid Measurement of Pseudocontact Shifts in Metalloproteins by Proton-Detected Solid-State NMR Spectroscopy J. Am. Chem. Soc. 2012 134 (36) 14730-14733. https://doi.org/10.1021/ja306813j
Bertarello, A.; Schubeis, T.; Fuccio, C.; Ravera, E.; Fragai, M.; Parigi, G.; Emsley, L.; Pintacuda, G.; Luchinat, C. Paramagnetic Properties of a Crystalline Iron–Sulfur Protein by Magic-Angle Spinning NMR Spectroscopy Inorg. Chem. 2017 56, 6624-6629
Ravera, E.; Cerofolini, L.; Martelli, T.; Louka, A.; Fragai, M.; Luchinat, C. (1) H-detected solid-state NMR of proteins entrapped in bioinspired silica: a new tool for biomaterials characterization. Sci. Rep. 2016 6 27851
Ravera, E.; Ciambellotti, S.; Cerofolini, L.; Martelli, T.; Kozyreva, T.; Bernacchioni, C.; Giuntini, S.; Fragai, M.; Turano, P.; Luchinat, C. Solid‐State NMR of PEGylated Proteins. Angew. Chem. Int. Ed. 2016 55 2445-2449
 Giuntini, S.; Cerofolini, L.; Ravera, E.; Fragai, M.; Luchinat, C. Atomic structural details of a protein grafted onto gold nanoparticles. Sci. Rep. 2017 7 1
Bruno, F.; Francischello, R.; Bellomo, G.; Gigli, L.; Flori, A.; Menichetti, L.; Tenori, L.; Luchinat, C.; Ravera, E. Multivariate Curve Resolution for 2D solid-state NMR spectra. Anal. Chem. 2020 92 4451-4458

Kateb, F, and Piccioli, M., New routes to the detection of relaxation allowed coherence transfer in paramagnetic molecules., J. Am. Chem. Soc. 2003,125:14978-9.
Brancaccio D., Gallo A., Mikolaijczyk M., Zovo K., Palumaa P., Novellino E., Piccioli M., Ciofi-Baffoni S., Banci L., Formation of [4Fe-4S] clusters in the mitochondrial iron-sulfur cluster assembly machinery J. Am. Chem. Soc. 2014 Nov 19;136(46):16240-16250
Parigi G, Ravera E, Luchinat C. Magnetic susceptibility and paramagnetism-based NMR Prog Nucl Magn Reson Spectrosc. 2019;114-115:211-236
Parigi G, Benda L, Ravera E, Romanelli M, Luchinat C. Pseudocontact shifts and paramagnetic susceptibility in semiempirical and quantum chemistry theories. J Chem Phys. 2019 Apr 14;150(14):144101
Camponeschi F, Muzzioli R, Ciofi-Baffoni S, Piccioli M, Banci L. Paramagnetic 1H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes. J Mol Biol. 2019 Nov 8;431(22):4514-4522. doi: 10.1016/j.jmb.2019.08.018
Bertini, I., Jiménez, B, Piccioli, M. and Poggi, L., Asymmetry in 13C-13C COSY spectra identifies ligand geometry in paramagnetic proteins, J. Am. Chem. Soc. , 2005, 127, 12216-7.
Mori M, Kateb F, Bodenhausen G, Piccioli M, Abergel D. Toward structural dynamics: protein motions viewed by chemical shift modulations and direct detection of C'N multiple-quantum relaxation. J Am Chem Soc. 2010,132, 3594-3600
Banci L., Bertini I., Calderone V., Ciofi-Baffoni S., Giachetti A., Jaiswal D., Mikolajczyk M., Piccioli M., Winkelmann J, A molecular view of an electron transfer pathway essential for iron-sulfur protein biogenesis, Proc Natl Acad Sci U S A. 2013, 110(18), 7136-7141

Ciofi Baffoni S., Gallo A., Muzzioli R., Piccioli M., The IR-15N-HSQC-AP experiment: a new tool for NMR spectroscopy of paramagnetic molecules, J. Biomol. NMR 2014, 58, 123-128
Trindade I, Invernici M, Cantini, C, Louro R, Piccioli M. , NOE-less protein structures-an alternative paradigm in Highly Paramagnetic Systems arXiv:2002.01228
Invernici M, Trindade I, Cantini, C, Louro R, Piccioli M: Measuring Transverse Relaxation in highly paramagnetic systems (submitted)