Measuring transverse relaxation in highly paramagnetic systems
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ARTICLE
Measuring transverse relaxation in highly paramagnetic systems Michele Invernici2,3 · Inês B. Trindade1 · Francesca Cantini2,3 · Ricardo O. Louro1 · Mario Piccioli2,3 Received: 17 May 2020 / Accepted: 9 July 2020 © The Author(s) 2020
Abstract The enhancement of nuclear relaxation rates due to the interaction with a paramagnetic center (known as Paramagnetic Relaxation Enhancement) is a powerful source of structural and dynamics information, widely used in structural biology. However, many signals affected by the hyperfine interaction relax faster than the evolution periods of common NMR experiments and therefore they are broadened beyond detection. This gives rise to a so-called blind sphere around the paramagnetic center, which is a major limitation in the use of PREs. Reducing the blind sphere is extremely important in paramagnetic metalloproteins. The identification, characterization, and proper structural restraining of the first coordination sphere of the metal ion(s) and its immediate neighboring regions is key to understand their biological function. The novel HSQC scheme we propose here, that we termed R2-weighted, HSQC-AP, achieves this aim by detecting signals that escaped detection in a conventional HSQC experiment and provides fully reliable R2 values in the range of 1H R2 rates ca. 50–400 s−1. Independently on the type of paramagnetic center and on the size of the molecule, this experiment decreases the radius of the blind sphere and increases the number of detectable PREs. Here, we report the validation of this approach for the case of PioC, a small protein containing a high potential 4Fe-4S cluster in the reduced [ Fe4S4]2+ form. The blind sphere was contracted to a minimal extent, enabling the measurement of R2 rates for the cluster coordinating residues. Keywords Paramagnetic NMR · Iron sulfur proteins · Pulse sequences · NMR based structural restraints · Transverse relaxation · Paramagnetic relaxation enhancement
Introduction The hyperfine interaction between electron and nuclear spins gives rise to additional contributions to chemical shifts and nuclear relaxation, both of which can be used as a source of structural restraints (Piccioli and Turano 2015; Turner et al. 1998). Nowadays, NMR solution structures of paramagnetic macromolecules are obtained by a combination of * Ricardo O. Louro [email protected] * Mario Piccioli [email protected] 1
Instituto de Tecnologia Química e Biológica António Xavier (ITQB‑NOVA), Universidade Nova de Lisboa, Av. da República (EAN), 2780‑157 Oeiras, Portugal
2
Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy
3
Consorzio Interuniversitario Risonanze Magnetiche Di Metallo Proteine (CIRMMP), Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy
conventional restraints (Ab et al. 2006; Mori et al. 2008), such as NOE and residual dipolar couplings, and of paramagnetic-based restraints (Arnesano et al. 2006; Clore 2015; Kudhair, et al. 2280; Parigi e
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