1H, 13C and 15N assignment of the paramagnetic high potential iron–sulfur protein (HiPIP) PioC from Rhodopseudomonas palustris TIE-1

Trindade, Inês B.; Invernici, Michele; Cantini, Francesca; Louro, Ricardo O.; Piccioli, Mario

doi:10.1007/s12104-020-09947-6

Cited by 10 publications

(11 citation statements)

References 24 publications

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“…The complete resonance assignment of the protein was obtained combining the conventional approach based on triple resonance experiments (Table S1) with a non-systematic procedure using a combination of 1D NOEs, 13 C direct detection, double and triple resonance experiments recorded with parameters optimized a-la-carte (Table S2) [92]. These experiments provided the complete NMR assignment of PioC (BRMB entry 34487) [93]. We assigned (excluding the N-ter Val 1) 100% of backbone 1 H N , 13 C, and 15 N resonances, 98% of Hα, 86% and 91% of 1 H and 13 C side chains atoms, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Paramagnetic NMR experiments provided structural constraints for cluster-binding residues (through coordinative or hydrogen bonds) crucial to define their orientation: Cys βCH 2 hyperfine shifts were converted into four χ 2 dihedral angle constraints defining the cluster binding topology according to a procedure already described [95], seven crucial 1D NOEs provided distances between Cys βCH 2 and neighboring residues (Fig. S1), large 15 N contact shifts, observed for Gln27, Val37, and Leu49 [93] were taken as an evidence of three hydrogen bonds, respectively, linking H N to the Sγ atoms of the preceding (i-2 or i-3) cluster-bound cysteine residues [96]. Only fourteen constraints of this type are available but they are extremely important to frame the cluster within the protein and to provide restraints where other experimental approaches fail to provide information.…”

Section: Resultsmentioning

confidence: 99%

“…Data analysis and resonances assignment were performed using CARA 1.9[106]. The complete assignment has been submitted in BRMB entry 34487[93]. NOEs were analyzed and converted into upper distance limits and used for manual structure calculation in CYANA 2.1.…”

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confidence: 99%

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PRE‐driven protein NMR structures: an alternative approach in highly paramagnetic systems

et al. 2020

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The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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PRE‐driven protein NMR structures: an alternative approach in highly paramagnetic systems

et al. 2020

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“…Under favorable conditions, this approach succeeded to remove the blind sphere around a paramagnetic center and provided a complete resonance assignment for paramagnetic metalloproteins [83,84]. Based on the above criteria, triple-resonance experiments such as HNCA, HNCO, and CBCANH, characterized by many pulses and many polarization transfer steps, can be efficiently optimized for the identification of paramagnetic signals.…”

Section: New Experiments In Nmr Of Paramagnetic Molecules and Their Amentioning

confidence: 99%

Paramagnetic NMR Spectroscopy Is a Tool to Address Reactivity, Structure, and Protein–Protein Interactions of Metalloproteins: The Case of Iron–Sulfur Proteins

Piccioli¹

2020

Magnetochemistry

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The study of cellular machineries responsible for the iron–sulfur (Fe–S) cluster biogenesis has led to the identification of a large number of proteins, whose importance for life is documented by an increasing number of diseases linked to them. The labile nature of Fe–S clusters and the transient protein–protein interactions, occurring during the various steps of the maturation process, make their structural characterization in solution particularly difficult. Paramagnetic nuclear magnetic resonance (NMR) has been used for decades to characterize chemical composition, magnetic coupling, and the electronic structure of Fe–S clusters in proteins; it represents, therefore, a powerful tool to study the protein–protein interaction networks of proteins involving into iron–sulfur cluster biogenesis. The optimization of the various NMR experiments with respect to the hyperfine interaction will be summarized here in the form of a protocol; recently developed experiments for measuring longitudinal and transverse nuclear relaxation rates in highly paramagnetic systems will be also reviewed. Finally, we will address the use of extrinsic paramagnetic centers covalently bound to diamagnetic proteins, which contributed over the last twenty years to promote the applications of paramagnetic NMR well beyond the structural biology of metalloproteins.

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“…The backbone NMR assignment of PioC (Trindade et al 2020a ) has been published on Biomolecular NMR Assignment and deposited in the BMRB data bank (ID 34487).…”

Section: Methodsmentioning

confidence: 99%

Measuring transverse relaxation in highly paramagnetic systems

et al. 2020

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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.

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1H, 13C and 15N assignment of the paramagnetic high potential iron–sulfur protein (HiPIP) PioC from Rhodopseudomonas palustris TIE-1

Cited by 10 publications

References 24 publications

PRE‐driven protein NMR structures: an alternative approach in highly paramagnetic systems

PRE‐driven protein NMR structures: an alternative approach in highly paramagnetic systems

Paramagnetic NMR Spectroscopy Is a Tool to Address Reactivity, Structure, and Protein–Protein Interactions of Metalloproteins: The Case of Iron–Sulfur Proteins

Measuring transverse relaxation in highly paramagnetic systems

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