Towards a Protocol for Solution Structure Determination of Copper(II) Proteins: the Case of Cu<sup>II</sup>Zn<sup>II</sup> Superoxide Dismutase

Bertini, Ivano; Felli, Isabella C.; Luchinat, Claudio; Parigi, Giacomo; Pierattelli, Roberta

doi:10.1002/cbic.200700006

Cited by 26 publications

(24 citation statements)

References 52 publications

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“…[1][2][3] Amongt hem, the copper(II)-zinc(II) SOD is present in the plasma and in extracellular spaces, and it decomposess uperoxider adicals into molecular oxygen and hydrogen peroxide. [4][5][6] The latter molecules are subsequently removed by the catalase enzyme. [7] However,s upplementation of SOD is difficult owing to its high sensitivity to environmentalc onditions (e.g.,t emperature, pH, and complex nature in biofluids).…”

Section: Introductionmentioning

confidence: 99%

Immobilization of Superoxide Dismutase on Polyelectrolyte‐Functionalized Titania Nanosheets

2017

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The superoxide dismutase (SOD) enzyme was successfully immobilized on titania nanosheets (TNS) functionalized with the poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte. The TNS-PDADMAC solid support was prepared by hydrothermal synthesis followed by self-assembled polyelectrolyte layer formation. It was found that SOD strongly adsorbed onto oppositely charged TNS-PDADMAC through electrostatic and hydrophobic interactions. The TNS-PDADMAC-SOD material was characterized by light scattering and microscopy techniques. Colloidal stability studies revealed that the obtained nanocomposites possessed good resistance against salt-induced aggregation in aqueous suspensions. The enzyme kept its functional integrity upon immobilization; therefore, TNS-PDADMAC-SOD showed excellent superoxide radical anion scavenging activity. The developed system is a promising candidate for applications in which suspensions of antioxidant activity are required in the manufacturing processes.

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Section: Introductionmentioning

confidence: 99%

Immobilization of Superoxide Dismutase on Polyelectrolyte‐Functionalized Titania Nanosheets

2017

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“…In addition there were 25 1 H-15 N cross-peaks observable in the diamagnetic but not paramagnetic form. In these cases, it was assumed that this was due to broadening of the 1 H resonances beyond detection, and an upper distance limit to the Cu ion of 10 Å was used, without lower limit (39). The remaining residues corresponded to peaks for which accurate PRE determination was not possible.…”

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confidence: 99%

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

Knight

Pell

Bertini

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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175

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We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with 1 H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of 15 N and 13 C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu þ (diamagnetic) or Cu 2þ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to 1 H-1 H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.paramagnetism | nuclear relaxation rates | copper | microcrystal S tructure determination of proteins plays a central role in understanding key events in biology. Although the structure of many proteins can be obtained from single-crystal X-ray diffraction, or by solution-state nuclear magnetic resonance (NMR) spectroscopy, there is nevertheless a range of important substrates for which structures cannot be determined today. These include immobile systems lacking long-range order such as protein aggregates, large complexes and membrane-bound systems.Solid-state NMR has the unique potential to study, with atomic resolution, systems of this nature, and spectacular progress has been made in this area over the last decade (1). There are today a small handful of structures obtained by solid-state NMR, from microcrystalline samples to fibrils and membrane-associated systems (2). Additionally, solid-state NMR is uniquely sensitive to site-specific protein dynamics over a broad range of timescales, and a number of demonstration studies have recently appeared for model proteins (3).The use of perdeuterated proteins has very recently opened the way to highly sensitive proton-detected solid-state experiments (4). Despite early proof-of-principle papers (5), this approach only became popular with the realization that amide sites must be only partially reprotonated (typically 10-30% back exchange) (6-8) to yield well-resolved 1 H spectra. This represented a significant compromise in sensitivity to gain resolution, and effectively made the determination of internuclear distances impractical, with few exceptions (9-11). We have recently shown how this problem can be completely overcome by using 100% reprotonation of exchangeable sites, without loss of resolution, if perdeuteration is combined wit...

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“…After 6 days the cells were harvested by centrifugation. The NP2 protein was isolated and purified as described elsewhere, 39 but included the addition of hemin during the purification to complex any apo-NP2 that might be present (0.2 mL of ~2 mM hemin dissolved in a few drops of DMSO and diluted into pH 7.5 sodium phosphate buffer). A preliminary 1 H, 15 N-HSQC spectrum, recorded at pH 5.0 and 298 K, showed that there were only three amide 1 H, 15 N cross peaks in the normal protein amide window (5.5 – 11.0 ppm 1 H, 110 – 140 ppm 15 N) even down to the noise floor, and one additional amide cross peak, at 2.90 1 H, 116.0 15 N ppm, which confirmed that there was no measurable scrambling of the 15 N (and by implication, the 13 C) label.…”

Section: Methodsmentioning

confidence: 99%

Electron Spin Density on the Axial His Ligand of High-Spin and Low-Spin Nitrophorin 2 Probed by Heteronuclear NMR Spectroscopy

et al. 2013

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The electronic structure of heme proteins is exquisitely tuned by the interaction of the iron center with the axial ligands. NMR studies of paramagnetic heme systems have been focused on the heme signals, but signals from the axial ligands have been rather difficult to detect and assign. We report an extensive assignment of the 1H, 13C and 15N resonances of the axial His ligand in the NO-carrying protein nitrophorin 2 (NP2) in the paramagnetic high-spin and low-spin forms, as well as in the diamagnetic NO complex. We find that the high-spin protein has σ spin delocalization to all atoms in the axial His57, which decreases in size as the number of bonds between Fe(III) and the atom in question increase, except that within the His57 imidazole ring the contact shifts are a balance between positive σ and negative π contributions. In contrast, the low-spin protein has π spin delocalization to all atoms of the imidazole ring. Our strategy, adequately combined with a selective residue labeling scheme, represents a straightforward characterization of the electron spin density in heme axial ligands.

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Towards a Protocol for Solution Structure Determination of Copper(II) Proteins: the Case of Cu^IIZn^II Superoxide Dismutase

Cited by 26 publications

References 52 publications

Immobilization of Superoxide Dismutase on Polyelectrolyte‐Functionalized Titania Nanosheets

Immobilization of Superoxide Dismutase on Polyelectrolyte‐Functionalized Titania Nanosheets

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

Electron Spin Density on the Axial His Ligand of High-Spin and Low-Spin Nitrophorin 2 Probed by Heteronuclear NMR Spectroscopy

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Towards a Protocol for Solution Structure Determination of Copper(II) Proteins: the Case of CuIIZnII Superoxide Dismutase

Cited by 26 publications

References 52 publications

Immobilization of Superoxide Dismutase on Polyelectrolyte‐Functionalized Titania Nanosheets

Immobilization of Superoxide Dismutase on Polyelectrolyte‐Functionalized Titania Nanosheets

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

Electron Spin Density on the Axial His Ligand of High-Spin and Low-Spin Nitrophorin 2 Probed by Heteronuclear NMR Spectroscopy

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Towards a Protocol for Solution Structure Determination of Copper(II) Proteins: the Case of Cu^IIZn^II Superoxide Dismutase