Ultrafast MAS Solid-State NMR Permits Extensive <sup>13</sup>C and <sup>1</sup>H Detection in Paramagnetic Metalloproteins

Bertini, Ivano; Emsley, Lyndon; Lelli, Moreno; Luchinat, Claudio; Mao, Jiafei; Pintacuda, Guido

doi:10.1021/ja100398q

Cited by 110 publications

(126 citation statements)

References 23 publications

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“…Importantly, however, calculations known to provide good prediction of the long-range PCSs should also provide detailed spectral assignment aids and electronic-structure information closer to the metal center, in a region in which experimental determination of the pNMR shifts is much more challenging. Full pNMR shifts for nuclei closer to the metal center are evaluated and compared to available experimental data [5] in Tables S5, S6, and Figure S3 in SI. While these comparisons expose shortcomings in DFT-computed HFCs, they nevertheless may be helpful for assignment and spectral-range predictions of signals, as we move closer into the "blind sphere" around the paramagnetic metal center.…”

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Pseudo‐Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP‐12 from First Principles

et al. 2016

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Long-range pseudo-contact NMR shifts (PCSs) provide important restraints for the structure refinement of proteins when a paramagnetic metal center is present, either naturally or introduced artificially. Here we show that ab initio quantum-chemical methods and a modern version of the Kurland-McGarvey approach for paramagnetic NMR (pNMR) shifts in the presence of zero-field splitting (ZFS) together provide accurate predictions of all PCSs in a metalloprotein (high-spin cobalt-substituted MMP-12 as a test case). Computations of 314 13 C PCSs via g-and ZFS-tensors based on multi-reference methods provide a reliable bridge between EPRparameter-and susceptibility-based pNMR formalisms. Due to the high sensitivity of PCSs to even small structural differences, local structures based either on X-ray diffraction or on various DFT optimizations could be evaluated critically by comparing computed and experimental PCSs. Many DFT functionals provide insufficiently accurate structures. We also found the available 1RMZ PDB X-ray structure to exhibit deficiencies related to binding of a hydroxamate inhibitor. This has led to a newly refined PDB structure for MMP-12 (5LAB) that provides a more accurate coordination arrangement and PCSs.The anisotropic magnetic susceptibility [1] of paramagnetic metal ions induces the so-called pseudo-contact shifts (PCSs) in NMR spectra, which can be observed for nuclei between 5 Å and 40 Å from the metal center. [2] PCSs provide precious structural information on the biomolecules on which they are measured, both in solution and in the solid state. [3] PCS-based structural restraints have also become important for protein NMR crystallography. [4] Their importance is further enhanced by recent developments in fast magic-angle spinning (MAS) combined with high-field instruments. [4d, 5] While PCSs can thus provide crucial information on the structure of a metalloprotein as a whole, NMR is typically blind to nuclei near the paramagnetic metal center due to fast paramagnetic relaxation. The computation of pNMR shifts by first-principles quantum-chemical (QC) methods, on the other hand, has recently progressed appreciably, in particular by inclusion of the non-contact terms in small to medium-sized molecules, with no fundamental limitations close to the metal center. [6] There has so far been no attempt to access the longrange PCSs in larger biological systems by first-principles calculations, as the molecular sizes needed for an explicit treatment of the hyperfine coupling (HFC) anisotropies appeared prohibitive.Here we show that introduction of the point-dipole approximation (PDA), appropriate for the long-range spin-dipolar HFCs, into modern quantum-chemical pNMR shift machinery can be used to compute long-range PCSs based on accurate multireference ab initio calculations of g-and zero-field splitting (ZFS) D-tensors. Using such a combined approach, we have computed the entire set of 314 previously measured 13 C long-range PCSs [4a] (and further shifts from nuclei closer to the meta...

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Pseudo‐Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP‐12 from First Principles

et al. 2016

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“…For dilute spins, the NMR conditions can be selected such that the signal from nuclei near the paramagnetic centre is "invisible" owing to rapid relaxation and only the longer-range throughspace pseudocontact shifts are observed. 1 These are generally on the order of a few ppm and occur over distances such that the unpaired electron can be treated as a point spin, resulting in a simple 1/r 3 relationship with the shift. For dense spin networks such as transition metal oxides, it may be possible to assign the NMR spectra by analysis of bonding pathways (via oxygen).…”

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Calculation and experimental measurement of paramagnetic NMR parameters of phenolic oximate Cu(ii) complexes

Dawson

Mack

et al. 2017

Chem. Commun.

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We present a strategy for predicting the unusual 1 H and 13 C shifts in NMR spectra of paramagnetic bisoximato copper(II) complexes using DFT. We demonstrate good agreement with experimental measurements, although 1 H-13 C correlation spectra show that a combined experimental and theoretical approach remains necessary for full assignment.In recent years, paramagnetic NMR (or "pNMR") has developed greatly, with systems from metalloproteins 1 (dilute, isolated spins) to metal-organic frameworks 2-4 (denser networks of potentially coupled spins) and metal oxides 5 (very dense networks of highly coupled spins) being studied. For dilute spins, the NMR conditions can be selected such that the signal from nuclei near the paramagnetic centre is "invisible" owing to rapid relaxation and only the longer-range throughspace pseudocontact shifts are observed. 1 These are generally on the order of a few ppm and occur over distances such that the unpaired electron can be treated as a point spin, resulting in a simple 1/r 3 relationship with the shift. For dense spin networks such as transition metal oxides, it may be possible to assign the NMR spectra by analysis of bonding pathways (via oxygen). 5 However, in the intermediate regime, including materials such as catalytically-active transition metal complexes and metal-organic frameworks (MOFs), both the through-bond Fermi contact and through-space pseudocontact interactions affect the observed NMR spectrum and assignment can be both nontrivial and counterintuitive. 2,6-9 For example, in the 13 C NMR spectrum of the MOF HKUST-1 (Cu3btc2, btc = benzene-1,3,5-tricarboxylate), 10 the broadest resonance, shifted most by paramagnetic interactions, is not the carboxylate C (separated from Cu 2+ by just two bonds), but rather the adjacent quaternary C (three bonds from Cu). This assignment was confirmed using the relatively costly and timeconsuming approach of specific 13 C labelling in conjunction with 1 H-13 C cross polarisation (CP) NMR, which is generally inefficient for paramagnetic materials. 2 It would, therefore, be desirable to have a more general assignment method that does not rely on the development of bespoke synthetic pathways for efficient isotopic enrichment. Owing to the rapid MAS rates (necessitating the use of small rotors with, consequently, small sample volumes) required for highresolution pNMR spectra, sensitivity is inherently low and it would, therefore, also be advantageous to be able to predict shifts prior to the experimental measurement, particularly as resonances can be several hundred ppm away from their typical diamagnetic range.Periodic density functional theory (DFT) calculations have enjoyed great success in solid-state NMR, allowing the optimisation of experimental structures to an energy minimum and the subsequent calculation of highly accurate NMR parameters [11][12][13] However, pNMR DFT calculations are still in their relative infancy, particularly for periodic solids. The field is more advanced for molecular calculations, which have successfull...

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“…These effects act up to very long distances, and carry a well-defined dependence on the nuclear position with respect to the paramagnetic center. In solids, measuring pseudocontact shifts is relatively straightforward, and when they occur, they can be used as invaluable structural constraints (20)(21)(22). However, measuring PREs in solids is a prime example where traditional detection methods have difficulty to provide sufficient sensitivity.…”

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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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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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Ultrafast MAS Solid-State NMR Permits Extensive ¹³C and ¹H Detection in Paramagnetic Metalloproteins

Cited by 110 publications

References 23 publications

Pseudo‐Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP‐12 from First Principles

Pseudo‐Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP‐12 from First Principles

Calculation and experimental measurement of paramagnetic NMR parameters of phenolic oximate Cu(ii) complexes

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

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Ultrafast MAS Solid-State NMR Permits Extensive 13C and 1H Detection in Paramagnetic Metalloproteins

Cited by 110 publications

References 23 publications

Pseudo‐Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP‐12 from First Principles

Pseudo‐Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP‐12 from First Principles

Calculation and experimental measurement of paramagnetic NMR parameters of phenolic oximate Cu(ii) complexes

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

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Ultrafast MAS Solid-State NMR Permits Extensive ¹³C and ¹H Detection in Paramagnetic Metalloproteins