Paramagnetic Ions Enable Tuning of Nuclear Relaxation Rates and Provide Long-Range Structural Restraints in Solid-State NMR of Proteins

Nadaud, Philippe S.; Helmus, Jonathan; Kall, Stefanie L.; Jaroniec, Christopher P.

doi:10.1021/ja900224z

Cited by 123 publications

(135 citation statements)

References 121 publications

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“…However, mixing of Co-4MMDPA (or Yb/Tm-4MMPDA) protein mutants and their diamagnetic counterpart Zn-4MMDPA (or Lu-4MMDPA) in solution required for the microcrystal formation suffers from the problem of rapid exchange of paramagnetic ions in U- 13 C, 15 N labeled protein and diamagnetic ions in natural abundance protein, resulting in undesirable diamagnetic U- 13 C, 15 N labeled and paramagnetic natural abundance proteins. 38 We observed by 1 H- 15 N HSQC spectra that 30 min of mixing of U-13 C, 15 N labeled Co42-4MMDPA and na-Zn42-4MMDPA at a molar ratio of 1:4 results in an almost even distribution of Co 2+ and Zn 2+ in all protein molecules (enriched and not-enriched). The time for the full redistribution of Co 2+ and Zn 2+ between 4MMDPA binding sites is much shorter than that required for the microcrystal formation.…”

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confidence: 85%

“…To overcome this problem, in the subsequent sample preparations, we utilized wt-na-GB1 to dilute the paramagnetic U- 13 C, 15 N GB1 to prevent exchange of metal ions; there are not high affinity metal binding sites in wt-GB1. 38 Journal of the American Chemical Society High-ratio dilution is favorable to obtain pure intramolecular PCSs and avoid intermolecular interaction. However, high-ratio dilution results in lower signal-to-noise ratio since the volume of a MAS rotor is limited, and hence a lower amount of U- 13 C, 15 N enriched proteins (the only species giving rise to the NMR signal) can be loaded in the MAS rotor.…”

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confidence: 99%

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Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

Pilla

et al. 2013

J. Am. Chem. Soc.

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Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromolecules which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is shown that high-resolution structure of proteins in solid phase can be determined without the use of traditional dipolar−dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomethyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunoglobulin-binding domain of protein G (GB1). The long-range structural restraints with metal-nucleus distance of up to ∼20 Å are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances backcalculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic susceptibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 Å root-mean-square deviation relative to X-ray structure.

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confidence: 85%

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confidence: 99%

Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

Pilla

et al. 2013

J. Am. Chem. Soc.

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“…Unpaired electrons in paramagnetic ions or organic radicals enhance nuclear-spin T2 and T1 relaxation in a distance-dependent fashion. The PRE effect is sensitive to electron-nuclear distances up to approximately 2 nm (Nadaud et al, 2009;Jaroniec, 2015), which is much longer than the distances measurable from nuclear-spin dipolar couplings. PRE solidstate NMR has been used to refine protein structure (Sengupta et al, 2012(Sengupta et al, , 2015, measure the depth of insertion of membrane proteins in lipid bilayers (Buffy et al, 2003;Su et al, 2012;Marbella et al, 2013;Maltsev et al, 2014), and study metal ion complexation in bacterial CWs (Kern et al, 2010).…”

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confidence: 93%

The Target of β-Expansin EXPB1 in Maize Cell Walls from Binding and Solid-State NMR Studies

et al. 2016

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The wall-loosening actions of b-expansins are known primarily from studies of EXPB1 extracted from maize (Zea mays) pollen. EXPB1 selectively loosens cell walls (CWs) of grasses, but its specific binding target is unknown. We characterized EXPB1 binding to sequentially extracted maize CWs, finding that the protein primarily binds glucuronoarabinoxylan (GAX), the major matrix polysaccharide in grass CWs. This binding is strongly reduced by salts, indicating that it is predominantly electrostatic in nature. For direct molecular evidence of EXPB1 binding, we conducted solid-state nuclear magnetic resonance experiments using paramagnetic relaxation enhancement (PRE), which is sensitive to distances between unpaired electrons and nuclei. By mixing 13 C-enriched maize CWs with EXPB1 functionalized with a Mn 2+ tag, we measured Mn 2+ -induced PRE. Strong 1 H and 13 C PREs were observed for the carboxyls of GAX, followed by more moderate PREs for carboxyl groups in homogalacturonan and rhamnogalacturonan-I, indicating that EXPB1 preferentially binds GAX. In contrast, no PRE was observed for cellulose, indicating very weak interaction of EXPB1 with cellulose. Dynamics experiments show that EXPB1 changes GAX mobility in a complex manner: the rigid fraction of GAX became more rigid upon EXPB1 binding while the dynamic fraction became more mobile. Combining these data with previous results, we propose that EXPB1 loosens grass CWs by disrupting noncovalent junctions between highly substituted GAX and GAX of low substitution, which binds cellulose. This study provides molecular evidence of b-expansin's target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously.

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“…While enhanced relaxation caused by a paramagnetic center can be exploited for fast recycling and condensed data collection approaches relying on 13 C detection (23)(24)(25), the addition of paramagnetic dopants is not appropriate for the quantitative measurement of relaxation times as a source of structural and dynamic information. As a result, site-specific PREs in the solid state have only been reported on one small model protein (GB1) by the use of cysteine-containing mutants with paramagnetic tags attached (26,27). …”

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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.

179

244

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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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Paramagnetic Ions Enable Tuning of Nuclear Relaxation Rates and Provide Long-Range Structural Restraints in Solid-State NMR of Proteins

Cited by 123 publications

References 121 publications

Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

The Target of β-Expansin EXPB1 in Maize Cell Walls from Binding and Solid-State NMR Studies

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

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