Nanomole-scale protein solid-state NMR by breaking intrinsic 1H T1 boundaries

Wickramasinghe, Nalinda P.; Parthasarathy, Sudhakar; Jones, Christopher R.; Bhardwaj, Chhavi; Long, Fei; Kotecha, Mrignayani; Mehboob, Shahila; Fung, Leslie W.‐M.; Past, Jaan; Samoson, Ago; Ishii, Yoshitaka

doi:10.1038/nmeth.1300

Cited by 191 publications

(246 citation statements)

References 16 publications

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“…To exploit PRE (Paramagnetic Relaxation Enhancement), Cu(edta) was added to the monomeric Ab 1-40 peptide, prior to fibrilization, at a concentration of 75 mM. 40,41 Growth and quality of the fibrils were monitored using EM. For each sample, typically 10 mg of Ab 1-40 fibrils were packed into a 3.2 mm MAS solid-state NMR rotor.…”

Section: Methodsmentioning

confidence: 99%

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

Agarwal

Linser

Dasari

et al. 2013

Phys. Chem. Chem. Phys.

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The amyloid b-peptide (Ab) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Ab peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.Alzheimer's disease (AD) is a slowly progressive neurological disorder which is associated with memory loss, cognitive impairment and finally dementia and death. The amyloid b-peptide (Ab) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Ab is a cleavage product resulting from Alzheimer Precursor Protein (APP) processing. 1 The g-secretase cut yields Ab fragments of different lengths of which Ab40 and Ab42 are most abundant. 2,3 In vitro, the Ab peptide aggregates over time into oligomers and fibrillar structures. 4 In the past, several Ab fibril structure models have been suggested. These are based on MAS solidstate NMR experiments, 5-11 and cryo-electron microscopic image reconstructions. 12 Even though there is some controversy concerning the conformational space that Ab fibrils can adopt, all published models, including the 2-fold 7,10 and 3-fold 9 symmetric Ab 1-40 fibril structure suggested by Tycko, and Bertini and co-workers, agree on the basic building block which involves a b-sheet (b1, residues 12-24), a turn, and a second b-sheet (b2, residues 28-40). Biophysical studies indicate a certain degree of conformational plasticity for the N-terminal b-sheet. 13,14 Whereas b2 is present in all the studies, b1 can be truncated or does not adopt a regular b-sheet structure. Depending on the preparation conditions, amide protons in the region of the peptide where the first b-strand is expected show differential H/D protection factors. 13 Similarly, a certain degree of conformational variability in the N-terminus is suggested using cryo-electron microscopy. 14 In all structural models, Ab peptides are arranged in a parallel fashion. An antiparallel arrangement is only observed for the Iowa mutant Ab-D23N, 15 and for truncated forms of the peptide such as Ab [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . 16 The fibril structure is stabilized in particular by hydrogen bonding interactions. Therefore, observation of exchangeable and titratable groups is of particular interest to identify hydrogen bonding patterns. We and others could show in the past that high resolution proton spectra can be obtained for...

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Section: Methodsmentioning

confidence: 99%

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

Agarwal

Linser

Dasari

et al. 2013

Phys. Chem. Chem. Phys.

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“…However, measuring PREs in solids is a prime example where traditional detection methods have difficulty to provide sufficient sensitivity. 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.

175

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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“…MEIOSIS can also be applied with faster spinning speeds 29 and/or perdeuteration. 30 When applied in concert with other fast acquisition and sensitivity enhancement techniques (e.g., dynamic nuclear polarization, 31 paramagnetic relaxation enhancements, 32 and 1 H detection 33 ), this approach will dramatically increase the throughput of high-resolution structure determination by MAS ssNMR.…”

Section: Discussionmentioning

confidence: 99%

Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra

Gopinath

Veglia

2013

The Journal of Chemical Physics

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We propose a general method that enables the acquisition of multiple 2D and 3D solid-state NMR spectra for U-13 C, 15 N-labeled proteins. This method, called MEIOSIS (Multiple ExperIments via Orphan SpIn operatorS), makes it possible to detect four coherence transfer pathways simultaneously, utilizing orphan (i.e., neglected) spin operators of nuclear spin polarization generated during 15 N-13 C cross polarization (CP). In the MEIOSIS experiments, two phase-encoded free-induction decays are decoded into independent nuclear polarization pathways using Hadamard transformations. As a proof of principle, we show the acquisition of multiple 2D and 3D spectra of U-13 C, 15 N-labeled microcrystalline ubiquitin. Hadamard decoding of CP coherences into multiple independent spin operators is a new concept in solid-state NMR and is extendable to many other multidimensional experiments. The MEIOSIS method will increase the throughput of solid-state NMR techniques for microcrystalline proteins, membrane proteins, and protein fibrils. © 2013 AIP Publishing LLC.

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Nanomole-scale protein solid-state NMR by breaking intrinsic 1H T1 boundaries

Cited by 191 publications

References 16 publications

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

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

Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra

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