The Accuracy of Distance Measurements in Solid-State NMR

Hodgkinson, Paul; Emsley, Lyndon

doi:10.1006/jmre.1999.1759

Cited by 73 publications

(113 citation statements)

References 22 publications

Supporting

Mentioning

106

Contrasting

Order By: Relevance

“…2D PAIN-CP spectra (Figure 4a) can be acquired in a relatively short experimental time (1À2 days), leading to highly resolved 15 NÀ 13 C spectra. The low sensitivity of the TSAR mechanism to the dipolar truncation phenomenon 36 combined with the sparse labeling allows for efficient polarization transfer simultaneously to intraresidue and sequential 13 C atoms. The vast majority of the sequential contacts can already be observed at short mixing times (5 ms).…”

mentioning

confidence: 99%

¹³C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

et al. 2011

View full text Add to dashboard Cite

A strategy for simplified and complete resonance assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-13 C]-or [2-13 C]glucose are very sparsely labeled, and the resulting 2D ssNMR spectra exhibit smaller line widths (by a factor of ∼2 relative to uniformly labeled proteins) and contain a reduced number of crosspeaks. This allows for an accelerated and straightforward resonance assignment without the necessity of time-consuming 3D spectroscopy or sophisticated pulse sequences. The strategy aims at complete backbone and side-chain resonance assignments based on bidirectional sequential walks. The approach was successfully demonstrated with the de novo assignment of the Type Three Secretion System PrgI needle protein. Using a limited set of simple 2D experiments, we report a 97% complete resonance assignment of the backbone and side-chain 13 C atoms.R ecent developments in magic-angle-spinning (MAS) solidstate NMR (ssNMR) methodology, 1À4 isotope labeling schemes, 5,6 structure calculation protocols, 7,8 and access to highfield instrumentation have allowed details at atomic resolution and, in the most favorable cases, high-resolution structures of microcrystalline proteins, 6À11 fibrillar aggregates, 12À17 oligomeric complexes, 18À22 and membrane proteins in nativelike environments 23À26 to be obtained. However, achieving a sufficient (and if possible, nearly complete) assignment of the NMR signals still remains as a major obstacle in obtaining site-specific structural information. The lack of resolution and the spectral overlap observed in uniformly ([U-13 C]glucose) labeled proteins requires the use of several 2D experiments 27 and, in the case of remaining assignment ambiguities, 3D or 4D spectroscopy. This renders the assignment step extremely time-consuming and demanding in terms of instrument performance and access to high-field spectrometers. An alternative to uniform labeling is the use of 13 C alternate labeling schemes, 5,28,29 which reduce spectral crowding and facilitate the assignment and the collection of distance restraints, as previously demonstrated 5,6 with glycerolbased labeling schemes.We recently reported 30 the use of mixtures of [1-13 C]-5 and [2-13 C]glucose (Glc) 29 for ssNMR studies of supramolecular protein interfaces. The high spectral quality observed for [1-13 C]-and [2-13 C]Glc-labeled R-synuclein already suggested that this labeling scheme could be useful for resonance assignments. Here we demonstrate that [1-13

show abstract

mentioning

confidence: 99%

¹³C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

et al. 2011

View full text Add to dashboard Cite

show abstract

“…However, the deleterious effects of multiple-spin interactions interfere with the efficiency and analysis of zeroth-order broadband homonuclear dipolar recoupling schemes. 11,15,[49][50][51][52] In particular, the recoupling of a small dipolar coupling between a distant spin pair is severely attenuated in the presence of a third spin strongly coupled to one of the first two, an effect known as dipolar truncation. 52 As a result of dipolar truncation, the multiple-spin recoupled dipolar interactions are dominated by the strong dipolar coupling, prohibiting the measurement of the long-range distances that are important in constraining structures.…”

Section: Introductionmentioning

confidence: 99%

Dipolar truncation in magic-angle spinning NMR recoupling experiments

Bayro¹,

Huber²,

Ramachandran³

et al. 2009

The Journal of Chemical Physics

172

169

View full text Add to dashboard Cite

Quantitative solid-state NMR distance measurements in strongly coupled spin systems are often complicated due to the simultaneous presence of multiple noncommuting spin interactions. In the case of zeroth-order homonuclear dipolar recoupling experiments, the recoupled dipolar interaction between distant spins is attenuated by the presence of stronger couplings to nearby spins, an effect known as dipolar truncation. In this article, we quantitatively investigate the effect of dipolar truncation on the polarization-transfer efficiency of various homonuclear recoupling experiments with analytical theory, numerical simulations, and experiments. In particular, using selectively 13 C-labeled tripeptides, we compare the extent of dipolar truncation in model three-spin systems encountered in protein samples produced with uniform and alternating labeling. Our observations indicate that while the extent of dipolar truncation decreases in the absence of directly bonded nuclei, two-bond dipolar couplings can generate significant dipolar truncation of small, long-range couplings. Therefore, while alternating labeling alleviates the effects of dipolar truncation, and thus facilitates the application of recoupling experiments to large spin systems, it does not represent a complete solution to this outstanding problem.

show abstract

“…Distances between backbone and side chain carbons or between side chain and side chain carbons provide information about the tertiary structure. The detection of structure-defining long-range carbon-carbon restraints is only possible when so-called dipolar truncation effects are suppressed (13,14). This can be accomplished by employing a reduced labeling scheme, in which chemically bonded carbons are not simultaneously labeled and hence the number of strong dipolar couplings between connected nuclei is reduced.…”

mentioning

confidence: 99%

“…This methodology includes the measurement of multiple 13 C- 13 C distance restraints on biosynthetically site-directed 13 C-enriched samples, obtained by growing bacteria on [2-13 C]glycerol and [1,3-13 C]glycerol. 3D 15 N-13 C-13 C dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C R -C R , CO-CO, C R -CO, and CO-C R type appear.…”

mentioning

confidence: 99%

Determination of Solid-State NMR Structures of Proteins by Means of Three-Dimensional ¹⁵N−¹³C−¹³C Dipolar Correlation Spectroscopy and Chemical Shift Analysis

et al. 2003

View full text Add to dashboard Cite

In this paper, a three-dimensional (3D) NMR-based approach for the determination of the fold of moderately sized proteins by solid-state magic-angle spinning (MAS) NMR is presented and applied to the R-spectrin SH3 domain. This methodology includes the measurement of multiple 13 C- 13 C distance restraints on biosynthetically site-directed 13 C-enriched samples, obtained by growing bacteria on [2-13 C]glycerol and [1,3-13 C]glycerol. 3D 15 N-13 C-13 C dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C R -C R , CO-CO, C R -CO, and CO-C R type appear. Additional restraints for confining the structure were obtained from φ and ψ backbone torsion angles of 29 residues derived from C R , C , CO, NH, and H R chemical shifts. Using both distance and angular restraints, a refined structure was calculated with a backbone root-mean-square deviation of 0.7 Å with respect to the average structure.Many biological systems, such as membrane proteins and amyloid fibrils, remain a challenge in structural biology because of difficulties with crystallization and solubility. In the past years, solid-state NMR 1 has become a promising method for obtaining structural information about these systems, via the measurement of accurate distances (1-7), φ and backbone torsion angles (8-10), and chemical shift anisotropy (11,12). In these studies, samples labeled only in the positions of interest were investigated. For the determination of the complete protein folds, however, a different approach that allows the collection of a large number of structural restraints from a small number of samples has to be followed. The quality of the structures increases with the number of restraints, and the more that are measured, the lower the accuracy of the individual restraints may be. From a close analysis of the topology of helical and -sheet structures, it transpires that carboncarbon distances are very important in defining the fold of a protein. For example, distances between backbone carbons, i.e., R-carbons and carbonyl carbons, define the secondary structure of a protein (Table 1). Distances between backbone and side chain carbons or between side chain and side chain carbons provide information about the tertiary structure. The detection of structure-defining long-range carbon-carbon restraints is only possible when so-called dipolar truncation effects are suppressed (13,14). This can be accomplished by employing a reduced labeling scheme, in which chemically bonded carbons are not simultaneously labeled and hence the number of strong dipolar couplings between connected nuclei is reduced. For proteins expressed in bacterial systems, this can be achieved by using [2-13 C]glycerol or [1,3-13 C]glycerol as the only carbon source in the media (15)(16)(17). In combination with this labeling pattern, long-range 13 C-13 C distance restraints may be collected by using a broad-band recoupling method like the proton-driven spindiffusion (PDSD...

show abstract

The Accuracy of Distance Measurements in Solid-State NMR

Cited by 73 publications

References 22 publications

¹³C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

¹³C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

Dipolar truncation in magic-angle spinning NMR recoupling experiments

Determination of Solid-State NMR Structures of Proteins by Means of Three-Dimensional ¹⁵N−¹³C−¹³C Dipolar Correlation Spectroscopy and Chemical Shift Analysis

Contact Info

Product

Resources

About

The Accuracy of Distance Measurements in Solid-State NMR

Cited by 73 publications

References 22 publications

13C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

13C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

Dipolar truncation in magic-angle spinning NMR recoupling experiments

Determination of Solid-State NMR Structures of Proteins by Means of Three-Dimensional 15N−13C−13C Dipolar Correlation Spectroscopy and Chemical Shift Analysis

Contact Info

Product

Resources

About

¹³C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

¹³C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

Determination of Solid-State NMR Structures of Proteins by Means of Three-Dimensional ¹⁵N−¹³C−¹³C Dipolar Correlation Spectroscopy and Chemical Shift Analysis