Synthesis of a <sup>13</sup>C‐Methyl‐Group‐Labeled Methionine Precursor as a Useful Tool for Simplifying Protein Structural Analysis by NMR Spectroscopy

Fischer, Michael B.; Kloiber, Karin; Häusler, Johannes; Ledolter, Karin; Konrat, Robert; Schmid, Walther

doi:10.1002/cbic.200600551

Cited by 70 publications

(34 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Initial strategies focused on methyls of Ile (d1), as well as Leu/Val, where in the latter case only one of the two prochiral methyl groups is 13 CH 3 (the other is 12 CD 3 ), using commercially available precursors [25]. More recently, biosynthetic strategies for producing proteins with 13 CH 3 labeling at Ala [26,27], Met [28], Ile (C c2 ) [29] as well as stereospecific incorporation of methyl label into Leu, Val proR or proS positions [30] have emerged. The reason why methyl groups are so useful as probes in supra-molecules can be understood by considering the energy level diagram for an isolated 13 CH 3 spin system, Fig.…”

Section: A Simple Strategy For Preserving Magnetization Emerges From mentioning

confidence: 99%

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Kay

2011

Journal of Magnetic Resonance

View full text Add to dashboard Cite

Section: A Simple Strategy For Preserving Magnetization Emerges From mentioning

confidence: 99%

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Kay

2011

Journal of Magnetic Resonance

View full text Add to dashboard Cite

“…53 Methods for efficient 13 C 1 H 3 labeling of Ala, Met, and/or Thr methyls have also been developed, which yield additional NOE data and more NMR probes. 35,36,54–56 …”

Section: Discussionmentioning

confidence: 99%

Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2

et al. 2015

View full text Add to dashboard Cite

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly 13C/15N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for 13C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of 13C1H3-labeled methyls by comparing distance information obtained from three-dimensional methyl–methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl–methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed “NOE-methyl cluster” onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV 13C1H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.

show abstract

“…Thus, the mixing time in the NOESY experiment should be optimized to provide such information. In addition to Leu, Ile(δ1), and Val, new biosynthetic strategies have been introduced for producing proteins with 13 CH 3 labeling at Ala, Met, Ile(γ2), and Thr [17–19]. The combination of these labeling schemes will provide more dense clusters of methyl-methyl NOE network, which should dramatically increase the performance of our approach.…”

Section: Discussionmentioning

confidence: 99%

FLAMEnGO: A fuzzy logic approach for methyl group assignment using NOESY and paramagnetic relaxation enhancement data

Chao¹,

Shi²,

Masterson³

et al. 2012

Journal of Magnetic Resonance

View full text Add to dashboard Cite

Building on a recent method by Matthews and coworkers [1], we developed a new and efficient algorithm to assign methyl resonances from sparse and ambiguous NMR data. The new algorithm (FLAMEnGO: Fuzzy Logic Assignment of MEthyl GrOups) uses Monte Carlo sampling in conjunction with fuzzy logic to obtain the assignment of methyl resonance at high fidelity. Furthermore, we demonstrate that the inclusion of paramagnetic relaxation enhancement (PRE) data in the assignment strategy increases the percentage of correct assignments with sparse NOE data. Using synthetic tests and experimental data we show that this new approach provides up to ~80% correct assignments with only 30% of methyl-methyl NOE data. In the experimental case of ubiquitin, PRE data from two spin labeled sites improve the percentage of assigned methyl groups up to ~91%. This new strategy promises to further expand methyl group NMR spectroscopy to very large macromolecular systems.

show abstract

Synthesis of a ¹³C‐Methyl‐Group‐Labeled Methionine Precursor as a Useful Tool for Simplifying Protein Structural Analysis by NMR Spectroscopy

Cited by 70 publications

References 34 publications

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2

FLAMEnGO: A fuzzy logic approach for methyl group assignment using NOESY and paramagnetic relaxation enhancement data

Contact Info

Product

Resources

About

Synthesis of a 13C‐Methyl‐Group‐Labeled Methionine Precursor as a Useful Tool for Simplifying Protein Structural Analysis by NMR Spectroscopy

Cited by 70 publications

References 34 publications

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view

Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2

FLAMEnGO: A fuzzy logic approach for methyl group assignment using NOESY and paramagnetic relaxation enhancement data

Contact Info

Product

Resources

About

Synthesis of a ¹³C‐Methyl‐Group‐Labeled Methionine Precursor as a Useful Tool for Simplifying Protein Structural Analysis by NMR Spectroscopy