Publication year: 2012 Source:Journal of Magnetic Resonance
Jie Wen, Jihui Wu, Pei Zhou
The methyl-methyl NOESYexperimentplays an important role in determiningthe global folds of large proteins. Despite the high sensitivity of this experiment, the analysisof methyl-methyl NOEs is frequently hindered by the limited chemical shift dispersion of methyl groups, particularly methyl protons. Thismakes it difficult to unambiguously assign all of the methyl-methyl NOE crosspeaksusing 3-D spectroscopy.The recent development ofsparse sampling methodsenables highly efficient acquisitionof high-resolution 4-D spectra, which provides an excellent solution to resolving the degeneracy of methyl signals.However, many reconstruction algorithms for processing sparsely-sampled NMR data do not provide adequate suppression of aliasing artifacts in the presence of strongNOE diagonal signals. In order to overcome this limitation, we presenta 4-D diagonal-suppressed methyl-methyl NOESY experimentspecifically optimized for ultrasparse sampling and evaluateitusingadeuterated, ILV methyl-protonatedsample of the 42kDaEscherichia coli maltose binding protein (MBP). Suppression of diagonal signals removes the dynamic range barrier of the methyl-methyl NOESY experimentsuch thatresidual aliasing artifacts in the CLEAN-reconstructed high-resolution 4-D spectrum can be further reduced. At an ultrasparse sampling rate of less than 1%, we were able to identify and unambiguouslyassign the vast majority of expected NOE crosspeaks between methyl groups separated by less than 5 Å and to detect very weak NOE crosspeaksfrom methyl groups that areover 7Å apart. Graphical Abstract
Graphical abstract Highlights
? Diagonal suppression reduces the dynamic range of signals in the 4-D CH3-CH3 NOESY experiment. ? Diagonal suppression reduces aliasing artifacts left over by CLEAN for ultrasparse sampling. ? Diagonalsuppressionenables detection of the majority of NOEsfor methyl groups within 5 Å of MBP. ? Diagonal suppression enables detection of NOE crosspeaks for methyl groups over 7 Å apart in MBP.
Simultaneous measurement of 1Hâ??15N and Methyl 1Hmâ??13Cm residual dipolar couplings in large proteins
Simultaneous measurement of 1Hâ??15N and Methyl 1Hmâ??13Cm residual dipolar couplings in large proteins
Abstract A two-dimensional TROSY-based SIM-13Cmâ??1Hm/1Hâ??15N NMR experiment for simultaneous measurements of methyl 1 D CH and backbone amide 1 D NH residual dipolar couplings (RDC) in {U-; Ileδ1-; Leu,Val-}-labeled samples of large proteins is described. Significant variation in the alignment tensor of the 82-kDa enzyme Malate synthase G is observed as a function of only slight changes in experimental conditions. The SIM-13Cmâ??1Hm/1Hâ??15N data sets provide convenient means...
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Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Abstract Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic...
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09-26-2011 06:42 AM
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
Chem Commun (Camb). 2011 Jul 26;
Authors: Ayala I, Hamelin O, Amero C, Pessey O, Plevin MJ, Gans P, Boisbouvier J
An efficient synthetic route is proposed to produce 2-hydroxy-2-ethyl-3-oxobutanoate for the specific labelling of Ile methyl-?(2) groups in proteins. The (2)H,...
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07-28-2011 10:51 AM
Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Abstract Methyl 13CHD2 isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from -glucose/D2O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Alaβ, Thrγ2) is possible using simple â??out-and-backâ?? NMR methodology. Such selective methyl-detected â??out-and-backâ?? NMR experiments allow complete assignments of threonine γ2...
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01-09-2011 12:46 PM
Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in 15N-13C-ILV methyl labeled proteins
Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in 15N-13C-ILV methyl labeled proteins
Abstract We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record 13C- and 15N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that...
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01-09-2011 12:46 PM
High-resolution methyl edited GFT NMR experiments for protein resonance assignments a
High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination
Abstract Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile, Leu, Met, Thr and Val) there is a significant overlap of 13C and 1H chemical shifts. Such overlap can be resolved using the recently proposed (3,2)D HCCH-COSY, a G-matrix Fourier transform (GFT) NMR based experiment, which facilitates editing...
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09-18-2010 04:53 AM
High-resolution methyl edited GFT NMR experiments for protein resonance assignments a
High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination.
Related Articles High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination.
J Biomol NMR. 2010 Sep 14;
Authors: Jaipuria G, Thakur A, D'Silva P, Atreya HS
Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile,...
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09-15-2010 02:26 PM
High Resolution 1H Detected 1H,13C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1H,2H Isotopic Labeling of Methyl Groups
High Resolution <SUP>1</SUP>H Detected <SUP>1</SUP>H,<SUP>13</SUP>C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective <SUP>1</SUP>H,<SUP>2</SUP>H Isotopic Labeling of Methyl Groups
Vipin Agarwal, Anne Diehl, Nikolai Skrynnikov, and Bernd Reif
J. Am. Chem. Soc.; 2006; 128(39) pp 12620 - 12621;
Abstract:
MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme...
Efficient Acquisition of High-Resolution 4-D Diagonal-Suppressed Methyl-Methyl NOESY for Large Proteins
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