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 relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methylâ??methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.
Content Type Journal Article
Category Article
Pages 1-10
DOI 10.1007/s10858-011-9559-4
Authors
Vincenzo Venditti, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA
Nicolas L. Fawzi, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA
G. Marius Clore, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA
Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy
Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy
Abstract Relaxation violated coherence transfer NMR spectroscopy (Tugarinov et al. in J Am Chem Soc 129:1743â??1750, 2007) is an established experimental tool for quantitative estimation of the amplitudes of side-chain motions in methyl-protonated, highly deuterated proteins. Relaxation violated coherence transfer experiments monitor the build-up of methyl proton multiple-quantum coherences that can be created in magnetically equivalent...
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02-11-2012 10:31 AM
Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins
Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins
Abstract We report enhanced sensitivity NMR measurements of intrinsically disordered proteins in the presence of paramagnetic relaxation enhancement (PRE) agents such as Ni2+-chelated DO2A. In proton-detected 1H-15N SOFAST-HMQC and carbon-detected (H-flip)13CO-15N experiments, faster longitudinal relaxation enables the usage of even shorter interscan delays. This results in higher NMR signal intensities per units of experimental time, without adverse line...
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10-21-2011 10:04 PM
A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins
A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins
Abstract A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-glucose and subsaturating amounts of 2-methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional 1H-13C correlation spectra. Protein samples are conveniently prepared using the same media composition as the...
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02-06-2011 07:42 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
[NMR paper] NMR resonance assignment of selectively labeled proteins by the use of paramagnetic l
NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands.
Related Articles NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands.
J Biomol NMR. 2004 Oct;30(2):205-10
Authors: Cutting B, Strauss A, Fendrich G, Manley PW, Jahnke W
Selective isotopic labeling of larger proteins greatly simplifies protein NMR spectra and reduces signal overlap, but selectively labeled proteins cannot be easily assigned since the sequential assignment method is not applicable. Here we describe...
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11-24-2010 10:01 PM
[NMR paper] Cross-correlated relaxation enhanced 1H[bond]13C NMR spectroscopy of methyl groups in
Cross-correlated relaxation enhanced 1H13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes.
Related Articles Cross-correlated relaxation enhanced 1H13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes.
J Am Chem Soc. 2003 Aug 27;125(34):10420-8
Authors: Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE
A comparison of HSQC and HMQC pulse schemes for recording (1)H(13)C correlation maps of protonated methyl groups in highly deuterated proteins is presented....
Automated sequence-specific protein NMR assignment using the memetic algorithm MATCH
Automated sequence-specific protein NMR assignment using the memetic algorithm MATCH
Jochen Volk, Torsten Herrmann and Kurt Wüthrich
Journal of Biomolecular NMR; 2008; 41(3); pp 127 - 138
Abstract:
MATCH (Memetic Algorithm and Combinatorial Optimization Heuristics) is a new memetic algorithm for automated sequence-specific polypeptide backbone NMR assignment of proteins. MATCH employs local optimization for tracing partial sequence-specific assignments within a global, population-based search environment, where the simultaneous application of local and global optimization heuristics...
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
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