A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme
A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme
Available online 4 January 2013
Publication year: 2013 Source:Journal of Magnetic Resonance
The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-13C]Glucose ([2-13C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-13C]pyruvate produced mainly by glycolysis and [3-13C]/[1,3-13C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs of 13C?-13C?2 and 13C?-13C?1 in Val, and 13C?-13C?2 and 13C?-13C?1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin. Graphical abstract
Highlights
? Stereospecific assignments can improve the accuracy and precision of NMR structures. ? New strategy for solid-state NMR stereospecific resonance assignment of Val and Leu. ? Based on protein production in Escherichia coli media containing [2-13C]Glucose. ? Spectral simplification in the often congested methyl region. ? Applications to PrgI needles and ubiquitin are shown.
Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR.
Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR.
Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR.
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We describe a simple yet highly effective optimization strategy for SPINAL-64 (1)H decoupling conditions for magic-angle spinning solid-state NMR. With...
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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
Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR
Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR
Publication year: 2010
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 31 December 2010</br>
Gemma, Comellas , Jakob J., Lopez , Andrew J., Nieuwkoop , Luisel R., Lemkau , Chad M., Rienstra</br>
We describe a simple yet highly effective optimization strategy for SPINAL-64 1H decoupling conditions for magic-angle spinning solid-state NMR. With adjustment of the phase angles in a coupled manner,...
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01-01-2011 08:57 AM
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
J Biomol NMR. 2010 Dec 18;
Authors: Hefke F, Bagaria A, Reckel S, Ullrich SJ, Dötsch V, Glaubitz C, Güntert P
We present a computational method for finding optimal labeling patterns for the backbone...
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12-21-2010 01:00 PM
Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm
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Abstract We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273â??6279 (1982)), types of amino acids are labeled with 13C or/and 15N such that cross peaks between 13CO(i â?? 1) and 15NH(i) result only for pairs...
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[NMR paper] A combinatorial selective labeling method for the assignment of backbone amide NMR re
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A combinatorial selective labeling (CSL) method is presented for the assignment of backbone amide NMR resonances, which has a particular application in the identification of protein-ligand interaction sites. The method builds on the dual...
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A general method is described for the...
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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...
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A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme
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