Title: Ile, Leu, and Val Methyl Assignments of the 723-Residue Malate Synthase G Using a New Labeling Strategy and Novel NMR Methods
Abstract: New NMR experiments are presented for the assignment of methyl 13C and 1H chemical shifts from Ile, Leu, and Val residues in high molecular weight proteins. The first class of pulse schemes transfers magnetization from the methyl group to the backbone amide spins for detection, while the second more sensitive class uses an "out-and-back" transfer scheme in which side-chain carbons or backbone carbonyls are correlated with methyl 13C and 1H spins. Both groups of experiments benefit from a new isotopic labeling scheme for protonation of Leu and Val methyl groups in large deuterated proteins. The approach makes use of α-ketoisovalerate that is 13C-labeled and protonated in one of its methyl groups (13CH3), while the other methyl is 12CD3. The use of this biosynthetic precursor leads to production of Leu and Val residues that are 13CH3-labeled at only a single methyl position. Although this labeling pattern effectively reduces by 2-fold the concentration of Leu and Val methyls in NMR samples, it ensures linearity of Val and Leu side-chain 13C spin-systems, leading to higher sensitivity and, for certain classes of experiments, substantial simplification of NMR spectra. Very near complete assignments of the 276 Ile (δ1 only), Leu, and Val methyl groups in the single-chain 723-residue enzyme malate synthase G (MSG, molecular tumbling time 37 ± 2 ns at 37 °C) have been obtained using the proposed isotopic labeling strategy in combination with the new NMR experiments.
Publication Year: 2003
Publication Date: 2003-10-16
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 346
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