期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 125, 期 45, 页码 13868-13878出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja030345s
关键词
-
New NMR experiments are presented for the assignment of methyl C-13 and H-1 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 C-13 and H-1 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 alpha-ketoisovalerate that is C-13-Iabeled and protonated in one of its methyl groups ((CH3)-C-13), while the other methyl is (CD3)-C-12. The use of this biosynthetic precursor leads to production of Leu and Val residues that are (CH3)-C-13-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 C-13 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 (delta1 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 degreesC) have been obtained using the proposed isotopic labeling strategy in combination with the new NMR experiments.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据