Tugarinov Vitali, Kay Lewis E
Contribution from the Protein Engineering Network Centres of Excellence and the Departments of Medical Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
J Am Chem Soc. 2003 Nov 12;125(45):13868-78. doi: 10.1021/ja030345s.
New NMR experiments are presented for the assignment of methyl (13)C and (1)H 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 (13)C and (1)H 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 (13)C-labeled and protonated in one of its methyl groups ((13)CH(3)), while the other methyl is (12)CD(3). The use of this biosynthetic precursor leads to production of Leu and Val residues that are (13)CH(3)-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 (13)C 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 (delta 1 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 degrees C) have been obtained using the proposed isotopic labeling strategy in combination with the new NMR experiments.
本文介绍了用于确定高分子量蛋白质中异亮氨酸（Ile）、亮氨酸（Leu）和缬氨酸（Val）残基甲基（¹³C）和氢（¹H）化学位移的新核磁共振（NMR）实验。第一类脉冲序列将甲基的磁化转移至主链酰胺自旋以进行检测，而第二类更灵敏的脉冲序列采用“往返”转移方案，其中侧链碳或主链羰基与甲基¹³C和¹H自旋相关联。这两类实验都受益于一种新的同位素标记方案，用于在大型氘代蛋白质中使Leu和Val甲基质子化。该方法利用α-酮异戊酸，其一个甲基被¹³C标记并质子化（¹³CH₃），而另一个甲基为¹²CD₃。使用这种生物合成前体可产生仅在单个甲基位置被¹³CH₃标记的Leu和Val残基。尽管这种标记模式有效地将NMR样品中Leu和Val甲基的浓度降低了2倍，但它确保了Val和Leu侧链¹³C自旋系统的线性，从而提高了灵敏度，并且对于某些类型的实验，大大简化了NMR谱图。使用所提出的同位素标记策略结合新的NMR实验，已几乎完全确定了单链723个残基的苹果酸合酶G（MSG，37℃时分子翻滚时间为37±2纳秒）中276个Ile（仅δ1）、Leu和Val甲基的化学位移。
