Production and incorporation of N-15, C-13, H-2 (H-1-delta 1 methyl) isoleucine into proteins for multidimensional NMR studies
Production and incorporation of N-15, C-13, H-2 (H-1-delta 1 methyl) isoleucine into proteins for multidimensional NMR studies
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DOI：
10.1021/ja9706514
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发表时间：
1997-08-13
影响因子：
15
通讯作者：
Kay, LE
中科院分区：
文献类型：
--
作者：
Gardner, KH;Kay, LE
用NMR活性同位素标记的大分子的生产极大地增加了适用于多维溶液NMR研究的系统的范围。均匀的15N，13C标记促进了三重共振（15N，13C，1H）实验的发展，使详细的结构和动力学研究系统高达约20 kDa的分子量。较大大分子的1NMR谱通常由于交叉峰数目的增加和线宽的伴随增长而变得复杂，这降低了信噪比和分辨率。这些问题可以显着减少氘化脂肪族碳网站，提高灵敏度和分辨率的许多实验，依赖于磁化转移通过13 C核。2-7不幸的是，氘化也去除了许多质子，这些质子是基于核奥弗豪泽效应（NOE）的距离限制的来源，而距离限制对于确定高分辨率结构至关重要。考虑到这一点，我们已经开发了一种策略，其中蛋白质在2H2O中以（13 C，1H）-丙酮酸盐作为唯一碳源生长的细菌中过表达。[8]这些蛋白质在几乎所有的脂肪族位置上都是高度氘化的，同时在四个氨基酸中保留了40 - 80%的甲基质子：Ala、瓦尔、Leu和Ile（仅γ 2）。骨架和甲基核的化学位移分配使用最近开发的脉冲方案，3，4，9，10，而甲基甲基，甲基-NH，NH-NH距离的限制，建立了一系列的4D NOE实验。相对于仅使用骨架酰胺质子之间的NOE产生的那些结构，来自这些基于甲基的NOE的消除距离限制提高了结构的精确度和准确度。15 - 17然而，几种氨基酸的甲基没有使用这种基于琥珀酸盐的策略质子化，8包括异亮氨酸δ 1甲基。在Ile δ 1甲基位置的质子化是期望的，因为即使在大于40 kDa的蛋白质中，在13 C-1H位移相关光谱中，来自这些甲基的交叉峰通常比来自其他甲基类型的信号显著更好地分辨。这在41.0 kDa麦芽糖结合蛋白（MBP）-麦芽糖复合物的13 C-1H相关光谱中得到了说明（图1）。此外，与缬氨酸γ和亮氨酸δ甲基相反，单独的异亮氨酸δ 1甲基不必立体特异性地指定。从结构的角度来看，异亮氨酸是一个重要的残基，因为它富集在蛋白质的疏水核心。18异亮氨酸δ 1 Me基团位于距离蛋白质骨架相当远的位置（约3.7 nm），便于观察Ile δ 1甲基质子与一级序列中较远的氨基酸侧链之间的NOE。在对超过290个非同源蛋白质结构的分析中，我们发现平均有5.1（1.9）个骨架酰胺质子，总共有2.8（1.5）个Ile δ 1、瓦尔γ和Leu δ Me基团，它们与给定的Ile δ 1 Me基团相距6.0 μ m。甲基-甲基NOE对于结构测定特别重要，因为它们涉及具有30个氨基酸的中值分离的残基，而酰胺-酰胺和酰胺-甲基NOE分别为2个和3个。（15 N，13 C）标记的高度氘代δ 1甲基质子化异亮氨酸遵循大肠杆菌的生物合成路线，从15 N，13 C，1H苏氨酸（方案1）。异亮氨酸是由原养型E.大肠杆菌在2H2O为基础的基本培养基与15 NH4Cl和50 mg/L的（3，3 - 2H2）13 C 2-酮丁酸，如步骤...
The production of macromolecules labeled with NMR-active isotopes has greatly increased the range of systems that are amenable to multidimensional solution NMR studies. Uniform 15N, 13C labeling has facilitated the development of triple resonance (15N, 13C, 1H) experiments, making possible detailed structural and dynamics studies of systems up to approximately 20 kDa molecular mass. 1 NMR spectra of larger macromolecules are often complicated by both increasing numbers of cross-peaks and a concomitant growth in line widths that decreases signal-to-noise and resolution. These problems can be significantly reduced by deuterating aliphatic carbon sites, improving the sensitivity and resolution of many experiments that rely on magnetization transfer through 13C nuclei. 2-7 Unfortunately, deuteration also removes many of the protons that are the source of nuclear Overhauser effect (NOE)-based distance restraints that are essential to determining highresolution structures. With this in mind, we have developed a strategy whereby proteins are overexpressed in bacteria grown in 2H2O with (13C, 1H)-pyruvate as the sole carbon source. 8 Such proteins are highly deuterated in virtually all aliphatic positions while retaining between 40-80% of the methyl protons in four amino acids: Ala, Val, Leu, and Ile (γ2 only). Chemical shift assignments of backbone and methyl group nuclei are obtained using recently developed pulse schemes, 3, 4, 9, 10 while methylmethyl, methyl-NH, and NH-NH distance restraints are established from a series of 4D NOE experiments. 11-14 Incorporating distance restraints from these methyl-based NOEs improves the precision and accuracy of structures relative to those generated using NOEs solely between backbone amide protons. 15-17 However, the methyl groups of several amino acids are not protonated using this pyruvate-based strategy, 8 including the isoleucine δ1 methyl group. Protonation at the Ile δ1 methyl position is desirable since cross-peaks from these methyl groups are often significantly better resolved than signals from other methyl types in 13C-1H shift correlation spectra, even in proteins larger than 40 kDa. This is illustrated in a 13C-1H correlation spectrum of a 41.0 kDa maltose binding protein (MBP)-maltose complex (Figure 1). Additionally, in contrast to valine γ and leucine δ methyl groups, the lone isoleucine δ1 methyl does not have to be stereospecifically assigned. From a structural perspective, isoleucine is an important residue because of its enrichment in protein hydrophobic cores. 18 The location of the isoleucine δ1 Me group at a substantial distance (approximately 3.7 Å) from the protein backbone facilitates the observation of NOEs between Ile δ1 methyl protons and amino acid side chains that are distant in the primary sequence. In an analysis of over 290 nonhomologous protein structures solved to better than 2.5 Å resolution, 19 we have found an average of 5.1 (1.9 backbone amide protons and a total of 2.8 (1.5 Ile δ1, Val γ, and Leu δ Me groups within 6.0 Å of a given Ile δ1 Me group. Methyl-methyl NOEs are particularly important for structure determination as they involve residues with a median separation of 30 amino acids, as opposed to two and three for amide-amide and amide-methyl NOEs, respectively.Our approach to generate uniformly (15N, 13C)-labeled, highly deuterated, δ1 methyl-protonated isoleucine follows the biosynthetic route of Escherichia coli starting with 15N, 13C, 1H threonine (Scheme 1). Isoleucine is produced by growing prototrophic E. coli in a 2H2O-based minimal medium with 15NH4Cl and 50 mg/L of (3, 3-2H2) 13C 2-ketobutyrate as generated in steps …