Two-dimensional NMR methods for determining (chi 1) angles of aromatic residues in proteins from three-bond J(C'C gamma) and J(NC gamma) couplings
Two-dimensional NMR methods for determining (chi 1) angles of aromatic residues in proteins from three-bond J(C'C gamma) and J(NC gamma) couplings
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DOI：
10.1021/ja963625z
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发表时间：
1997-02-19
影响因子：
15
通讯作者：
Bax, A
中科院分区：
文献类型：
--
作者：
Hu, JS;Grzesiek, S;Bax, A
蛋白质中的芳香残基通常在NMR结构测定过程中非常重要，因为它们增加1H共振色散，并且它们提供大量的长程NOE约束。芳香族基团相对于多肽主链的取向由扭转角1和2限定。2角通常等于+90或-90，其中符号对于Phe和Tyr残基无关紧要。1最常见于-60或180旋转异构态。1虽然原则上1可以从3 JHRH偶联和残基内和连续NOE中获得，2对于缓慢翻滚的蛋白质，这些参数的定量测量往往是困难的。在这里，我们证明，在13 C/15 N富集的蛋白质的1角可以很容易地确定从两个简单的定量J相关实验，产生残基内3 JC ′ Cγ和3 JNC γ耦合常数。蛋白质中3 JCC和3 JNC偶联的测量在很大程度上限于涉及甲基基团的偶联3 -6，由于其有利的弛豫特性和3倍简并质子共振，提供了卓越的分辨率和灵敏度。在这里，我们利用主链15 N和13 C ′的长横向弛豫时间来测量芳香残基的侧链Cγ共振的3 J耦合。这些13 C γ共振落在一个相对较窄的区域内，范围从Trp的~ 110 ppm到Phe的~ 140 ppm，这允许使用两个简单的2D自旋回波差实验测量这些残基的3 JC ′ Cγ和3 JNC γ。用于测量3 JC ′ Cγ和3 JNC γ的脉冲方案如图1所示。在图1A的脉冲方案中，HN磁化被转移到其15 N，并且在随后的半恒定时间演化周期之后，它被转换成先前羰基的反相C′ yNz磁化。在随后的自旋回波延迟2δ的中点，一个选择性的180 C′脉冲使JC′ N、JC′ C和JC′ H耦合的效应重新相位化（除了羰基-羰基/羧基耦合，它会使C′ yNz衰减）。如果在自旋回波延迟结束时（位置a）施加13 Carom选择性180脉冲，芳香残基中JC′ Cγ偶联的效应也将重新聚焦。然而，当在位置B处施加13 Carom 180脉冲时，JC′ Cγ失相在整个周期2δ内是有效的。因此，在后一种情况下，在2δ周期结束时的C′ yNz磁化强度，以及由此在2D 15 N-1HN相关谱中的强度，被cos（2πJC′ Cγδ）衰减。由于δ是已知的，JC′ Cγ可以由JC′ Cγ）cos-1（Ib/Ia）/2πδ计算，其中Ia和
Aromatic residues in proteins typically are very important in the NMR structure determination process because they increase 1H resonance dispersion and they provide large numbers of longrange NOE constraints. The orientation of an aromatic group relative to the polypeptide backbone is defined by the torsion angles 1 and 2. The 2 angle usually equals+ 90 or-90, where the sign is of no consequence for Phe and Tyr residues. 1 is most commonly found in either-60 or 180 rotameric states. 1 Although, in principle, 1 can be obtained from 3JHRH couplings and intraresidue and sequential NOEs, 2 for slowly tumbling proteins, quantitative measurement of these parameters tends to be difficult. Here, we demonstrate that in 13C/15N-enriched proteins the 1 angle can readily be determined from two simple quantitative J correlation experiments which yield the intraresidue 3JC′ Cγ and 3JNCγ coupling constants. Measurement of 3JCC and 3JNC couplings in proteins has largely been restricted to couplings involving methyl groups3-6 which, as a result of their favorable relaxation properties and 3-fold degenerate proton resonance, offer exceptional resolution and sensitivity. Here, we exploit the long transverse relaxation times of the backbone 15N and 13C′ to measure 3J couplings to side-chain Cγ resonances of aromatic residues. These 13Cγ resonances fall in a relatively narrow region, ranging from∼ 110 ppm for Trp to∼ 140 ppm for Phe, which permits 3JC′ Cγ and 3JNCγ for these residues to be measured using two simple 2D spin-echo difference experiments. The pulse schemes used for measurement of 3JC′ Cγ and 3JNCγ are shown in Figure 1. In the pulse scheme of Figure 1A, HN magnetization is transferred to its 15N, and after a subsequent semi-constant-time evolution period, 7 it is converted into antiphase C′ yNz magnetization of the preceding carbonyl. At the midpoint of the subsequent spin-echo delay, 2δ, a selective 180 C′ pulse rephases the effect of JC′ N, JC′ C, and JC′ H couplings (except for carbonyl-carbonyl/carboxyl couplings, which will attenuate C′ yNz). If the 13Carom selective 180 pulse is applied at the end of the spin-echo delay (position a), the effect of JC′ Cγ couplings in aromatic residues will also refocus. However, when the 13Carom 180 pulse is applied at position b, JC′ Cγ dephasing is active for the full period 2δ. Therefore, in this latter case, the C′ yNz magnetization at the end of the 2δ period, and thereby the intensity in the 2D 15N-1HN correlation spectrum, is attenuated by cos (2πJC′ Cγδ). As δ is known, JC′ Cγ can be calculated from JC′ Cγ) cos-1 (Ib/Ia)/2πδ, where Ia and