Farmer B T, Venters R A
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.
J Biomol NMR. 1996 Jan;7(1):59-71. doi: 10.1007/BF00190457.
The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain 13C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D 15N/15N-separated NOESY, as many main-chain and side-chain 1HN/15N resonances as possible must be assigned. Traditionally, only backbone amide 1HN/15N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain 1HN/15N resonances to side-chain 13C resonances with high sensitivity: NH2-filtered 2D 1H-15N HSQC(H2N-HSQC), 3D H2N(CO)C gamma/beta and 3D H2N(COC gamma/beta)C beta/alpha for glutamine and asparagine side-chain amide groups; 2D refocused H(N epsilon/zeta)C delta/epsilon and H(N epsilon/zeta C delta/epsilon)C gamma/delta for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N epsilon)C zeta and nonrefocused H(N epsilon, eta)C zeta for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated 13C-/15N-labeled human carbonic anhydrase II (2H-HCA II). Because more than 95% of all side-chain 13C resonances in 2H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain 1HN/15N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain HN protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only 1HN-1HN NOE restraints. In these studies, the inclusion of NOE restraints to side-chain HN protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592-9593].
已证明，在大型蛋白质中对脂肪族位点进行全氘代可极大地促进主链和侧链13C顺序归属的过程，并且还可用于获取用于结构计算的远程NOE距离限制。为了从4D 15N/15N分离的NOESY中获取最大信息，必须尽可能多地归属主链和侧链1HN/15N共振。传统上，仅通过相关实验归属主链酰胺1HN/15N共振，而通过NOE与侧链脂肪族质子的相互作用归属缓慢交换的侧链酰胺、氨基和胍基质子。然而，在全氘代蛋白质中，这类质子的数量极少。因此，我们开发了几种包含水翻转脉冲的梯度增强和灵敏度增强脉冲序列，以高灵敏度提供脂肪族侧链1HN/15N共振与侧链13C共振的通过键相关：用于谷氨酰胺和天冬酰胺侧链酰胺基团的NH2过滤二维1H-15N HSQC（H2N-HSQC）、三维H2N(CO)Cγ/β和三维H2N(COCγ/β)Cβ/α；用于精氨酸侧链氨基的二维重聚焦H(Nε/ζ)Cδ/ε和H(Nε/ζCδ/ε)Cγ/δ以及用于赖氨酸侧链氨基的非重聚焦版本；用于精氨酸侧链胍基的二维重聚焦H(Nε)Cζ和非重聚焦H(Nε,η)Cζ。这些脉冲序列已应用于全氘代13C-/15N标记的人碳酸酐酶II（2H-HCA II）。由于使用C(CC)(CO)NH实验已归属了2H-HCA II中超过95%的所有侧链13C共振，因此使用上述脉冲序列直接归属侧链1HN/15N共振变得很容易。最近的研究证明了归属这些侧链HN质子的重要性，在这些研究中，仅使用1HN-1HN NOE限制模拟蛋白质全局折叠的计算。在这些研究中，包含侧链HN质子的NOE限制显著提高了可确定的全氘代蛋白质全局折叠的质量[R.A. Venters等人（1995年）《美国化学会志》，117，9592 - 9593]。
