Measurement of eight scalar and dipolar couplings for methine-methylene pairs in proteins and nucleic acids
Measurement of eight scalar and dipolar couplings for methine-methylene pairs in proteins and nucleic acids
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DOI：
10.1007/s10858-005-0175-z
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发表时间：
2005-03-01
影响因子：
2.7
通讯作者：
Bax, A
中科院分区：
文献类型：
--
作者：
Miclet, E;Boisbouvier, J;Bax, A
一个新的三维，自旋状态选择相干转移核磁共振实验描述，产生精确测量八个标量或偶极偶联在一个自旋系统组成的亚甲基相邻的甲基。对蛋白质和核酸进行了优化的实验实施。实验证实了来自链球菌蛋白G （GB3）的第三个igg结合域的C-β-C-α部分和24 nt RNA低聚物中的C5‘-C4’基团。C-α、C-β和H-β（分别为C4′、C5′和H5′）的化学位移分散在三个正交维度上，并且由于不存在异核解耦，导致E.COSY多重模式清晰且分辨率高。在一个各向同性的示例中,E.COSY位移对应(1)J (C Hαα),(2)J (H Cαα)+ (2)J (H Cαβ3),(2)J (C Hαβ),(1)J (H Cββ2)+ (1)JC (Hββ3),(1)J (H Cββ2),(2)J (Hβ2 Hβ3)(1)J (H Cββ3)- (2)J (Hβ2 Hβ3),(3)J (HαHβ2)和(3)J蛋白(HαHβ3),和(1)J (C4'H4”),(2)JC (4 'h5”)+ 2 J (C4'H5 '), (2) (C5'H4”),(1)J (C5'H5”)+ (1)(C5'H5”),(1)J (C5'H5”)(2)(H5'H5”),(1)J (C5'H5”)(2)(H5'H5”),(3)J (H4'H5”),和(3)J在核酸(H4'H5”)。该实验基于弛豫优化光谱，当应用于甲基-亚甲基基对应于一个合理孤立的自旋系统的残基时，如适用于蛋白质中的C， F， Y， W， D， N和H残基，或核酸中的C5‘-C4’基团时，可以获得最佳结果。劈裂可以在各向同性或弱排列条件下测量，通过(3)J耦合和一键、二键和三键偶极相互作用获得有价值的结构信息。发现GB3中13个侧链中有10个侧链的偶极耦合与其x射线结构非常吻合，而一个残基采用不同的骨干几何形状，两个残基受到广泛的x(1)旋转体平均。偶极偶联的丰度也可以产生非等效亚甲基质子的立体定向分配。对于RNA低聚物，偶极数据得到了该低聚物环区8个C5'H-2基团中的6个的立体定向分配，在所有情况下均由(1)J(C5'H5')>证实；(1)J（C5‘H5 ‘）和H-5’在H-5’下场共振。
A new 3D, spin-state-selective coherence transfer NMR experiment is described that yields accurate measurements for eight scalar or dipolar couplings within a spin system composed of a methylene adjacent to a methine group. Implementations of the experiment have been optimized for proteins and for nucleic acids. The experiments are demonstrated for C-β-C-α moieties of the third IgG-binding domain from Streptococcal Protein G (GB3) and for C5'-C4' groups in a 24-nt RNA oligomer. Chemical shifts of C-α, C-β and H-β (respectively C4', C5' and H5') are dispersed in the three orthogonal dimensions, and the absence of heteronuclear decoupling leads to distinct and well-resolved E.COSY multiplet patterns. In an isotropic sample, the E.COSY displacements correspond to (1)J(Cα Hα), (2)J(Cα Hα) + (2)J(Cα Hβ 3), (2)J(Cβ Hα), (1)J(Cβ Hβ 2) + (1)JC(β Hβ 3), (1)J(Cβ Hβ 2)-(2)J(Hβ 2Hβ 3,) (1)J(Cβ Hβ 3)-(2)J(Hβ 2Hβ 3), (3)J(Hα Hβ 2) and (3)J(Hα Hβ 3) for proteins, and (1)J(C4'H4'), (2)JC(4'H5')+2J(C4'H5'), (2)J(C5'H4'), (1)J(C5'H5')+(1)J(C5'H5"), (1)J(C5'H5')-(2)J(H5'H5'), (1)J(C5'H5')-(2)J(H5'H5'), (3)J(H4'H5'), and (3)J(H4'H5") in nucleic acids. The experiment, based on relaxation-optimized spectroscopy, yields best results when applied to residues where the methine-methylene group corresponds to a reasonably isolated spin system, as applies for C, F, Y, W, D, N and H residues in proteins, or the C5'-C4' groups in nucleic acids. Splittings can be measured under either isotropic or weakly aligned conditions, yielding valuable structural information both through the (3)J couplings and the one-, two- and three-bond dipolar interactions. Dipolar couplings for 10 out of 13 sidechains in GB3 are found to be in excellent agreement with its X-ray structure, whereas one residue adopts a different backbone geometry, and two residues are subject to extensive x(1) rotamer averaging. The abundance of dipolar couplings can also yield stereospecific assignments of the non-equivalent methylene protons. For the RNA oligomer, dipolar data yielded stereospecific assignments for six out of the eight C5'H-2 groups in the loop region of the oligomer, in all cases confirmed by (1)J(C5'H5')>(1)J(C5'H5"), and H-5' resonating downfield of H-5".