Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR
Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR
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DOI：
10.1529/biophysj.106.082735
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发表时间：
2006-08-15
影响因子：
3.4
通讯作者：
Fujiwara, Toshimichi
中科院分区：
文献类型：
--
作者：
Todokoro, Yasuto;Yumen, Ikuko;Fujiwara, Toshimichi
通过固态核磁共振波谱法测定了与阴离子磷脂双层紧密结合的马斯托帕兰-X (MP-X) 的结构，这是一种来自黄蜂毒液的 G 蛋白激活肽。均匀标记的 MP-X 的碳 13 和氮 15 NMR 信号通过多维残基内 C-C、N-(CC β)-C-α 和 N-C-α-C' 以及残基间 C-α-(CC β)-C-α、N-(CC β)-C-α 和 N-C'-C-α 相关实验完全归属。借助蛋白质 NMR 数据库程序，根据 C-13'、C-13(α)、C-13(β) 和 N-15 信号的化学位移预测主链扭转角。此外，分别通过旋转共振和REDOR实验精确测量了主链核之间的两个C-13-C-13和三个C-13-N-15距离。 MP-X 的主干结构由 26 个二面角约束和 5 个距离确定，平均均方根偏差为 0.6 埃。处于双层结合状态的肽 MP-X 形成残基 Trp(3) - Leu(14) 的两亲性 α 螺旋，并采用 Asn(2) 的扩展构象。讨论了这种膜结合构象与肽在膜中形成孔和激活 G 蛋白的活性。这项研究展示了均匀同位素标记分子的多维固态核磁共振和距离测量在确定与脂膜结合的肽结构方面的能力。
The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-(CC beta)-C-alpha, and N-C-alpha-C', and interresidue C-alpha-(CC beta)-C-alpha, N-(CC beta)-C-alpha, and N-C'-C-alpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of C-13', C-13(alpha), C-13(beta), and N-15 signals with the aid of protein NMR database programs. In addition, two C-13-C-13 and three C-13-N-15 distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 angstrom. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp(3) - Leu(14) and adopted an extended conformation for Asn(2). This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.