Widmer H, Billeter M, Wüthrich K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
Proteins. 1989;6(4):357-71. doi: 10.1002/prot.340060403.
With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.
借助于1H核磁共振（NMR）光谱法，确定了海葵Anemonia sulcata的46个残基多肽神经毒素ATX Ia在水溶液中的三维结构。结构计算的输入数据包括来自核Overhauser效应（NOE）的263个距离约束和76个邻位耦合常数。为了进行结构计算，在一个由五个连续计算步骤组成的修订策略中使用了几个新的或改进的程序。首先，使用程序HABAS对所有与残基内和序列NMR约束兼容的主链和χ1构象进行全面搜索。其次，使用程序DISMAN，通过对所有与局部和中程NMR约束一致的构象进行广泛采样，将这种方法扩展到五肽。这两个步骤都导致了额外二面角约束的定义以及对一些β-亚甲基的立体专一性归属。在接下来的两个步骤中，使用DISMAN获得一组八个构象异构体，它们没有明显的NMR约束或范德华接触的残余违反情况。最后，使用AMBER分子力学模块的修改版本对这些结构进行受限能量精修，该版本除了能量力场之外，还包括NOE距离约束和二面角约束的势能。对于定义明确的分子核心（包含35个残基的主链原子和20个内部侧链），计算得到的最终精修构象异构体之间的成对最小RMS距离的平均值为1.5±0.3 Å。这个核心由一个四链β-折叠片层组成，通过两个定义明确的环连接，并且还有一个由11个残基8-18组成的额外柔性环。蛋白质的核心由三个二硫键稳定，这些二硫键被疏水残基包围，并在一侧被亲水残基屏蔽。
