Andersen N H, Chen C P, Marschner T M, Krystek S R, Bassolino D A
Department of Chemistry, University of Washington, Seattle 98195.
Biochemistry. 1992 Feb 11;31(5):1280-95. doi: 10.1021/bi00120a003.
The conformational features of endothelin-1 (ET-1) in mixed water/ethylene glycol media have been studied by two-dimensional 1H NMR experiments throughout the pH range 3.2-7.2. At pH less than 5 all backbone NH signals can be observed, and NOESY experiments provided a large set of dipolar cross-peaks. Cross-peak intensities from each experiment (different mixing times and H2O versus D2O) were converted to distance constraints using a novel algorithm (program DISCON) for removing spin diffusion effects and thus obtain cross-rates rather than cross-peak intensities. A set of 168 nonstereospecific distance bounds (average experimental precision, +/- 0.38 A) was used in dynamics simulated annealing refinements. Two consensus structural features were found--a reverse turn at Ser5----Asp8 and an alpha-helical stretch from Lys9 to Cys15; however, after constraint-free minimization, structures generated using XPLOR-1.5, CONGEN, and DISCOVER all violated at least 32% of the bounds by more than 0.2 A, which we ascribe to conformational isomerism. When the constraints were modified to reflect subsequent experimental data and to eliminate constraints that could not be obeyed by any single conformer structure, the relaxed structures still violated at least 15% of this more limited and looser set of constraints. Therefore, a modified procedure for constrained dynamics refinement (using XPLOR-2.1), which allows for conformational isomerism outside of the central helical core region, was developed. This "conformer search procedure" produced structures which fell into five tightly defined conformational clusters. The two most populated clusters correspond to a rotation of the 8,9-amide unit. The conformer which we propose as the major contributor at pH 3.2-5.8 was defined to a backbone rmsd of 0.51 A over residues 1----15. An alternative description of the motional averaging in segments of the endothelin structure as extensive randomization rather than rapid interconversion between a small number of discreet conformers was ruled out by an analysis of NH shift-temperature gradients and exchange rates. This analysis suggests that small delta delta/delta T values need not correlate with H-bonding for conformational mixtures. In ET-1 the greatest motional averaging occurs from Ser2 through Ser5 (not in the C-terminus) and may be so extensive as to approximate a flexible random coil population as high as 30%. The C-terminus shows less rapid and less extensive conformational averaging, but no definitive structures for individual conformers could be derived in the absence of stereospecific constraints. The pharmacological implications of the consensus structural features are discussed.
通过二维¹H NMR实验，在pH值范围为3.2 - 7.2的混合水/乙二醇介质中研究了内皮素-1（ET-1）的构象特征。在pH小于5时，可以观察到所有主链NH信号，并且NOESY实验提供了大量的偶极交叉峰。使用一种新颖的算法（程序DISCON）将每个实验（不同混合时间以及H₂O与D₂O）的交叉峰强度转换为距离约束，以消除自旋扩散效应，从而获得交叉速率而非交叉峰强度。在动力学模拟退火精修中使用了一组168个非立体特异性距离界限（平均实验精度，±0.38 Å）。发现了两个一致的结构特征——Ser5至Asp8处的反向转角以及从Lys9到Cys15的α-螺旋伸展；然而，在无约束最小化之后，使用XPLOR-1.5、CONGEN和DISCOVER生成的结构均至少有32%的界限被违反超过0.2 Å，我们将此归因于构象异构现象。当修改约束以反映后续实验数据并消除任何单个构象异构体结构都无法遵守的约束时，松弛后的结构仍然至少违反了这组更有限且更宽松约束的15%。因此，开发了一种用于约束动力学精修的修改程序（使用XPLOR-2.1），该程序允许在中央螺旋核心区域之外存在构象异构现象。这种“构象异构体搜索程序”产生的结构落入五个紧密定义的构象簇中。两个数量最多的簇对应于8,9-酰胺单元的旋转。我们提出在pH 3.2 - 5.8时作为主要贡献者的构象异构体，在残基1至15上的主链均方根偏差定义为0.51 Å。通过对NH位移-温度梯度和交换速率的分析，排除了将内皮素结构片段中的运动平均描述为广泛随机化而非少数离散构象异构体之间快速相互转换的另一种说法。该分析表明，对于构象混合物，小的δδ/δT值不一定与氢键相关。在ET-1中，最大的运动平均发生在Ser2至Ser5之间（不在C末端），并且可能广泛到近似高达30%的柔性无规卷曲群体。C末端显示出较慢且较不广泛的构象平均，但在没有立体特异性约束的情况下，无法得出单个构象异构体的确切结构。讨论了一致结构特征的药理学意义。
