Wolff N, Guenneugues M, Gilquin B, Drakopoulou E, Vita C, Ménez A, Zinn-Justin S
CEA, Département d'Ingénierie et d'Etudes des Protéines, CE Saclay, Gif-sur-Yvette, France.
Eur J Biochem. 2000 Nov;267(22):6519-33. doi: 10.1046/j.1432-1327.2000.01723.x.
By transferring the central curaremimetic beta hairpin of the snake toxin alpha into the scaffold of the scorpion charybdotoxin, a chimeric protein was constructed that reproduced the three-dimensional structure and partially reproduced the function of the parent beta hairpin, without perturbing the three-dimensional structure of the scaffold [1]. Picosecond to hour time scale motions of charybdotoxin and the engineered protein were observed, in order to evaluate the dynamic consequences of the six deletions and eight mutations differentiating the two molecules. The chimeric protein dynamics were also compared to that of toxin alpha, in order to examine the beta hairpin motions in both structural contexts. Thus, 13C R1, R1rho and 1H-->13C nOe were measured for all the CalphaHalpha and threonine CbetaHbeta vectors. As the proteins were not labeled, accordion techniques combined to coherence selection by pulsed field gradients and preservation of magnetization following equivalent pathways were used to considerably reduce the spectrometer time needed. On one hand, we observed that the chimeric protein and charybdotoxin are subjected to similar picosecond to nanosecond time scale motions except around the modified beta sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its beta structure is less stable on a minute to hour time scale. On the other hand, when the beta hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin alpha, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the beta hairpin sequence, and the beta sheet seems more stable in toxin alpha than in the chimera. The slower microsecond to hour time scale motions seem to be extremely sensitive to the structural context, and thus poorly transferred from one protein to another.
通过将蛇毒素α的中央箭毒样β发夹结构转移到蝎毒查卡毒素的支架结构中，构建了一种嵌合蛋白，该蛋白重现了母体β发夹的三维结构并部分重现了其功能，同时不干扰支架的三维结构[1]。观察了查卡毒素和工程蛋白从皮秒到小时时间尺度的运动，以评估区分这两种分子的六个缺失和八个突变所产生的动力学影响。还将嵌合蛋白的动力学与毒素α的动力学进行了比较，以研究β发夹在两种结构环境中的运动。因此，对所有的α碳原子-氢原子和苏氨酸的β碳原子-氢原子向量测量了13C纵向弛豫率（R1）、旋转坐标系下的纵向弛豫率（R1ρ）和1H→13C核Overhauser效应（nOe）。由于蛋白未进行标记，采用了将手风琴技术与脉冲场梯度相干选择以及等效路径下的磁化保留相结合的方法，以大幅减少所需的光谱仪时间。一方面，我们观察到，除了在修饰的β折叠区域周围，嵌合蛋白和查卡毒素经历了相似的皮秒到纳秒时间尺度的运动。嵌合蛋白在其整个序列上还表现出额外的毫秒时间尺度运动，并且其β结构在分钟到小时时间尺度上不太稳定。另一方面，当在两种不同的结构环境中比较β发夹的动力学时，即在嵌合蛋白和箭毒样毒素α中，皮秒到纳秒时间尺度的运动相当保守。然而，在β发夹的大部分序列上，微秒到毫秒时间尺度的运动是不同的，并且β折叠在毒素α中似乎比在嵌合体中更稳定。较慢的微秒到小时时间尺度的运动似乎对结构环境极其敏感，因此很难从一种蛋白转移到另一种蛋白。
