使用定点自旋标记和电子顺磁共振对人波形蛋白中连接子2区域进行表征。
Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance.
作者信息
Hess John F, Budamagunta Madhu S, Shipman Rebecca L, FitzGerald Paul G, Voss John C
机构信息
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616, USA.
出版信息
Biochemistry. 2006 Oct 3;45(39):11737-43. doi: 10.1021/bi060741y.
Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405.
定点自旋标记和电子顺磁共振被用于探测人类波形蛋白的281 - 304位残基，该区域被预测为非α螺旋连接区以及卷曲螺旋结构域2B的起始部分。尽管从未对连接区结构进行过直接测试，但该区域被假设为柔性的，多肽链彼此环绕。对自旋标记突变体的电子顺磁共振分析表明：（a）几个残基彼此靠近，这表明二聚体中相邻的连接区是平行排列的；（b）多肽主链在该区域相对刚性且不易弯曲。然而，该区域并未表现出分子中其他部位已确定的卷曲螺旋结构特征。在该区域内，283位和291位的光谱与迄今为止所检测的所有其他位置的光谱都不同。预计处于非卷曲螺旋结构的这些位置，显示出比卷曲螺旋区域的α - δ接触位置更强的相互作用。通过从8 M尿素中透析和逐步热变性对组装早期阶段的分析表明，283位和291位残基处的紧密并列和结构刚性在组装过程中很早就出现了，且相对突然，远早于分子其他部位的卷曲螺旋形成。这些特征与将连接区设想为柔性区域或彼此环绕的假设不一致，并增加了连接区可能是二聚体排列和/或形成起始位点的可能性。在更下游位置放置的自旋标记产生的光谱表明，杆状结构域2的第一个规则七肽起始于302位。结合我们之前对305 - 336区域的表征以及杆状结构2B从328 - 405位的解析结构，现在已知杆状结构2B中卷曲螺旋结构域的完整范围，从波形蛋白的302 - 405位。
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