Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human α-tropomyosin
Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human α-tropomyosin
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DOI：
10.1111/j.1432-1033.2004.04351.x
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发表时间：
2004-10-01
期刊：
影响因子：
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通讯作者：
Bertolini, MC
中科院分区：
文献类型：
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作者：
Hilario, E;da Silva, SLF;Bertolini, MC
蛋白质 α-原肌球蛋白 (Tm) 的突变可导致一种称为家族性肥厚性心肌病的疾病。为了了解此类突变如何导致蛋白质功能障碍，将三个点突变引入编码人骨骼原肌球蛋白的 cDNA 中，并在酵母毕赤酵母中产生高水平的重组 Tms。两个突变（A63V 和 K70T）位于 Tm 的 N 末端区域，一个突变（E180G）位于钙依赖性肌钙蛋白 T 结合域附近。将突变体 Tms 的功能和结构特性与野生型蛋白进行比较。尽管在突变体 Tm K70T 中观察到肌动蛋白结合略高（如共沉测定中所证明），但没有任何突变改变头尾聚合。这些突变也没有通过增加 Ca2+ 浓度来改变细丝激活的协同性。然而，在缺乏肌钙蛋白的情况下，所有突变型 Tms 在调节肌动球蛋白亚片段 1 Mg2+ ATP 酶活性方面均不如野生型有效。圆二色光谱显示 Tms 的二级结构没有差异。然而，通过圆二色性或差示扫描量热法监测的热诱导解折叠表明突变体不如野生型稳定。这些结果表明，突变的主要影响与Tm作为一个整体的整体稳定性有关，并且突变对构成细丝的蛋白质之间的协作相互作用仅产生较小的影响。
Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.