Department of Biomedical Engineering, University of Arizona, Tucson, Arizona 85721, United States.
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States.
Biochemistry. 2022 Jul 5;61(13):1229-1242. doi: 10.1021/acs.biochem.2c00091. Epub 2022 Jun 13.
The structural analysis of large protein complexes has been greatly enhanced through the application of electron microscopy techniques. One such multiprotein complex, the cardiac thin filament (cTF), has cyclic interactions with thick filament proteins to drive contraction of the heart that has recently been the subject of such studies. As important as these studies are, they provide limited or no information on highly flexible regions that in isolation would be characterized as inherently disordered. One such region is the extended cardiac troponin T (cTnT) linker between the regions of cTnT which have been labeled TNT1 and TNT2. It comprises a hinge region (residues 158-166) and a highly flexible region (residues 167-203). Critically, this region modulates the troponin/tropomyosin complex's position across the actin filament. Thus, the cTnT linker structure and dynamics are central to the regulation of the function of cardiac muscles, but up to now, it was ill-understood. To establish the cTnT linker structure, we coupled an atomistic computational cTF model with time-resolved fluorescence resonance energy transfer measurements in both ±Ca conditions utilizing fully reconstituted cTFs. We mapped the cTnT linker's positioning across the actin filament, and by coupling the experimental results to computation, we found mean structures and ranges of motion of this part of the complex. With this new insight, we can now address cTnT linker structural dynamics in both myofilament activation and disease.
通过应用电子显微镜技术，对大型蛋白质复合物的结构分析有了很大的提高。这样的多蛋白复合物之一，即心脏细肌丝（cTF），与粗肌丝蛋白周期性相互作用，驱动心脏收缩，最近的研究就集中在这个方面。这些研究非常重要，但它们提供的关于高度灵活的区域的信息有限或没有，这些区域在孤立的情况下会被认为是固有无序的。其中一个这样的区域是位于 cTnT1 和 cTnT2 区域之间的心脏肌钙蛋白 T（cTnT）延伸连接区。它包括一个铰链区（残基 158-166）和一个高度灵活的区域（残基 167-203）。至关重要的是，这个区域调节了肌钙蛋白/原肌球蛋白复合物在肌动蛋白丝上的位置。因此，cTnT 连接区的结构和动力学是调节心脏肌肉功能的核心，但到目前为止，它还没有被很好地理解。为了建立 cTnT 连接区的结构，我们将原子计算的 cTF 模型与在±Ca 条件下利用完全重组的 cTF 进行的时间分辨荧光共振能量转移测量相结合。我们绘制了 cTnT 连接区在肌动蛋白丝上的定位图，通过将实验结果与计算相结合，我们发现了该复合物这一部分的平均结构和运动范围。有了这个新的认识，我们现在可以研究肌球蛋白激活和疾病中 cTnT 连接区的结构动力学。
