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首页> 外文期刊>Biochemistry >The structure and dynamics of partially folded actin.
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The structure and dynamics of partially folded actin.

机译：部分折叠肌动蛋白的结构和动力学。

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Steady-state and time-resolved intrinsic fluorescence, fluorescence quenching by acrylamide, and surface testing by hydrophobic label ANS were used to study the structure of inactivated alpha-actin. The results are discussed together with that of earlier experiments on sedimentation, anisotropy of fluorescence, and CD spectrum in the near- and far-UV regions. A dramatic increase in ANS binding to inactivated actin in comparison with native and unfolded protein indicates that the inactivated actin has solvent-exposed hydrophobic clusters on the surface. It results in specific association of actin macromolecules (sedimentation constants for native and inactivated actin are 3 and 20 S, respectively) and, consequently, in irreversibility of native-inactivated actin transition. It was found that, though the fluorescence spectrum of inactivated actin is red-shifted, the efficiency of the acrylamide collision quenching is even lower than that of the intact protein. It suggests that tryptophan residues of inactivated actin are located in the inner region of protein formed by polar groups, which are highly packed. It correlates with the pronounced near-UV CD spectrum of inactivated actin. The experimentally found tryptophan fluorescence lifetimes allowed evaluation rotational correlation times on the basis of Perrin plots. It is found that oscillations of tryptophan residues in inactivated actin are restricted in comparison with native one. The inactivated actin properties were invariant with experimental conditions (ionic strength, the presence of reducing agents), the way of inactivation (Ca2+ and/or ATP removal, heating, 3-5 M urea or 1.5 M GdmCl treatment), and protein concentration (within the limits 0.005-1.0 mg/mL). The same state of actin appears on the refolding from the completely unfolded state. Thermodynamic stability, pronounced secondary structure, and the existing hydrophobic clusters, tested by ANS fluorescence and reversibility of transition inactivated-unfolded forms, allowed us to suggest that inactivated actin can be intermediate in the folding-unfolding pathway.
机译：稳态和时间分辨的固有荧光，丙烯酰胺的荧光猝灭以及疏水性标记ANS的表面测试用于研究灭活的α-肌动蛋白的结构。讨论了结果以及早期在近紫外和远紫外区域的沉积，荧光各向异性和CD光谱的实验结果。与天然和未折叠的蛋白质相比，ANS与失活的肌动蛋白的结合显着增加表明该失活的肌动蛋白在表面具有溶剂暴露的疏水簇。它导致肌动蛋白大分子的特异性结合（天然和失活肌动蛋白的沉降常数分别为3和20 S），因此，天然失活肌动蛋白的转变不可逆。已经发现，尽管灭活的肌动蛋白的荧光光谱是红移的，但是丙烯酰胺碰撞猝灭的效率甚至低于完整蛋白的效率。这表明失活的肌动蛋白的色氨酸残基位于由极性基团形成的蛋白质的内部区域，该基团高度堆积。它与灭活的肌动蛋白的明显的近紫外CD光谱有关。实验上发现的色氨酸荧光寿命允许根据Perrin图评估旋转相关时间。发现与天然的肌动蛋白相比，失活的肌动蛋白中色氨酸残基的振荡受到限制。失活的肌动蛋白性质随实验条件（离子强度，还原剂的存在），失活方式（Ca2 +和/或ATP的去除，加热，3-5 M尿素或1.5 M GdmCl处理）不变。在极限范围内0.005-1.0 mg / mL）。肌动蛋白的相同状态出现在从完全展开状态开始的重新折叠中。热力学稳定性，明显的二级结构，和现有的疏水簇，通过ANS荧光测试和过渡失活-未折叠形式的可逆性，使我们建议失活的肌动蛋白可以在折叠-展开途径的中间。

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