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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Molecular crowding enhances native structure and stability of α/β protein flavodoxin
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Molecular crowding enhances native structure and stability of α/β protein flavodoxin

机译：分子拥挤增强了α/β蛋白黄酮毒素的天然结构和稳定性

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摘要

To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue single-domain α/β protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (ΔT_m of 20℃ at 400 mg/ml; pH 7). As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble (pH 7, 20℃) but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model and hard-sphere particles, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25% volume occupancy of spheres, native flavodoxin is thermally stabilized, and the free energy landscape shifts to favor more compact structures in both native and denatured states. The difference contact map reveals that the native-state compaction originates in stronger interactions between the helices and the central β-sheet, as well as by less fraying in the terminal helices. This study demonstrates that macromolecular crowding has structural effects on the folded ensemble of polypeptides.
机译：为了研究大分子拥挤对球蛋白行为的影响，我们对148个残基的单域α/β蛋白Desulfovibrio desulfuricans apoflavodoxin进行了组合实验和计算研究。在各种量的惰性球形拥挤剂Ficoll 70的存在下，通过使用远紫外CD探测体外热展开实验以及评估天然和变性结构。 Ficoll 70对脱附黄酮毒素的热稳定性具有浓度依赖性（在400 mg / ml，pH 7时，ΔT_m为20℃）。根据CD的判断，添加Ficoll 70会使原始状态集合（pH 7、20℃）中二级结构的数量增加，但对变性状态的影响很小。基于非晶格模型和硬球粒子的理论计算与体外数据非常吻合。模拟表明，在球体体积占有率为25％的情况下，天然黄酮毒素是热稳定的，自由能格局发生变化，有利于天然和变性状态下更紧凑的结构。差异接触图揭示了原始状态的压实起源于螺旋与中心β-折叠之间的较强相互作用，以及末端螺旋的较少磨损。这项研究表明，大分子拥挤对多肽的折叠整体具有结构作用。

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