NanoScience Technology Center , University of Central Florida , Orlando , Florida 32826 , United States.
Burnett School of Biomedical Sciences, College of Medicine , University of Central Florida , Orlando , Florida 32827 , United States.
J Phys Chem B. 2019 Apr 4;123(13):2770-2779. doi: 10.1021/acs.jpcb.8b12320. Epub 2019 Mar 19.
The cellular environment is crowded with high concentrations of macromolecules that significantly reduce accessible volume for biomolecular interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions, including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament mechanics and structure is unknown. In this study, we investigate the effects of solution crowding on the modulations of actin filament bending stiffness and conformations both in vitro and in silico. Direct visualization of thermally fluctuating filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging. Biophysical analysis indicates that molecular crowding enhances filament's effective bending stiffness and reduces average filament lengths. Utilizing the all-atom molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and intersubunit contacts that are directly coupled to the mechanical properties of filaments. Taken together, our study suggests that the interplay between excluded volume effects and nonspecific interactions raised from molecular crowding may modulate actin filament mechanics and structure.
细胞环境中充斥着高浓度的生物大分子，这大大降低了生物分子相互作用的可用体积。细胞体积的减少会产生耗竭力，影响蛋白质的组装和稳定性。肌动蛋白丝的机械和结构特性在各种细胞功能中起着关键作用，包括结构支撑、细胞运动、分裂和细胞内运输。虽然已经证明了分子拥挤对肌动蛋白聚合的影响，但拥挤环境如何影响丝的力学和结构尚不清楚。在这项研究中，我们通过体外和计算机模拟研究了溶液拥挤对肌动蛋白丝弯曲刚度和构象的调节作用。通过荧光显微镜成像实现了在拥挤剂存在下热波动丝的直接可视化。生物物理分析表明，分子拥挤增强了丝的有效弯曲刚度并减少了平均丝长度。利用全原子分子动力学模拟，我们证明了分子拥挤改变了丝构象和亚基间的接触，这些接触与丝的力学性质直接相关。总之，我们的研究表明，排除体积效应和分子拥挤引起的非特异性相互作用的相互作用可能调节肌动蛋白丝的力学和结构。
