Laboratory of Single-Molecule Biophysics and Polymer Physics, COMSET, Clemson University, Clemson, South Carolina 29631, USA.
ACS Nano. 2011 Aug 23;5(8):6306-14. doi: 10.1021/nn201331n. Epub 2011 Jul 25.
Fullerene derivative C(60)(OH)(20) inhibited microtubule polymerization at low micromolar concentrations. The inhibition was mainly attributed to the formation of hydrogen bonding between the nanoparticle and the tubulin heterodimer, the building block of the microtubule, as evidenced by docking and molecular dynamics simulations. Our circular dichroism spectroscopy measurement indicated changes in the tubulin secondary structures, while our guanosine-5'-triphosphate hydrolysis assay showed hindered release of inorganic phosphate by the nanoparticle. Isothermal titration calorimetry revealed that C(60)(OH)(20) binds to tubulin at a molar ratio of 9:1 and with a binding constant of 1.3 ± 0.16 × 10(6) M(-1), which was substantiated by the binding site and binding energy analysis using docking and molecular dynamics simulations. Our simulations further suggested that occupancy by the nanoparticles at the longitudinal contacts between tubulin dimers within a protofilament or at the lateral contacts of the M-loop and H5 and H12 helices of neighboring tubulins could also influence the polymerization process. This study offered a new molecular-level insight on how nanoparticles may reshape the assembly of cytoskeletal proteins, a topic of essential importance for illuminating cell response to engineered nanoparticles and for the advancement of nanomedicine.
富勒烯衍生物 C(60)(OH)(20) 在低微摩尔浓度下抑制微管聚合。这种抑制主要归因于纳米颗粒与微管的组成部分微管蛋白二聚体之间形成氢键，这一点通过对接和分子动力学模拟得到了证实。我们的圆二色性光谱测量表明微管蛋白二级结构发生了变化，而我们的鸟苷-5'-三磷酸水解测定表明纳米颗粒阻碍了无机磷酸盐的释放。等温滴定量热法显示，C(60)(OH)(20) 与微管蛋白的摩尔比为 9:1，结合常数为 1.3±0.16×10(6)M(-1)，这一点通过对接和分子动力学模拟的结合位和结合能分析得到了证实。我们的模拟还表明，纳米颗粒在原纤维内微管蛋白二聚体之间的纵向接触处或相邻微管蛋白的 M 环和 H5 和 H12 螺旋的横向接触处的占据也可能影响聚合过程。这项研究提供了一个新的分子水平的见解，即纳米颗粒如何重塑细胞骨架蛋白的组装，这对于阐明细胞对工程纳米颗粒的反应以及推进纳米医学具有重要意义。
