Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota.
Department of Molecular Medicine, University of South Florida, Tampa, Florida.
Biophys J. 2018 Feb 6;114(3):550-561. doi: 10.1016/j.bpj.2017.12.013.
We used time-resolved Förster resonance energy transfer, circular dichroism, and molecular dynamics simulation to investigate the structural dependence of synaptotagmin 1's intrinsically disordered region (IDR) on phosphorylation and dielectric constant. We found that a peptide corresponding to the full-length IDR sequence, a ∼60-residue strong polyampholyte, can sample structurally collapsed states in aqueous solution, consistent with its κ-predicted behavior, where κ is a sequence-dependent parameter that is used to predict IDR compaction. In implicit solvent simulations of this same sequence, lowering the dielectric constant to more closely mimic the environment near a lipid bilayer surface promoted further sampling of collapsed structures. We then examined the structural tendencies of central region residues of the IDR in isolation. We found that the exocytosis-modulating phosphorylation of Thr disrupts a local disorder-to-order transition induced by trifluoroethanol/water mixtures that decrease the solution dielectric constant and stabilize helical structure. Implicit solvent simulations on these same central region residues testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric. In these helical conformers, lysine-aspartic acid salt bridges contribute to stabilization of transient secondary structure. In contrast, phosphorylation results in formation of salt bridges unsuitable for helix formation. Collectively, these results suggest a model in which phosphorylation and compaction of the IDR sequence regulate structural transitions that in turn modulate neuronal exocytosis.
我们使用时间分辨Förster 共振能量转移、圆二色性和分子动力学模拟来研究突触融合蛋白 1 的无规卷曲结构域 (IDR) 的结构依赖性与其磷酸化和介电常数的关系。我们发现，全长 IDR 序列对应的肽段，一个约 60 个残基的强两性聚电解质，可以在水溶液中采样结构塌陷状态，与 κ 预测行为一致，其中 κ 是一个依赖于序列的参数，用于预测 IDR 紧缩。在相同序列的隐溶剂模拟中，降低介电常数更接近模拟脂质双层表面附近的环境，可促进进一步采样塌陷结构。然后，我们研究了 IDR 中心区域残基的结构趋势。我们发现，Thr 的促分泌作用的磷酸化破坏了三氟乙醇/水混合物引起的局部无序到有序的转变，降低了溶液介电常数并稳定了螺旋结构。对这些相同的中心区域残基进行的隐溶剂模拟，仅测试介电常数的影响，得出了类似的结果，表明在降低介电常数时，螺旋结构形成的概率更高。在这些螺旋构象中，赖氨酸-天冬氨酸盐桥有助于稳定瞬时二级结构。相比之下，磷酸化导致不适合形成螺旋的盐桥的形成。总之，这些结果表明，IDR 序列的磷酸化和紧缩调节结构转变，进而调节神经元的胞吐作用。
