Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Nanoscale. 2020 May 28;12(20):11070-11078. doi: 10.1039/d0nr01577f.
Post-translational modifications (PTMs) of proteins are recognized as crucial components of cell signaling pathways through modulating folding, altering stability, changing interactions with ligands, and, therefore, serving multiple regulatory functions. PTMs occur as covalent modifications of the protein's amino acid side chains or the length and composition of their termini. Here we study the functional consequences of PTMs for α-synuclein (αSyn) interactions with the nanopore of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. PTMs were mimicked by a divalent Alexa Fluor 488 sidechain attached separately at two positions on the αSyn C-terminus. Using single-channel reconstitution into planar lipid membranes, we find that such modifications change interactions drastically in both efficiency of VDAC inhibition by αSyn and its translocation through the VDAC nanopore. Analysis of the on/off kinetics in terms of an interaction "quasipotential" allows the positions of the C-terminal modifications to be determined with an accuracy of about three residues. Moreover, our results uncover a previously unobserved mechanism by which cytosolic proteins control β-barrel channels and thus a new regulatory function for PTMs.
蛋白质的翻译后修饰（PTMs）被认为是细胞信号通路的关键组成部分，通过调节折叠、改变稳定性、改变与配体的相互作用，从而发挥多种调节功能。PTMs 表现为蛋白质氨基酸侧链的共价修饰或其末端的长度和组成。在这里，我们研究了 PTMs 对 α-突触核蛋白（αSyn）与线粒体外膜电压依赖性阴离子通道（VDAC）纳米孔相互作用的功能影响。通过在 αSyn C 末端的两个位置分别连接一个二价 Alexa Fluor 488 侧链来模拟 PTMs。利用单通道重组到平面脂质膜中，我们发现这种修饰在 αSyn 抑制 VDAC 的效率及其通过 VDAC 纳米孔的转运方面都极大地改变了相互作用。根据相互作用“准势”对开/关动力学进行分析，可以确定 C 末端修饰的位置，精度约为三个残基。此外，我们的结果揭示了一种以前未观察到的机制，即胞质蛋白控制 β-桶状通道，从而发现了 PTMs 的一种新的调节功能。
