Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
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单分子成像和计算显微镜方法阐明了绿色荧光蛋白二聚化和膜相互作用的机制
DOI：
10.3390/ijms20061410
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发表时间：
2019-03
影响因子：
5.6
通讯作者：
Deng Xin
中科院分区：
文献类型：
--
作者：
Wang Xiaohua;Song Kai;Li Yang;Tang Ling;Deng Xin
绿色荧光蛋白(GFP)被广泛用作生命系统的生物标记物，但GFP及其变异体在生理条件下容易形成低亲和力的二聚体。当荧光蛋白(FP)被限制在膜上、与天然寡聚蛋白融合或在细胞中高水平表达时，这种不良趋势会加剧。FPS的寡聚化将伪影引入亚基化学计量学的测量，以及融合到FPS的蛋白质之间的相互作用。单一突变A206K的引入已被证明破坏了负责GFP二聚化的区域的疏水相互作用，从而促进了GFP的单聚化。然而，对于这种单一氨基酸依赖的GFP二聚化抑制是如何在原子水平上发生的详细了解仍然缺乏。单分子实验结合计算显微镜(原子分子动力学)表明，A206的氨基通过多价静电相互作用促进了GFP二聚体的形成。我们进一步证明肉豆蔻基修饰是促进GFP膜附着的一种有效机制。基于分子动力学的定点突变已被用于鉴定FPS中的关键功能残基。本文提供的数据已被用作下游单分子研究的单体对照，有助于更准确地对活细胞中的功能蛋白质复合体进行化学计量量化。
Green fluorescent protein (GFP) is widely used as a biomarker in living systems; however, GFP and its variants are prone to forming low-affinity dimers under physiological conditions. This undesirable tendency is exacerbated when fluorescent proteins (FP) are confined to membranes, fused to naturally-oligomeric proteins, or expressed at high levels in cells. Oligomerization of FPs introduces artifacts into the measurement of subunit stoichiometry, as well as interactions between proteins fused to FPs. Introduction of a single mutation, A206K, has been shown to disrupt hydrophobic interactions in the region responsible for GFP dimerization, thereby contributing to its monomerization. Nevertheless, a detailed understanding of how this single amino acid-dependent inhibition of dimerization in GFP occurs at the atomic level is still lacking. Single-molecule experiments combined with computational microscopy (atomistic molecular dynamics) revealed that the amino group of A206 contributes to GFP dimer formation via a multivalent electrostatic interaction. We further showed that myristoyl modification is an efficient mechanism to promote membrane attachment of GFP. Molecular dynamics-based site-directed mutagenesis has been used to identify the key functional residues in FPs. The data presented here have been utilized as a monomeric control in downstream single-molecule studies, facilitating more accurate stoichiometry quantification of functional protein complexes in living cells.
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影响因子：
4.3
作者：
Song, Kai;Xue, Yiqun;Lin, Jinxing
通讯作者：
Lin, Jinxing
单分子荧光成像可量化活植物细胞中的膜蛋白动力学和寡聚化
影响因子：
14.8
作者：
Wang, Xiaohua;Li, Xiaojuan;Lin, Jinxing
通讯作者：
Lin, Jinxing
L1198F 突变通过改变抑制剂和 ATP 结合位点的构象使克唑替尼对 ALK 重新敏感
影响因子：
5.6
作者：
Li J;Sun R;Wu Y;Song M;Li J;Yang Q;Chen X;Bao J;Zhao Q
通讯作者：
Zhao Q
影响因子：
6.8
作者：
Meirovitch, Hagai
通讯作者：
Meirovitch, Hagai
DOI：
10.1073/pnas.97.22.11984
发表时间：
2000-10-24
影响因子：
11.1
作者：
Baird, GS;Zacharias, DA;Tsien, RY
通讯作者：
Tsien, RY