Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

doi:10.3390/ijms20061410

CORC > 植物研究所 > 中国科学院植物研究所 > 中科院北方资源植物重点实验室

	Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
	Wang, Xiaohua ; Song, Kai 2; Li, Yang 1; Tang, Ling 1; Deng, Xin
刊名	INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
	2019
卷号	20 期号:6
关键词	single molecule stoichiometry molecular dynamics N-myristoylation
ISSN号	1661-6596
DOI	10.3390/ijms20061410
文献子类	Article
英文摘要	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.
学科主题	Biochemistry & Molecular Biology ; Chemistry, Multidisciplinary
电子版国际标准刊号	1422-0067
出版地	BASEL
WOS关键词	HIGH-THROUGHPUT ; FREE-ENERGY ; DYNAMICS ; SIMULATION ; OLIGOMERIZATION ; TRANSITION ; DEPENDENCE
WOS研究方向	Biochemistry & Molecular Biology ; Chemistry
语种	英语
出版者	MDPI
WOS记录号	WOS:000465523400050
资助机构	National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31770400, 31270412] ; Chinese Academy of SciencesChinese Academy of Sciences
内容类型	期刊论文
源URL	[http://ir.ibcas.ac.cn/handle/2S10CLM1/19818]
专题	中科院北方资源植物重点实验室
作者单位	1.Changan Univ, Middle Sect South Er Huan Rd, Xian 710064, Shaanxi, Peoples R China 2.Chinese Acad Sci, Inst Bot, Key Lab Plant Resources, Beijing 100093, Peoples R China 3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Wang, Xiaohua,Song, Kai,Li, Yang,et al. Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein[J]. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES,2019,20(6).
APA	Wang, Xiaohua,Song, Kai,Li, Yang,Tang, Ling,&Deng, Xin.(2019).Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein.INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES,20(6).
MLA	Wang, Xiaohua,et al."Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein".INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 20.6(2019).