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

被引:10
作者
Wang, Xiaohua [1 ]
Song, Kai [2 ]
Li, Yang [1 ,3 ]
Tang, Ling [1 ,3 ]
Deng, Xin [1 ]
机构
[1] Chinese Acad Sci, Inst Bot, Key Lab Plant Resources, Beijing 100093, Peoples R China
[2] Changan Univ, Middle Sect South Er Huan Rd, Xian 710064, Shaanxi, Peoples R China
[3] Univ Chinese Acad Sci, Beijing 100049, Peoples R China
基金
中国国家自然科学基金;
关键词
single molecule; stoichiometry; molecular dynamics; N-myristoylation; HIGH-THROUGHPUT; FREE-ENERGY; DYNAMICS; SIMULATION; OLIGOMERIZATION; TRANSITION; DEPENDENCE;
D O I
10.3390/ijms20061410
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
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.
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页数:15
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共 43 条
[1]   Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral [J].
Baird, GS ;
Zacharias, DA ;
Tsien, RY .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (22) :11984-11989
[2]   Analysis of Hydrogen-Bond Interaction Potentials from the Electron Density: Integration of Noncovalent Interaction Regions [J].
Contreras-Garcia, Julia ;
Yang, Weitao ;
Johnson, Erin R. .
JOURNAL OF PHYSICAL CHEMISTRY A, 2011, 115 (45) :12983-12990
[3]   Assessing the Tendency of Fluorescent Proteins to Oligomerize Under Physiologic Conditions [J].
Costantini, Lindsey M. ;
Fossati, Matteo ;
Francolini, Maura ;
Snapp, Erik Lee .
TRAFFIC, 2012, 13 (05) :643-649
[4]   Single-Particle Tracking for the Quantification of Membrane Protein Dynamics in Living Plant Cells [J].
Cui, Yaning ;
Yu, Meng ;
Yao, Xiaomin ;
Xing, Jingjing ;
Lin, Jinxing ;
Li, Xiaojuan .
MOLECULAR PLANT, 2018, 11 (11) :1315-1327
[5]   Biomolecular Simulation: A Computational Microscope for Molecular Biology [J].
Dror, Ron O. ;
Dirks, Robert M. ;
Grossman, J. P. ;
Xu, Huafeng ;
Shaw, David E. .
ANNUAL REVIEW OF BIOPHYSICS, VOL 41, 2012, 41 :429-452
[6]   Molecular simulation and modeling of complex I [J].
Hummer, Gerhard ;
Wikstrom, Marten .
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2016, 1857 (07) :915-921
[7]   VMD: Visual molecular dynamics [J].
Humphrey, W ;
Dalke, A ;
Schulten, K .
JOURNAL OF MOLECULAR GRAPHICS & MODELLING, 1996, 14 (01) :33-38
[8]   Reversible Dimerization of Aequorea victoria Fluorescent Proteins Increases the Dynamic Range of FRET-Based Indicators [J].
Kotera, Ippei ;
Iwasaki, Takuya ;
Imamura, Hiromi ;
Noji, Hiroyuki ;
Nagai, Takeharu .
ACS CHEMICAL BIOLOGY, 2010, 5 (02) :215-222
[9]   g_mmpbsa-A GROMACS Tool for High-Throughput MM-PBSA Calculations [J].
Kumari, Rashmi ;
Kumar, Rajendra ;
Lynn, Andrew .
JOURNAL OF CHEMICAL INFORMATION AND MODELING, 2014, 54 (07) :1951-1962
[10]   Molecular Insights into the Adsorption Mechanism of Human β-Defensin-3 on Bacterial Membranes [J].
Lee, Juho ;
Jung, Sang Won ;
Cho, Art E. .
LANGMUIR, 2016, 32 (07) :1782-1790