Cargando…

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

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 naturall...

Descripción completa

Detalles Bibliográficos
Autores principales: Wang, Xiaohua, Song, Kai, Li, Yang, Tang, Ling, Deng, Xin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471090/
https://www.ncbi.nlm.nih.gov/pubmed/30897814
http://dx.doi.org/10.3390/ijms20061410
_version_ 1783411946664493056
author Wang, Xiaohua
Song, Kai
Li, Yang
Tang, Ling
Deng, Xin
author_facet Wang, Xiaohua
Song, Kai
Li, Yang
Tang, Ling
Deng, Xin
author_sort Wang, Xiaohua
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6471090
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-64710902019-04-26 Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein Wang, Xiaohua Song, Kai Li, Yang Tang, Ling Deng, Xin Int J Mol Sci Article 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. MDPI 2019-03-20 /pmc/articles/PMC6471090/ /pubmed/30897814 http://dx.doi.org/10.3390/ijms20061410 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wang, Xiaohua
Song, Kai
Li, Yang
Tang, Ling
Deng, Xin
Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
title Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
title_full Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
title_fullStr Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
title_full_unstemmed Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
title_short Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein
title_sort single-molecule imaging and computational microscopy approaches clarify the mechanism of the dimerization and membrane interactions of green fluorescent protein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471090/
https://www.ncbi.nlm.nih.gov/pubmed/30897814
http://dx.doi.org/10.3390/ijms20061410
work_keys_str_mv AT wangxiaohua singlemoleculeimagingandcomputationalmicroscopyapproachesclarifythemechanismofthedimerizationandmembraneinteractionsofgreenfluorescentprotein
AT songkai singlemoleculeimagingandcomputationalmicroscopyapproachesclarifythemechanismofthedimerizationandmembraneinteractionsofgreenfluorescentprotein
AT liyang singlemoleculeimagingandcomputationalmicroscopyapproachesclarifythemechanismofthedimerizationandmembraneinteractionsofgreenfluorescentprotein
AT tangling singlemoleculeimagingandcomputationalmicroscopyapproachesclarifythemechanismofthedimerizationandmembraneinteractionsofgreenfluorescentprotein
AT dengxin singlemoleculeimagingandcomputationalmicroscopyapproachesclarifythemechanismofthedimerizationandmembraneinteractionsofgreenfluorescentprotein