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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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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 |
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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 |
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