Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids

Abstract Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeede...

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Autores principales:	Ilya D. Solovyev, Alexandra V. Gavshina, Aditya S. Katti, Alexey I. Chizhik, Leonid M. Vinokurov, Grigory D. Lapshin, Tatiana V. Ivashina, Maria G. Khrenova, Igor I. Kireev, Ingo Gregor, Jörg Enderlein, Alexander P. Savitsky
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Rational Mutagenesis Succinic Anhydride Single-molecule Localization Microscopy Hydrophobic Interface Constant Current Rate Medicine R Science Q
Acceso en línea:	https://doaj.org/article/976772660578499693c31861f28eb1e2
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spelling	oai:doaj.org-article:976772660578499693c31861f28eb1e22021-12-02T15:08:25ZMonomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids10.1038/s41598-018-33250-z2045-2322https://doaj.org/article/976772660578499693c31861f28eb1e22018-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-33250-zhttps://doaj.org/toc/2045-2322Abstract Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein’s hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.Ilya D. SolovyevAlexandra V. GavshinaAditya S. KattiAlexey I. ChizhikLeonid M. VinokurovGrigory D. LapshinTatiana V. IvashinaMaria G. KhrenovaIgor I. KireevIngo GregorJörg EnderleinAlexander P. SavitskyNature PortfolioarticleRational MutagenesisSuccinic AnhydrideSingle-molecule Localization MicroscopyHydrophobic InterfaceConstant Current RateMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-14 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Rational Mutagenesis Succinic Anhydride Single-molecule Localization Microscopy Hydrophobic Interface Constant Current Rate Medicine R Science Q
spellingShingle	Rational Mutagenesis Succinic Anhydride Single-molecule Localization Microscopy Hydrophobic Interface Constant Current Rate Medicine R Science Q Ilya D. Solovyev Alexandra V. Gavshina Aditya S. Katti Alexey I. Chizhik Leonid M. Vinokurov Grigory D. Lapshin Tatiana V. Ivashina Maria G. Khrenova Igor I. Kireev Ingo Gregor Jörg Enderlein Alexander P. Savitsky Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
description	Abstract Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein’s hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.
format	article
author	Ilya D. Solovyev Alexandra V. Gavshina Aditya S. Katti Alexey I. Chizhik Leonid M. Vinokurov Grigory D. Lapshin Tatiana V. Ivashina Maria G. Khrenova Igor I. Kireev Ingo Gregor Jörg Enderlein Alexander P. Savitsky
author_facet	Ilya D. Solovyev Alexandra V. Gavshina Aditya S. Katti Alexey I. Chizhik Leonid M. Vinokurov Grigory D. Lapshin Tatiana V. Ivashina Maria G. Khrenova Igor I. Kireev Ingo Gregor Jörg Enderlein Alexander P. Savitsky
author_sort	Ilya D. Solovyev
title	Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
title_short	Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
title_full	Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
title_fullStr	Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
title_full_unstemmed	Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
title_sort	monomerization of the photoconvertible fluorescent protein saasoti by rational mutagenesis of single amino acids
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/976772660578499693c31861f28eb1e2
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Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids

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