Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
2018 | journal article. A publication with affiliation to the University of Göttingen.
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Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids
Solovyev, I. D.; Gavshina, A. V.; Katti, A. S.; Chizhik, A. I. ; Vinokurov, L. M.; Lapshin, G. D. & Ivashina, T. V. et al. (2018)
Scientific Reports, 8(1) art. 15542. DOI: https://doi.org/10.1038/s41598-018-33250-z
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Details
- Authors
- Solovyev, Ilya D.; Gavshina, Alexandra V.; Katti, Aditya S.; Chizhik, Alexey I. ; Vinokurov, Leonid M.; Lapshin, Grigory D.; Ivashina, Tatiana V.; Khrenova, Maria G.; Kireev, Igor I.; Gregor, Ingo ; Enderlein, Jörg ; Savitsky, Alexander P.
- 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.
- Issue Date
- 2018
- Journal
- Scientific Reports
- Organization
- Fakultät für Physik
- Language
- English