Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids. Issue 1 (December 2018)

• Record Type: Journal Article
• Authors: Solovyev, Ilya; Gavshina, Alexandra; Katti, Aditya; Chizhik, Alexey; Vinokurov, Leonid; Lapshin, Grigory; Ivashina, Tatiana; Khrenova, Maria; Kireev, Igor; Gregor, Ingo; Enderlein, Jörg; Savitsky, Alexander
• Is Part Of: Scientific reports. Volume 8:Issue 1(2018)
• Page Start: 1
• Page End: 14
• Publication Date: 2018-12
• Subjects: Natural history -- Research -- Periodicals; Biology -- Research -- Periodicals; Physical sciences -- Research -- Periodicals; Earth sciences -- Research -- Periodicals; Environmental sciences -- Research -- Periodicals; 502.85
• DOI: 10.1038/s41598-018-33250-z
• Languages: English
• ISSNs: 2045-2322

Abstract

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 forin vivo cell studies.