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 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.

Novel Phototransformable Fluorescent Protein SAASoti with Unique Photochemical Properties

“…For different photoconvertible FPs, green-to-red PC was shown to proceed incompletely [20]. As it was demonstrated earlier [23], formation of the red form in the case of SAASoti is accomplished by its subsequent photobleaching under 400 nm. Increasing the 400 nm exposure time in the case of SAASoti leads to the photodestruction of both forms [22].…”

“…Previously, we investigated thermal relaxation and calculated corresponding constants (τ 1/2 = 30 min at 25 °C) as SAASoti can be irreversibly converted into the red fluorescent form by 400 nm illumination at pH 7.5 [24]. Figure 2 shows that ‘off’-switching with subsequent 400 nm illumination of the probe can be repeated with the same sample several times without PC to the red form at pH 9.2 (pH-dependency of SAASoti PC was done previously [23]). Green fluorescence photoswitching to the dark state (Figure 2A) at 470 nm (160 mW/cm 2 ) can be described by a bi-exponential model (Figure S1A).…”

“…V127T SAASoti was overexpressed in E. coli BL21 (DE3) as described previously [23]. Protein purity was analyzed by gel-electrophoresis.…”

“…Green-to-red PC occurs under 400 nm illumination and is accompanied by a peptide bond break, as it proceeds in Kaede-like proteins [11]. A 3D tetrameric model of SAASoti was constructed based on the known Kaede structure (PDB ID: 2GW3) [23]. While the wild type SAASoti exists as a tetramer tending towards aggregation, a V127T point mutation at its hydrophobic interface resulted in the monomeric variant of SAASoti [23].…”

“…A 3D tetrameric model of SAASoti was constructed based on the known Kaede structure (PDB ID: 2GW3) [23]. While the wild type SAASoti exists as a tetramer tending towards aggregation, a V127T point mutation at its hydrophobic interface resulted in the monomeric variant of SAASoti [23]. From here on, we will describe this monomeric form—V127T SAASoti.…”