The role of the correlated motion(s) of the chromophore in photoswitching of green and red forms of the photoconvertible fluorescent protein mSAASoti.

Wild-type SAASoti and its monomeric variant mSAASoti can undergo phototransformations, including reversible photoswitching of the green form to a nonfluorescent state and irreversible green-to-red photoconversion. In this study, we extend the photochemistry of mSAASoti variants to enable reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues.

The Role of the 145 Residue in Photochemical Properties of the Biphotochromic Protein mSAASoti: Brightness versus Photoconversion.

Photoswitchable fluorescent proteins (FPs) have become indispensable tools for studying life sciences. mSAASoti FP, a biphotochromic FP, is an important representative of this protein family. We created a series of mSAASoti mutants in order to obtain fast photoswitchable variants with high brightness.

Novel Phototransformable Fluorescent Protein SAASoti with Unique Photochemical Properties.

SAASoti is a unique fluorescent protein (FP) that combines properties of green-to-red photoconversion and reversible photoswitching (in its green state), without any amino acid substitutions in the wild type gene. In the present work, we investigated its ability to photoswitch between fluorescent red ('on') and dark ('off') states. Surprisingly, generated by 400 nm exposure, the red form of SAASoti (R1) does not exhibit any reversible photoswitching behavior under 550 nm illumination, while a combination of prior 470 nm and subsequent 400 nm irradiation led to the appearance of another-R2-form that can be partially photoswitched (550 nm) to the dark state, with a very fast recovery time.

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

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.