At present, it is believed that the synthesis of the chromophore of red fluorescent proteins passes through an intermediate “blue” form with an absorption maximum at 400 nm, which directly turns into “red” through the formation of a double bond in the side chain of the chromophore-forming residue Tyr. In the present work, using the example of a chromoprotein from Actinia equina (aeCP), it is shown for the first time that the synthesis of a DsRed-type chromophore can proceed through an intermediate “green” form with an absorption maximum at 530 nm.

-Extracellular mildly alkaline pH sensor named SypHerExtra was created, representing fusion of previously described SypHer3s sensor with the transmembrane domain of neurexin-1. 

-It was shown that using 445 nm excitation light the fluorescence lifetimes of both SypHer3s and SypHerExtra strongly depend on pH.

-These two sensors are suitable for quantitative measurements using the FLIM method to determine intracellular and extracellular pH in a range from pH 6.5 to 9.5 in different biological systems.

Through the examples of two highly homologous fluorescent proteins from Zoanthus sp. (zoanGFP and zoan2RFP), amino acid residues participating in the transformation of a protein with the green fluorescence (GFP) into the red fluorescent protein (RFP) were explored. As the result of zoanGFP mutagenesis, internal amino acid residues (a.a.r.) became identical to those of zoan2RFP. However, this mutant underwent only partial transformation into the red form. To elucidate the extra factors that might affect red chromophore biosynthesis, we used comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. As the result, additional a.a.r. were discovered on the surface of the protein that might influence both the arrangement and flexibility of the chromophore-surrounding a.a.r. Site-directed mutagenesis of these external a.a.r. confirmed the crucial role of these residues in red chromophore biosynthesis.

A new organic-compound-based fluorogenic marker has been created for live-cell membrane staining. Unlike current commercial cell markers, the obtained fluorogenic marker does not fluoresce in the aquatic environment, but acquires fluorescence immediately after being placed in a nonpolar medium, for example, in the cell membrane. This property allows one to instantly stain cells without further washing out the unbound dye. This marker can be applied in fluorescence microscopy for live-cell imaging and flow cytometry.