Goryacheva, E. A.Rossokhin, A. V.Ruchkin, D. A.Bogdanov, A. M.Artemyev, I. V.Pletneva, N. V.Plenev, V. Z.2024-09-242024-09-2420241068-16201608-330Xhttps://doi.org/10.1134/S1068162024040113https://hdl.handle.net/11147/14684Ruchkin, Dmitry/0009-0009-8925-9388Objective: Cell biology continuously shows the need for new fluorescent tags with advanced properties. The object of our current study is a new genetically encoded monomeric red fluorescent biomarker mKate2-K67R/R197H (lambda ex/lambda em 579/603 mn), designed from commercial biomarker mKate2 by two R197H/K67R mutations. The mKate2 precursor, a far-red fluorescent protein, is nearly 3-fold brighter than the previously designed mKate. Compared with commercial mKate2, the double mutant mKate2-K67R/R197H (alternative names FusionRed2 and Diogenes) exhibits an additional similar to 1.6-fold increase in fluorescence brightness and represents the next generation of extra-bright red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals. Methods: The paper presents the results of X-ray and molecular dynamics study of new bright biomarker mKate2-K67R/R197H. Results and Discussion: The three dimensional structure of new advanced red fluorescent biomarker mKate2-K67R/R197H has been studied by X-ray method at 1.5 angstrom resolution supported by molecular dynamics (MD) study The principal structural fold of the protein is an 11-stranded beta-barrel. The nearest chromophore environment (<= 4 angstrom) comprises 18 tightly packed residues. Conclusions: The MD study showed that the brightness of mKate2-K67R/R197H and its mKate2 precursor correlates with the dipole moments of the amino acid environments of the chromophores. The higher the dipole moment, the higher the brightness of biomarker.eninfo:eu-repo/semantics/closedAccessX-ray studymolecular dynamicsArticle2-s2.0-8520047693810.1134/S1068162024040113