E. A. Goryacheva, A. V. Rossokhin, D. A. Ruchkin, A. M. Bogdanov, I. V. Artemyev, N. V. Pletneva, V. Z. Plenev
{"title":"mKate2-K67R/R197H—Extra-Bright Red Fluorescent Biomarker of New Generation. X-Ray Structure and Molecular Dynamic Properties","authors":"E. A. Goryacheva, A. V. Rossokhin, D. A. Ruchkin, A. M. Bogdanov, I. V. Artemyev, N. V. Pletneva, V. Z. Plenev","doi":"10.1134/S1068162024040113","DOIUrl":null,"url":null,"abstract":"<p><b>Objective:</b> 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 (λex/λem 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 ~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. <b>Methods:</b> The paper presents the results of X-ray and molecular dynamics study of new bright biomarker mKate2-K67R/R197H. <b>Results and Discussion:</b> The three dimensional structure of new advanced red fluorescent biomarker mKate2-K67R/R197H has been studied by X-ray method at 1.5Å resolution supported by molecular dynamics (MD) study The principal structural fold of the protein is an 11-stranded β-barrel. The nearest chromophore environment (≤ 4 Å) comprises 18 tightly packed residues. <b>Conclusions:</b> 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.</p>","PeriodicalId":758,"journal":{"name":"Russian Journal of Bioorganic Chemistry","volume":"50 4","pages":"1306 - 1313"},"PeriodicalIF":1.1000,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Russian Journal of Bioorganic Chemistry","FirstCategoryId":"92","ListUrlMain":"https://link.springer.com/article/10.1134/S1068162024040113","RegionNum":4,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Objective: 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 (λex/λem 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 ~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 1.5Å resolution supported by molecular dynamics (MD) study The principal structural fold of the protein is an 11-stranded β-barrel. The nearest chromophore environment (≤ 4 Å) 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.
期刊介绍:
Russian Journal of Bioorganic Chemistry publishes reviews and original experimental and theoretical studies on the structure, function, structure–activity relationships, and synthesis of biopolymers, such as proteins, nucleic acids, polysaccharides, mixed biopolymers, and their complexes, and low-molecular-weight biologically active compounds (peptides, sugars, lipids, antibiotics, etc.). The journal also covers selected aspects of neuro- and immunochemistry, biotechnology, and ecology.