Fluorescent proteinsAs a molecular probe, it can specifically label target proteins, cell structures, and organelles, and plays a vital role in live-cell imaging and super-resolution microscopy. Its brightness and photostability are the core indicators to measure the application value of fluorescent proteins in fluorescence microscopy. Since the successful cloning of the first green fluorescent protein (GFP) in 1992, scientists have been working to improve these two key properties of fluorescent proteins in order to expand their application in imaging techniques. More recently, one derived from CThe green fluorescent protein of the Uchidae jellyfish was found to be particularly photostable and far superior to any known fluorescent protein, and was engineered to be as bright as mneongreen, hence the namestaygold。However, staygold itself is a dimer, limiting its scope of application as a fluorescent labeling tool. February 26, 2024, School of Life Sciences, Westlake Universitykiryl piatkevichThe research group is jointly with Moscow State Universityfedor subachThe research group is innature methodsThe magazine published an articlebright and stable monomeric fluorescent protein derived from staygold。They pickUsing the strategy of directed evolution, a monomeric version of StayGold was successfully developed, named MBAOJIN。This fluorescent monomer not only inherits the excellent properties of the original protein, but also has the potential for a wider range of applications. By analyzing the crystal structure of MBAOJIN at different pH conditions and comparing it with StayGold and other mainstream fluorescent proteins, they also revealed the key amino acid mutations necessary for monomerization and further elucidated the molecular mechanism of its excellent photostability. The high brightness and photostability of MBAOJIN, coupled with its excellent chemical stability, make it an ideal fluorescent protein tool for studying the morphological and dynamic changes of cells and subcellular structures, especially super-resolution microscopy and expansion microscopy. 
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In order to monomerize the dimeric green fluorescent protein StayGold, the authors constructed a protein expression vector in Figure 1A by fusing the mutant library of SatyGold with the DNA binding domain (aracDNA) of the ARAC protein. When the mutant was a monomer, the expression level of the mutant protein-driven reporter gene mtagbfp fused with ARAC was relatively low. When the mutant is a dimer, the expression level of the mutant protein-driven reporter gene mtagbfp fused to ARAC is relatively high. During each round of random mutations, the authors selected colonies with darker blue fluorescence and brightest green fluorescence and confirmed their oligomeric status by liquid chromatography. After eight rounds of directed evolution, a green fluorescent protein monomer with the brightest brightness was finally determined, and the N-terminal and C-terminus of mneongreen protein were used as linker peptides to ensure stable expression in mammalian cells, and it was named mbaojin. Aligned with the amino acid sequence of StayGold, mbaojin has the following mutations: S55T, H77R, E80G, Q140P, H141Q, C165Y, N171Y, and T201A. In order to detect whether mbaojin is monomeric in mammalian cells, the authors expressed mbaojin fused to the endoplasmic reticulum anchor domain (cyterm) in hela cells, and counted the proportion of healthy cells without vortex structures. Tandem and monomerized versions of StayGold (TD8OxSTAYGOLD, StayGold-E138D, and MstayGold, also known as QC2-6 FIQ) were used as comparisons, as well as validated monomers (MEGFP, MCLover3, MGreenLantern) and weak dimers (EGFP, Venus) were used for comparison. Mbaojin has a score of 937%, reaching the threshold of monomerism in cells. These results establish that mbaojin is a monomeric protein. 
Monomerized staygoldCrystal structure of mbaojinIn the article, the author parses the ph of mbaojin in. 6 and 8The X-ray crystal structure at 5 and compared with the DIMER structure of STAYGOLD, it was found that the structure of MBAOJIN was not significantly different at different pH values, the dimeric interface of MBAOJIN had less contact between subunits A and B, and the chloride ion pockets near the fluorophore were similar and similar to STAYGOLD in all structures of MBAOJIN. To further understand the molecular basis of MBAOJIN's enhanced photostability, the authors performed point mutations of amino acids at sites that interact with fluorophores (numbered in PDB). It was found that the photobleaching rate of mbaojin N137K, mbaojin S134p and mbaojin V152e mutants was 15-6.5 and 45 times. Comparing the fluorophore environment of photobleachable Mbaojin protein and less photobleachable Mneongreen and EGFP proteins, it was found that the photobleachable Mbaojin protein formed more hydrogen and hydrophobic bonds around the fluorophore. The physicochemical properties of mbaojin were characterized in cells and purified proteinsAfter verifying that mbaojin is a monomeric protein, the authors further characterized the spectral and biochemical properties of the purified mbaojin protein. It was found that compared with StayGold, the monomerization mutation generated by mBaojin did not change the absorption and fluorescence spectral characteristics. However, the molecular brightness of mbaojin is 1 less than that of staygold09 times, but 1 brighter than mneongreen24 times. At low excitation intensities (about 13 mW mm2, live-cell imaging conditions), the photostability of MBAOJIN is 15-fold, 9-fold, and 130-fold higher than that of EGFP, mneongreen, and mgreenlantern, respectively, although this difference is less significant at high power (about 120 mW mm2). mbaojin matures rapidly at 37 and has a maturation time of 75 minutes, 1 faster than StayGold, Mneongreen, and EGFP8 times, 22x and 18 times, one of the fastest maturing fluorescent proteins. In addition, the fluorescence of MBAOJIN exhibits extremely high chemical stability, with a high pH stability of 4 compared to other monomeric GFPs37。After 24 h incubation in 6 M GDNHCL, the fluorescence of MBAOJIN increased by 18%, while the fluorescence of HFYFP, one of the most chemically stable fluorescent proteins, increased by only 1%, the fluorescence of StayGold decreased by 2%, and the fluorescence of MneonGreen was completely quenched. In HEK and HELA cells, mBaojin was co-expressed with red fluorescent protein (Fusionred or mcherry) by P2A, and red fluorescence normalized the expression level of the protein. Compared to EGFP, MBAOJIN consistently has approximately 70-140% higher intracellular brightness in HEK and HeLa cells, while being comparable to (N1) StayGold (C4) and MstayGold. In addition, the brightness of mbaojin in HEK cells pretreated with expansion microscopy was 17 times higher than that of mneongreen, hfyfp and mstaygold, respectively4 times, 38 times. In photobleaching experiments performed on HEK cells, the authors used 35 times the lighting power (i.e. 50.)5 mW mm2) so that the difference in photobleaching rate can be observed in a shorter period of time. In addition to staygold and its derived fluorescent proteins are 19 to 2With 3x the photostability, the photobleaching speed of MBAOJIN is 4 times slower than other existing GFPS6 to 85 times. StayGold's tandem dimer, TD8OX2STAYGOLD, outperformed all tested GFPs in terms of brightness and photostability, performing best in HEK cells. Live-cell imaging and super-resolution microscopy of mbaojinMbaojin was imaged after fusion with structural proteins, including vimentin, mitochondrial presequence of human cytochrome c oxidase subunit viii, h2b, keratin, tubulin, actin, etc. The authors used super-resolution confocal microscopy to image the fusion proteins of mbaojin and mneogreen with -tubulin for up to 60 minutes, and mbaojin did not undergo photobleaching, while mneongreen reduced the initial fluorescence value by 25% under the same conditions. Actin fibers in living cells were then imaged using sparse deconvoluted structural illumination microscopy, and the photostability of Mbaojin was 2. higher than that of MneonGreen at higher illumination power (150-200 MW mm2).5-4 times. **In worms, mbaojin exhibits a 4-fold slower photobleaching rate than mneongreen in neurons with intracellular brightness in neurons, retaining more than 50% of initial fluorescence after 15 minutes of photobleaching. In living mouse brain tissue, mbaojin is brighter than mneongreen7 times, but 1 times darker than mstaygold7 times. However, in PFA-fixed brain tissue, MstayGold and Mbaojin exhibited comparable brightness and photostability, significantly better than MneonGreen. Due to its high chemical stability, MBAOJIN is used in expansive microscopy imaging of mitochondria, microfilament microtubules, and mouse brain tissue of HeLa cells. Importantly,MBAOJIN also maintains its high photostability in expanded brain tissue, enabling large-volume super-resolution imaging while reducing photobleaching. Kiryl D., Westlake UniversityResearcher Piatkevich and Moscow State University Fedor VProf. Subach is the co-corresponding author of **, Hanbin Zhang, a Ph.D. candidate at Westlake University, and Gleb D. at Moscow State UniversityDr. Lesnov is the co-first author of this paper, and Wenhao Zhang, a Ph.D. student at Westlake University, and St**rini Papadaki, a research assistant, participated in some of the research. **The plasmids used in the project can be obtained free of charge through the plasmid platform of the Westlake Experiment: . Also, Kiryl DIn 2023, Piatkevich published a paper in Nature Methods for the systematic characterization of near-infrared fluorescent proteins, and related plasmids are also available on the plasmid platform of the Westlake experiment. Wekwikgene is the first plasmid bank in China, initiated by Professor Piatkevich and funded by the Westlake Life Science and Biomedical Laboratory. All plasmids undergo rigorous quality control and testing. On **, you can find the complete sequence of each plasmid, while it provides a user-friendly experience, supports the creation of user accounts and lab pages, and provides the opportunity to increase the visibility of scientific work. Wekwikgene guarantees fast and free delivery of high-quality plasmids to support and promote open science. Original link:
References
1. a highly photostable and bright green fluorescent protein |nature biotechnology
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