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A dsDNA-lighted fluorophore for monitoring protein-ligand interaction through binding-mediated DNA protection
摘要： Because of their important roles in cellular functions, life activities, drug screening, and disease treatment, the development of efficient methods for monitoring protein-ligand interaction is essential. In this study, inspired by our previous studies on DNA conformation-selective fluorescent indicators, we developed a new sensing platform for monitoring protein-ligand interaction and detecting protein activity based on binding-mediated DNA protection and the dsDNA-lighted fluorophore, ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] butanoate (EBCB). The ligand was purposefully linked to the 3?-terminal of a hairpin DNA probe to selectively bind with the target protein and protect the DNA from cleavage by exonuclease III. By virtue of EBCB’s outstanding capacity to discriminate DNA conformation, the protein-ligand interaction could be effectively monitored through a fluorescence change in EBCB. A high fluorescence signal was detected when the hairpin DNA was protected in the presence of the target protein, whereas a much lower signal was observed in the presence of nontarget proteins. Our results demonstrated that the proposed strategy had high potential, such as high selectivity and relatively high sensitivity, for monitoring protein-ligand interaction and detecting protein activity. We believe these results will pave the way for applying dsDNA-lighted fluorophore EBCB as an effective signal transducer for DNA conformation transformation-mediated biochemical sensing.
关键词： DNA protection,protein-ligand interaction,dsDNA-lighted fluorophore,fluorescence detection
更新于2025-09-23 15:23:52
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A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser
摘要： The hydroxymethylation of cytosine bases plays a vital role in the phage DNA protection system inside the host Escherichia coli. This modification is known to be catalyzed by the dCMP hydroxymethylase from bacteriophage T4 (T4dCH); structural information on the complexes with the substrate, dCMP and the co-factor, tetrahydrofolate is currently available. However, the detailed mechanism has not been understood clearly owing to a lack of structure in the complex with a reaction intermediate. We have applied the X-ray free electron laser (XFEL) technique to determine a high-resolution structure of a T4dCH D179N active site mutant. The XFEL structure was determined at room temperature and exhibited several unique features in comparison with previously determined structures. Unexpectedly, we observed a bulky electron density at the active site of the mutant that originated from the physiological host (i.e., E. coli). Mass-spectrometric analysis and a cautious interpretation of an electron density map indicated that it was a dTMP molecule. The bound dTMP mimicked the methylene intermediate from dCMP to 5′-hydroxymethy-dCMP, and a critical water molecule for the final hydroxylation was convincingly identified. Therefore, this study provides information that contributes to the understanding of hydroxymethylation.
关键词： hydroxymethylation,XFEL,dCMP hydroxymethylase,X-ray free electron laser,T4dCH,hydroxylation,cytosine bases,phage DNA protection,dTMP,Escherichia coli,bacteriophage T4,methylene intermediate
更新于2025-09-12 10:27:22