A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer FormationReportar como inadecuado




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Pyrene-containing compounds are commonly used in a number of fluorescence-based applications because they can form excited-state dimers excimers by stacking interaction between excited-state and ground-state monomers. Their usefulness arises from the facts that excimer formation requires close proximity between the pyrenes and that the excimer emission spectrum is very different from that of the monomers. One of many applications is to assess proximity between specific sites of macromolecules labeled with pyrenes. This has been done using pyrene maleimide, a reagent that reacts with reduced thiols of cysteines, but its use for structural studies of proteins has been rather limited. This is because the introduction of two cysteines at sufficiently close distance from each other to obtain excimer fluorescence upon labeling with pyrene maleimide requires detailed knowledge of the protein structure or extensive site-directed mutagenesis trials. We synthesized and tested a new compound with a 4-carbon methylene linker placed between the maleimide and the pyrene pyrene-4-maleimide, with the aim of increasing the sampling distance for excimer formation and making the use of excimer fluorescence simpler and more widespread. We tested the new compound on thiol-modified oligonucleotides and showed that it can detect proximity between thiols beyond the reach of pyrene maleimide. Based on its spectroscopic and chemical properties, we suggest that pyrene-4-maleimide is an excellent probe to assess proximities between cysteines in proteins and thiols in other macromolecules, as well as to follow conformational changes.



Autor: Satomi Niwayama, Abdullatif S. Kassar, Tian Zhao, Roger B. Sutton, Guillermo A. Altenberg

Fuente: http://plos.srce.hr/



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