a pyrene maleimide with a flexible linker for sampling of longer inter-thiol distances by excimer formation一个芘马来酰亚胺与灵活的链接器采样时间inter-thiol距离的准分子的形成.pdfVIP

A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer Formation 1 2 1 2 2 Satomi Niwayama , Abdullatif S. Kassar , Tian Zhao , Roger B. Sutton , Guillermo A. Altenberg * 1 Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, United States of America, 2 Department of Cell Physiology and Molecular Biophysics, and Center for Membrane Protein Research, Texas Tech Health Sciences Center, Lubbock, Texas, United States of America Abstract 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 excim