Angelw: bismaleimide BODIPY fluorescent probe for protein specific labeling

Labeling specific target proteins (POI) with fluorescent probes has become one of the main ways to study protein expression, localization and transport However, most of the fluorescent probes used for labeling are "always on" type, which limits its wide application Therefore, the design and synthesis of "turn on" fluorescent probes for specific protein labeling has become a research hotspot Recently, Professor Jeffrey W Keillor of the University of Ottawa, Canada, designed and synthesized the bismaleimide fluorescence group 4 (also known as yc23) The molecule is based on the basic framework of dibromopyrrole (BODIPY), which is linked to the bismaleimide, and its photoinduced electron transfer (PET) characteristics cause the fluorescence quenching of BODIPY Only when the sulfhydryl group of a specific protein reacts with maleimide, the fluorescence of BODIPY can be restored (Fig 1) The molecule was also applied to fluorescent labeling of specific proteins in lysates and mammals Relevant achievements were published on a gew Chem Int ed (DOI: 10.1002 / anie 201805482) under the title of "a green BODIPY based, super flexible, protein specific labeling agent" Figure 1 The mechanism of yc23 acting on a specific protein (source: a gew Chem Int ed.) the author selected maltose binding protein (MBP) as the test protein, and the genetically coded peptide label DC10 α was labeled at the C-terminal to obtain mbp-dc10 α Then, the author studied the reaction of mbp-dc10 α with yc23 The results showed that yc23 showed negligible background fluorescence (Figure 2, black line on the x-axis) before the reaction with the test protein With the increase of mbp-dc10 α, the fluorescence intensity of yc23 increased significantly (Fig 2) Figure 2 The relationship between the amount of mbp-dc10 α and the fluorescence intensity of yc23 (source: a gew Chem Int ed.) Then, the author evaluated the kinetics of the reaction between 25 μ m mbp-dc10 α and 25 μ m yc23, and explored the function model fitted by the two addition reactions The results showed that it was mainly matched with the second-order model, and the rate constant was 868 ± 118 M-1 min-1 (Figure 3) Fig 3 Fitting diagram of fluorescence intensity with time after reaction of quantitative yc23 and quantitative mbp-dc10 α (source: a gew Chem Int ed.) Next, in order to explore the selectivity of mbp-dc10 α and yc23 binding, the author labeled mbp-dc10 α with yc23 in bacterial lysate and raised the temperature to 95 The temperature was maintained at 15 min and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) The results showed that the labeled test protein maintained its bright fluorescence all the time, even under denaturing conditions (Fig 4b) In the complex biological mixture of cell lysate, yc23 as low as 10 μ m is enough to mark the test protein visually The comparison between figure 4A and figure 4B reveals the selectivity of yc23, only the target protein is clearly labeled and emits strong fluorescence Figure 4 The selectivity analysis of mbp-dc10 α combined with yc23 (source: a gew Chem Int ed.) explored the selectivity of yc23 in bacterial lysate, and then the author studied its related characteristics in mammalian cells The histone H2B, which only exists in the nucleus, was selected as the specific protein, and dC10 α was labeled at its C end Then, Hela cells were transfected with plasmids that can express related proteins, and YC23 was incubated with Hela cells The results showed that the fluorescence of yc23 only existed in the nucleus, which proved that yc23 had good selectivity in mammalian cells (Fig 5) Figure 5 Protein labeling imaging of HeLa cells (source: a gew Chem Int ed.) Summary: the author has developed a new type of Bismaleimide BODIPY fluorescent probe for protein labeling, which can label specific proteins in bacterial lysate and mammalian cells, and has high selectivity and stability, which is useful for exploring the expression, location and transport of proteins It has important application value.