Development of Dimaleimide Based Fluorogens
An Application for Protein Labelling
- Development of Dimaleimide Based Fluorogens - An Application for Protein Labelling
- Fig. 1: Strategy for protein labelling.
- Fig. 2: Fluorogenic Addition Reaction (FIARe) labelling strategy.
- Fig. 3: Fluorescence enhancement of fluorogens upon protein labelling. 25 µM fluorogen (black) and 25 µM fluorogen reacted with one equivalent of test protein MBP-dC10α (red). λex = 440 nm. Insert: model of MBP-dC10α.
- Fig. 4: Intracellular labelling of test protein RFP-α with fluorogen. Images D-F show cells transfected with the test protein, while images A-C show cells transfected with empty (mock) vector. Images A & D were obtained in the green channel (fluorogen emission) and images B & E were obtained in the red channel (RFP emission). Images C & F were under bright field (visible) illumination.
We have developed a fluorescent protein labelling strategy where a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. In this work, novel fluorogens were developed to improve the labelling specificity. The breakthrough represented by this improvement was demonstrated herein, in the rapid and specific labelling of an intracellular POI.
Need for Direct Labelling of Specific Proteins
A huge number of putative genes have been identified through the sequencing of the human genome ; however, the functional role of only a few gene products can be inferred from their primary sequences. For the others, assigning functional roles will require new methods for monitoring the functions and interactions of proteins in different environment. Visualizing and following proteins, with minimal disruption of their function and distribution, is one of the foremost challenges in protein studies. Fluorescent labelling is one of the most widely used methods to study proteins function, localization and trafficking. Some labelling techniques are based on the use of fluorescent dyes bearing reactive functional groups such as succinimidyl esters or maleimides, known to react with amines or thiols, respectively . However, these methods are typically non-specific. Therefore, there is a need to develop fluorogenic and highly specific methods to label proteins, more importantly, to allow the direct labelling of a specific protein in vivo.
Criteria for Satisfying Labelling Results
Our lab has developed a two-step strategy for protein labelling the protein of interest is genetically tagged with a short peptide sequence presenting two Cys residues separated by two turns of an α-helix, which can selectively undergo covalent bond formation with synthetic fluorogenic reagents bearing two maleimide groups. The specificity of the recognition is determined by the distance, which is ~10 Å between the two cysteine residues on the dicysteine tag (dC10α tag) and also between the two maleimide groups on the labelling reagent (dM10) (fig.
2). To achieve the best labelling results, certain criteria must be satisfied. For example, the labelling reagent needs to be non-fluorescent itself but highly fluorescent upon labelling. It must also be specific to the protein of interest but not sensitive to other components present in biological milieu. We have directed our recent efforts on the modification of our labelling strategy to make it work in vivo.
Our fluorogenic reagents have fluorophores with a reactive unit bearing two maleimide groups, such that their fluorescence is quenched by photoinduced electron transfer (PET) until both maleimide groups undergo specific thiol addition reactions . Based on our understanding of the quenching mechanism, we carried out studies of the effect of spacer length and conformation on the fluorescence quenching and thus confirmed that the direct linkage of fluorophore and the dimaleimide scaffold is critical for quenching efficiency [4-6]. We then applied these principles to a widely used fluorophore (coumarin) to design fluorogenic reagents that have negligible background fluorescence, but show strong fluorescence after reacting with our target peptide tag (fig. 3).
Experiment and Results
Various proteins were successfully labelled by these reagents in vitro and kinetic studies were performed to elucidate the mechanism of the labelling process. Furthermore, we modified our dimaleimide reactive unit to achieve high selectivity for our peptide tag over the other thiol sources present in biological milieu. The strategy for this modification was based on the design of an asymmetric dimaleimide moiety, wherein the intrinsic reactivity of one maleimide was greatly reduced, effectively suppressing its intermolecular reaction. However, in the reaction with an appropriate dithiol, the reaction of the less reactive maleimide was intramolecular, and efficiently accelerated by the high effective molarity of the adjacent second thiol . We tested the reaction rate of our modified fluorogenic labelling reagent with tagged target protein and glutathione, the most abundant intracellular thiol that leads to non-specific labelling. Our results showed that for the improved fluorogen, the reaction with GSH was completely suppressed, while the reaction with MBP-dC10α was still rapid.
With this improvement, we were able to employ this method for intracellular labelling, demonstrating a rapid and specific labelling of a protein-of-interest expressed in mammalian cells. Red fluorescent protein (RFP) was chosen as a target protein so that its intrinsic fluorescence would serve as a control for the transfection and expression of the target protein. As shown in figure 4, HEK293T cells transfected with the plasmid coding for expression of the test protein showed red fluorescence throughout the cell (cytosol and nucleus), indicative of test protein expression (fig. 4E), figure 4A shows that minimal background fluorescence was observed, indicative of the low level of non-specific fluorescent labelling. Furthermore, fluorescent labelling is most intense and almost exclusively limited to cells (fig. 4D) that express the target red fluorescent test protein (fig. 4E).
Taken together, these results confirm the selectivity of fluorescent labelling of a dC10α-tagged target protein in living cells. Overall, our new strategy has shown the potential to label proteins with complex functions with precise temporal and spatial resolution. To explore and further extend the application of this strategy, we are making fluorogenic reagents with a wide range of excitation wavelengths (from UV to far-red) and testing our strategy with proteins of diverse distribution and function in different cell lines.
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Dr. Yingche Chen
Prof. Jeffrey W. Keillor
University of Ottawa