A Fluorescence Assay for Leucine Zipper Dimerization: Avoiding Unintended Consequences of Fluorophore Attachment

Formation of R-helical coiled-coil dimers represents one of the simplest examples of a specific protein-protein interaction. This dimerization mode is commonly observed among transcription regulator proteins, where the process is referred to as leucine zipper formation. Inhibitors of leucine zipper dimerization would allow one to control gene expression. As a first step toward identifying such inhibitors, we have developed a fluorescence-based assay for homodimerization of peptides corresponding to the leucine zipper region of the Jun protein. The assay involves attachment of 7-hydroxycoumarin to the N-terminus of the Jun peptide. Upon dimerization, pairs of fluorophores are held near one another, which leads to self-quenching. Disruption of the dimer is signaled by an increase of 7-hydroxycoumarin fluorescence. Development of this assay proved to be more complex than expected, because we found that two commonly used fluorescent tags, pyrene and fluorescein, induce high-order aggregation of leucine zipper-fluorophore conjugates. This aggregation was demonstrated to result from cooperativity between the fluorophore and peptide portions. A third common fluorophore, 7-diethylaminocoumarin, significantly stabilized the Jun homodimer. Of the four fluorophores we examined, 7-hydroxycoumarin caused the least perturbation of leucine zipper peptide behavior.