Site-Directed Fluorescence Labeling (SDFL): TrIQ Methods Provide Insights Using the Fluorescent Probe Bimane

This review is in honor of Professor Wayne Hubbell’s 80th birthday, and his many contributions to the philosophy and practice of science. He continues to pioneer the use of site-directed spin labeling (SDSL) as a powerful tool for studying protein structure and dynamics. Inspired by his efforts, our lab has worked to develop site-directed fluorescence labeling (SDFL) as a complementary approach to SDSL. Throughout this process, we have emulated many of the novel methods and concepts Dr. Hubbell and his colleagues used during their development of SDSL. Some of our approaches are discussed here, not in a comprehensive or exhaustive way, but to highlight SDFL methods we have found to be robust, reproducible, and relatively easy to use. Specifically, we will focus on the development and application of SDFL using the fluorescent probe bimane, and how the unique properties of bimane can be exploited to glean information about protein structure and function. We also review past examples of how we have used bimane and SDFL to investigate the activation of G protein-coupled receptors (GPCRs) and their interaction with key binding partners.