Small-molecule fluorophores are indispensable tools in chemical biology, including, for probing biomolecular structure, dynamics and microenvironments. Boron-dipyrromethene (BODIPY) dyes are such a class of fluorophores that have emerged as versatile agents in recent years due to their tunable emission profiles, high quantum yields, and multiple spectroscopic advantages. Particularly, solvatochromic BODIPYs have garnered significant interest due to their ability to exhibit exceptional sensitivity to both macro and micro-environmental polarity. This phenomenon, described as solvatofluorochromism, has enabled their application in polarity mapping, probing cellular processes, developing novel sensors and imaging live cells and tissues.
The function of proteins depends not only on their static architecture but also on dynamic conformational changes. This is particularly true for G protein-coupled receptors (GPCRs), whose conformational landscapes underpin >35% of marketed therapeutics, even though only a small proportion of the ~800 human GPCRs have been translated into clinical targets. Therefore, to understand how their dyanmic conformational states change, which are poorly addressed by traditional techniques such as cryo-electron microscopy and crystallography that capture only static receptor structures, we focus on utilizing solvatofluorchromic BODIPYs showing large changes in emission wavelength, quantum yield and fluorescence lifetime depending on local polarity and molecular conformation.
This work focuses on the design and application of a polarity-sensitive BODIPY fluorophore with pronounced charge-transfer character, covalently conjugated to human and salmon calcitonin (hCT,sCT) to generate environment-responsive peptide ligands for the human calcitonin receptor (hCTR), a Class B GPCR. Upon confirmation that these fluorophore-labelled ligands retain pharmacology of their parent hormones, multiple advanced techniques such as fluorescence spectroscopy, confocal microscopy and fluorescence lifetime imaging microscopy (FLIM) were used to observe differences in conformational dynamics upon binding of hCT and sCT to hCTR. This study thereby provides direct evidence supporting previous indirect observations that ligand-dependent conformational dynamics directly correlate with G protein activation.