Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

Abstract

The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by electrophysiological techniques from protein heterologously expressed in Xenopus oocytes and mammalian cells. On the other hand, expression and purification of these proteins for biophysical and structural analyses in vitro is problematic and expensive and, accordingly, only limited information on the purified channels is available in the literature. Here we describe a protocol for binding studies of fluorescently labeled cyclic nucleotides to a homologue of eukaryotic CNG channels. Furthermore, we describe how to directly probe binding of unlabeled cyclic nucleotides in a competition assay. The use of fluorescence as a sensitive probe for ligand binding reduces the amount of protein needed and enables fast and easy measurements using standard laboratory equipment.

Discovery of periplasmic solute binding proteins with specificity for ketone bodies: β‐hydroxybutyrate binding proteins

“…For pre-incubation experiments, protein samples were incubated in Protein LoBind ® Tubes (Eppendorf) with different concentrations of ligand for 30 min at room temperature, and the fluorescence spectra (excitation: 295 nm, emission sweep: 315-360 nm) of the mixtures were measured sequentially inside of a microvolume cuvette with the JASCO FP-8300 spectrofluorometer. For the titration experiments, we adapted a protocol from Schmidpeter and Nimigean, where 2 mL of a blank protein solution was titrated with stock containing an excess of protein/ligand complex (Schmidpeter & Nimigean, 2018). The solution was stirred inside of the cuvette for 2 min at 1500 rpm with a 2 mm diameter bar after each injection.…”

“…As a result, it was difficult to distinguish changes below the saturation point with the pre-incubation assay in a microvolume cuvette. Directly titrating the ligands in a 2 mL cuvette was a suitable experimental vehicle to confirm the results from the pre-incubation experiments and obtain more points to fit binding constants (Schmidpeter & Nimigean, 2018). These experiments were performed with respect to a reference cuvette, which helped to mitigate time and volume dependent changes in the fluorescence signal.…”

Design principles for cyclin K molecular glue degraders

“…b , In vitro TR-FRET complex formation assay performed as a compound titration with 50 nM CDK12-cyclin K (left) or 10 nM CDK12-cyclin K (right) for several compounds (n = 1). Data were fitted with a quadratic equation appropriate for a case where the expected K d value is lower than the protein concentration used 69 . Lowering the protein concentration resulted in a much smaller assay window but yielded K d * values in the sub-nanomolar range for the top compounds (DS17, DS73), while showing no difference for the weak recruiter roscovitine, indicating that the tightest glues lie below the limit of detection of the TR-FRET assay (this is also highlighted by the spurious fit observed for DS17).…”