Fig. 3
From: NanoBRET in C. elegans illuminates functional receptor interactions in real time
Affinity and specificity of TAM-labeled peptides at their corresponding receptors. BRET binding assays were performed to determine the binding of TAM-labeled peptides to the Nluc-fused receptor with slightly incised Nluc-expressing worms after 25 min incubation. A Schematic depiction of Nluc::NPR-11 (top, left) and Nluc::LAT-1 (bottom, left) with the amino acid sequence of the respective TAM-labeled FLP-34-1 and pLAT-1 and their corresponding scrambled (scr) versions (right). In TAM-pLAT-1 and TAM-scrpLAT-1, the fluorophore is attached to the side chain of the additional amino acid Dap (diaminopropionic acid). B TAM-FLP-34-1 binding to Nluc::NPR-11 shows a BRET window with a Kd value of 1.4 μM (left panel). Although there is no significant difference between the values from a) and b) (two-way ANOVA), the specific window appears clearly when calculating the netBRET between agonistic and scrambled control peptide (right panel). C Displacement assay based on NanoBRET. 1.6 μM TAM-FLP-34-1 were displaced with increasing concentrations of unlabeled FLP-34-1 at Nluc::NPR-11. FLP-34-1 is able to displace TAM-FLP-34-1 with a Ki value of 1.1 μM. The background signal from filter overlap is 0.10 and is indicated as dashed line, representing complete displacement. D TAM-pLAT-1 binding to Nluc::LAT-1 generates a BRET window, which is even larger with TAM-scrpLAT-1. This BRET signal was not saturable up to 10 μM, and thus, the Kd was estimated to be > 10 μM for both peptides. E NanoBRET of Nluc::LAT-1 incubated with TAM-pLAT-1 (0.5 μM) without and with 10 μM unlabeled pLAT-1 was determined after 60 min incubation showing that pLAT-1 is able to displace TAM-pLAT-1. Depicted in B-E is the mean ± SEM of n ≥ 3 assays with N = 30 worms in triplicates; ** p ≤ 0.01; two-sided t-test. Data in B and D are baseline-corrected to the respective 0 μM value. Luminescence and fluorescence values are given in Table S1. Note that calculation of BRET ratios are based on the raw luminescence and fluorescence values of each performed experiment. For these values, see Table S4. Schematic images were created with BioRender.com