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. 2024 Dec 10;13:RP99854. doi: 10.7554/eLife.99854

Figure 3. Steady-state tmFRET data from tetrameric SthKCterm.

(A) Left: structure of one subunit of SthKCterm-S361Acd with [Fe(phenM)3]2+ acceptor incorporated at V416C (adapted from PDB: 4D7T; Kesters et al., 2015). Middle: Time course of average fluorescence upon addition of [Fe(phenM)3]2+ and then cAMP or cGMP (apo, n=8; cAMP n=6; and cGMP, n=2). Right: summary of the FRET efficiencies from individual experiments, with mean values as horizontal lines. (B) Left: Time course of average fluorescence for same site upon addition of [Ru(bpy)2phenM]2+ acceptor and then cAMP or cGMP (apo, n=8; cAMP, n=4; and cGMP, n=4). Right: summary of FRET efficiencies, with mean values as horizontal lines. (C) Left: structure of one subunit of SthKCterm-Q364Acd-417C with [Cu(TETAC)]2+ acceptor incorporated at R417C. Middle: Time course of averaged fluorescence upon addition of [Cu(TETAC)]2+ then cAMP or cGMP (apo, n=8; cAMP, n=5; and cAMP, n=3). Right: summary of FRET efficiencies, with mean values as horizontal lines. (D) Dose response relations of FRET efficiency change as a function of cAMP concentration normalized for comparison and fit with Hill equations (K1/2: 0.25±0.01 µM, 0.14±0.01 µM, 0.21±0.02 µM, and h:1.2±0.07, 1, and 1 for [Fe(phenM)3]2+, magenta; [Ru(bpy)2phenM]2+, orange; and [Cu(TETAC)]2+, cyan respectively, ± SD).

Figure 3—source data 1. Excel data for time courses and dot plots of steady-state FRET efficiencies and dose response (Figure 3A–D).

Figure 3.

Figure 3—figure supplement 1. Steady-state tmFRET of monomeric and tetrameric SthKCterm.

Figure 3—figure supplement 1.

(A) SEC chromatogram showing separation of monomeric (empty triangle, blue area) and tetrameric protein (solid triangle, gray area) fractions for SthKCterm-I359Acd and SthKCterm-I359Acd-R417C (no WT SthKC-term present) on a Superdex 75 increase 10/300 column (GE Healthcare). (B) Structure showing SthKCterm-I359Acd with [Cu(TETAC)]2+ acceptor at R417C (PDB:4D7T). (C) Fluorescence time-course of SthKCterm-I359Acd-417C upon addition of [Cu(TETAC)]2+, cAMP and TCEP in tetrameric protein (closed diamonds) and monomeric protein (open triangles). (D) Summary of FRET efficiencies for monomer and tetramer, with mean values shown as horizontal lines. Left: SthKCterm-I359Acd-R417C-[Cu(TETAC)]2+ (apo, *p=0.0005, cAMP, p=0.06). Right: SthKCterm-S361Acd-V416C-[Fe(phenM)3]2+ (apo, *p=0.006, cAMP: p=0.9).
Figure 3—figure supplement 1—source data 1. Excel data for size exclusion chromatography traces, monomer and tetramer steady-state time courses, and FRET efficiency summaries (Figure 3—figure supplement 1A–D).