Figure 1.
Figure 1. TRPV1-CT CaM-binding site. (A) Schematic diagram showing the domain organization of TRPV1. CaM and putative CaM-binding sites are shaded in light gray. (B) SEC elution profile of TRPV1-CT44 alone or in complex with Ca2+–CaM. Shown are representative traces of a TRPV1-CT44 preparation (gray) after cleavage of an MBP tag (*) and Ca2+-CaM–TRPV1-CT44 complex (black). (C) Tryptophan fluorescence emission spectra of 10 µM TRPV1-CT44 (residues 759–802) alone or incubated with equimolar amounts of CaM in the presence of CaCl2 or EDTA, excited at 295 nm. (D) Titration of 0.1 µM TRPV1-CT44 with CaM from 0.01 to 1 µM in the presence of CaCl2. Tryptophan fluorescence emission at 330 nm was plotted against CaM concentration. Data were obtained in triplicate and analyzed using one site-specific binding nonlinear regression analysis (Prism 5; GraphPad). Line shows the best fit to the data (Kd = 5.4 ± 0.6 × 10−8 M).

TRPV1-CT CaM-binding site. (A) Schematic diagram showing the domain organization of TRPV1. CaM and putative CaM-binding sites are shaded in light gray. (B) SEC elution profile of TRPV1-CT44 alone or in complex with Ca2+–CaM. Shown are representative traces of a TRPV1-CT44 preparation (gray) after cleavage of an MBP tag (*) and Ca2+-CaM–TRPV1-CT44 complex (black). (C) Tryptophan fluorescence emission spectra of 10 µM TRPV1-CT44 (residues 759–802) alone or incubated with equimolar amounts of CaM in the presence of CaCl2 or EDTA, excited at 295 nm. (D) Titration of 0.1 µM TRPV1-CT44 with CaM from 0.01 to 1 µM in the presence of CaCl2. Tryptophan fluorescence emission at 330 nm was plotted against CaM concentration. Data were obtained in triplicate and analyzed using one site-specific binding nonlinear regression analysis (Prism 5; GraphPad). Line shows the best fit to the data (Kd = 5.4 ± 0.6 × 10−8 M).

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