Figure 1.
Effect of ATP on a GFP based FRET construct.
Steady state fluorescence emission spectrum of a GFP-tdTomato construct ([15]) in the presence or absence of 10 mM ATP. Excitation of the GFP was at 480 nm excitation.
Figure 2.
Effect of ATP on a CFP based FRET construct.
Steady state fluorescence emission spectra of CFP-xa-YFP (A) and CFP alone (B) at different ATP concentrations. Excitation of the CFP was at 420 nm excitation.
Figure 3.
Fluorescence decay curves of CFP – YFP constructs.
Normalized experimental (dotted line) and fitted (solid line) fluorescence decay curves of CFP-xa-YFP (curve 1), CFP-xa-YFP in the presence of 10 mM MgATP (curve 2) or 10 mM ATP (curve 3). The excitation wavelength was 430 nm and the detection wavelength of CFP emission was 480 nm. Weighted residuals are shown in the bottom panel and the recovered parameters (α, τ) are collected in Table 1.
Figure 4.
Fluorescence decay curves of purified monomeric CFP.
Normalized experimental (dotted line) and fitted (solid line) fluorescence decay curves of CFP (curve 1), CFP in the presence of 10 mM MgATP (curve 2) or 10 mM ATP (curve 3). The excitation wavelength was 430 nm and the detection wavelength of CFP emission was 480 nm. Weighted residuals are shown in the bottom panel and the recovered parameters (α, τ) are collected in Table 2.
Table 1.
Fluorescence decay parameters of CFP – YFP constructs.
Table 2.
Fluorescence decay parameters of purified monomeric proteins.
Figure 5.
Fluorescence decay curves of purified CrFP and CrFP-xa-YFP.
Normalized experimental (dotted line) and fitted (solid line) fluorescence decay curves of CrFP (curve 1; no ATP, curve 2; 10 mM MgATP, curve 3; 10 mM ATP), and CrFP-xa-YFP (curve 4; no ATP, curve 5; 10 mM MgATP, curve 6; 10 mM ATP). The excitation wavelength was 430 nm and the detection wavelength of CFP emission was 480 nm. The recovered parameters (α, τ) are collected in Table 1 and 2.
Figure 6.
Fluorescence emission spectra of proteins expressed in Cos-1 cells.
Steady state fluorescence emission spectra of Cos-1 cells expressing CFP-xa-YFP (A, C) and CrFP-xa-YFP (B, D) in cell lysates (A, B) and of Ni-NTA purified protein (C, D) at different ATP concentrations. Under both conditions the YFP/CFP ratio shows a clear ATP dependent change in the CFP-xa-YFP construct whereas hardly any change was observed in the CrFP-xa-YFP construct (E, F). Excitation of the CFP was at 425 nm excitation.
Table 3.
YFP/CFP peak ratios of CFP – YFP constructs.