Table 2.
Parameters of substrate binding to CaiT bearing given substitutions determined by tryptophan fluorescence analyses.
| CaiT variant | L-carnitine | γ-butyrobetaine | ||||
|---|---|---|---|---|---|---|
| Kd [mM] | max ΔF/Foa | λmax [nm] (Δλmax) | Kd [mM] | max ΔF/Fo | λmax [nm] (Δλmax) | |
| solubilized CaiT | ||||||
| wild-type | 3.3 ± 0.2 | 0.43 ± 0.02 | 336 (+5) | 3.4 ± 0.1 | 0.42 ± 0.01 | 336 (+6) |
| D288A | not detectable | −0.05 ± 0.01 | 337 (−1) | not detectable | −0.18 ± 0.04 | 337 (−1) |
| D288R | not detectable | −0.04 ± 0.01 | 337 (+1) | not detectable | −0.07 ± 0.01 | 337 (+1) |
| CaiT reconstituted into proteoliposomes | ||||||
| wild-type | 2.4 ± 0.2 | 0.27 ± 0.01 | 338 (0) | 4.5 ± 0.5 | 0.38 ± 0.01 | 338 (+1) |
| D288A | 1.8 ± 0.9 | 0.08 ± 0.01 | 338 (0) | 3.3 ± 1.3 | 0.08 ± 0.01 | 338 (0) |
| D288R | 1.9 ± 1.0 | 0.07 ± 0.01 | 338 (0) | 11.4 ± 4.4 | 0.11 ± 0.01 | 338 (0) |
amax ΔF/Fo represents a saturation value of a hyperbolic fit and was determined using the kinetic module of the software GraphPad Prism.
Binding of L-carnitine and γ-butyrobetaine was measured with solubilized CaiT and with CaiT reconstituted into proteoliposomes (lipid to protein ratio of 100:1 (w/w)) as described1. Experiments were performed as described in the legends of Figs 7 and 8. The change of the fluorescence intensity measured at 338 nm was plotted using the kinetic module of the software GraphPad Prism. Shown are mean values and standard deviations calculated from three experiments.