 | Acta Crystallographica Section D Acta Crystallographica Section D BIOLOGICAL CRYSTALLOGRAPHY |
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![[Figure 4]](kw5082fig4mag.jpg)
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Figure 4
Nucleotide-binding kinetics measurements. The nucleotide-binding stopped-flow experiments were performed as described in § ![[link]](../../../../../../logos/arrows/d_arr.gif) 2.4 with subsequent global data-fitting analysis according to the model given in (1). The nucleotide-binding properties of wild-type ClpB NBD2 and the N709A, R806A and R621Q mutants for MANT-dADP, MANT-dATP, ADP and ATP were determined. The kinetic traces shown exemplarily in this figure correspond to direct mixing (a), displacement (b) and competition (c) experiments using ClpB NBD2 R806A, MANT-dADP and ADP. Coloured lines represent the fits from the global data-fitting procedure. Concentrations increase through blue, green, yellow, orange to red. The schematic experimental setup with the solutions being mixed in the different experiments is depicted next to the kinetic traces. (a) Direct mixing of 2 µM NBD2 and 5–25 µM MANT-dADP. (b) Displacement experiment: 2 µM ClpB NBD2 was incubated with 5 µM MANT-dADP and then mixed with 0.5–5 mM ADP. (c) ClpB NBD2 was mixed simultaneously with both MANT-dADP and ADP, giving final mixing concentrations of 0.5 µM protein, 2.5 µM MANT-dADP and 2.5–100 µM ADP. The time axis is plotted on a logarithmic scale to account for the large range of rates in this experimental series. (d) The discrimination between ADP and ATP binding is illustrated by the ratio Kd(ATP)/Kd(ADP). The active-site mutations R806A and R621Q significantly reduce the discrimination, primarily by impairing ADP binding. All nucleotide-binding parameters obtained from these experiments are listed in Table 2. |
![[Figure 4]](kw5082fig4.jpg)
| BIOLOGICAL CRYSTALLOGRAPHY |
ISSN: 1399-0047
