3.2.2 Example Protocol:
P i Release Under
Single-Turnover
Conditions—SufBC ATPase
SufC is an ABC-type ATPase involved in iron sulfur cluster assembly under oxidative stress or Fe starvation in bacteria (including
human pathogens like Staphylococcus aureus, Mycobacterium tuberculosis), archaea, and the plastid organelle of algae and plants [53].
The kinetic mechanism of SufC ATPase alone and the complex
with its regulatory protein SufB has been studied using tryptophan
fluorescence measurements and fluorescently labeled ATP and ADP
analogues, mant-ATP, and mant-ADP [54, 55]. For the SufBC
complex, rate constants were determined for mant-ATP and ATP
binding (k +1 ¼ 0.55 μM
À1 s
À1 and 0.13 μM
À1 s
À1 ), mant-ATP
cleavage (k +2 ¼ 0.088 s
À1
), mant-ADP and ADP binding
(k À4 ¼ 0.91 μM
À1 s
À1 and 0.27 μM
À1 s
À1 ), and mant-ADP dissociation (k +4 ¼ 0.038 s
À1 ), suggesting that ATP cleavage and ADP
dissociation are rate-limiting [55]. This was confirmed in a later
study, where P i release and ADP release were measured directly
using MDCC-PBP and an ADP biosensor (MDCC-ParM), respectively [11]. A detailed protocol for the single-turnover P i release
measurements is described here.
Buffer and Concentrations
Experiments are performed in buffer containing 50 mM Tris–HCl
pH 7.6, 100 mM KCl, 5 mM MgCl 2 , and 2 mM DTT (using
components with minimal P i , see Subheading 2.2) at 20
C. The
buffer is prepared freshly on the day and kept at room temperature
throughout the experiments (see Note 15). The final concentrations of assay components are 2 μM ATP, 10 μM SufBC, and 10 μM
MDCC-PBP.
In the stopped-flow experiments, the two reactants (ATP and
SufBC) are mixed in a 1:1 ratio, so all solutions are prepared at
2Â the final concentration (see Note 16). The stop syringe is set to
100 μl, so the experiments need 50 μl volume per solution per push
plus 150 μl for priming.
Stopped-Flow Setup
and Cleaning
1. Switch on the lamp at least 20 min before the first measurement
to allow for signal stabilization.
2. Switch on all the other parts of the stopped-flow instrument.
3. Set the water bath to 20
C.
4. Put a 455 nm long-pass filter in front of the photomultiplier
and set the entrance and exit slits of the monochromator to
2.5 mm (see Note 17).
5. The stop syringe is set to 100 μl.
In order to remove any contaminating phosphate from the
stopped-flow instrument, it is cleaned out with P i mop solution
(see Subheading 2.5) prior to the experiments.
6. Prepare ~10 ml solution of 200 μM 7-MEG and 1 U/ml
PNPase in P i -free buffer.
308
Simone Kunzelmann
P i Release Under
Single-Turnover
Conditions—SufBC ATPase
SufC is an ABC-type ATPase involved in iron sulfur cluster assembly under oxidative stress or Fe starvation in bacteria (including
human pathogens like Staphylococcus aureus, Mycobacterium tuberculosis), archaea, and the plastid organelle of algae and plants [53].
The kinetic mechanism of SufC ATPase alone and the complex
with its regulatory protein SufB has been studied using tryptophan
fluorescence measurements and fluorescently labeled ATP and ADP
analogues, mant-ATP, and mant-ADP [54, 55]. For the SufBC
complex, rate constants were determined for mant-ATP and ATP
binding (k +1 ¼ 0.55 μM
À1 s
À1 and 0.13 μM
À1 s
À1 ), mant-ATP
cleavage (k +2 ¼ 0.088 s
À1
), mant-ADP and ADP binding
(k À4 ¼ 0.91 μM
À1 s
À1 and 0.27 μM
À1 s
À1 ), and mant-ADP dissociation (k +4 ¼ 0.038 s
À1 ), suggesting that ATP cleavage and ADP
dissociation are rate-limiting [55]. This was confirmed in a later
study, where P i release and ADP release were measured directly
using MDCC-PBP and an ADP biosensor (MDCC-ParM), respectively [11]. A detailed protocol for the single-turnover P i release
measurements is described here.
Buffer and Concentrations
Experiments are performed in buffer containing 50 mM Tris–HCl
pH 7.6, 100 mM KCl, 5 mM MgCl 2 , and 2 mM DTT (using
components with minimal P i , see Subheading 2.2) at 20
C. The
buffer is prepared freshly on the day and kept at room temperature
throughout the experiments (see Note 15). The final concentrations of assay components are 2 μM ATP, 10 μM SufBC, and 10 μM
MDCC-PBP.
In the stopped-flow experiments, the two reactants (ATP and
SufBC) are mixed in a 1:1 ratio, so all solutions are prepared at
2Â the final concentration (see Note 16). The stop syringe is set to
100 μl, so the experiments need 50 μl volume per solution per push
plus 150 μl for priming.
Stopped-Flow Setup
and Cleaning
1. Switch on the lamp at least 20 min before the first measurement
to allow for signal stabilization.
2. Switch on all the other parts of the stopped-flow instrument.
3. Set the water bath to 20
C.
4. Put a 455 nm long-pass filter in front of the photomultiplier
and set the entrance and exit slits of the monochromator to
2.5 mm (see Note 17).
5. The stop syringe is set to 100 μl.
In order to remove any contaminating phosphate from the
stopped-flow instrument, it is cleaned out with P i mop solution
(see Subheading 2.5) prior to the experiments.
6. Prepare ~10 ml solution of 200 μM 7-MEG and 1 U/ml
PNPase in P i -free buffer.
308
Simone Kunzelmann