Monitoring ATP hydrolysis and ATPase inhibitor screening using 1H NMR

Article abstract of DOI:10.1039/c4cc04399e

We present a versatile method to characterize ATPase and kinase activities and discover new inhibitors of these proteins. The proton NMR-based assay directly monitors ATP turnover and is easy to implement, requires no additional reagents and can potentially be applied to GTP. We validated the method's accuracy, applied it to the monitoring of ATP turnover by actin and to the screening of ATPase inhibitors, and showed that it is also applicable for the monitoring of GTP hydrolysis.

Full text of DOI:10.1039/c4cc04399e

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Monitoring ATP hydrolysis and ATPase inhibitor
1
screening using H NMR†
Cite this: Chem. Commun., 2014,
50, 12037
Bingqian Guo,a Pinar S. Gurel,b Rui Shu,b Henry N. Higgs,b Maria Pellegrini*a and
Dale F. Mierke*a
Received 9th June 2014,
Accepted 22nd August 2014
DOI: 10.1039/c4cc04399e
www.rsc.org/chemcomm
We present a versatile method to characterize ATPase and kinase more readily available, is inherently more sensitive and provides
activities and discover new inhibitors of these proteins. The proton additional gain in sensitivity through the use of cryogenic probes.
NMR-based assay directly monitors ATP turnover and is easy to imple-
During the course of our research, we tested the method on
ment, requires no additional reagents and can potentially be applied to Bruker 600 MHz and 700 MHz spectrometers equipped with
GTP. We validated the method’s accuracy, applied it to the monitoring cryogenic probes as well as on a 500 MHz spectrometer with a
of ATP turnover by actin and to the screening of ATPase inhibitors, and standard room temperature probe (TBI probe). In each case this
showed that it is also applicable for the monitoring of GTP hydrolysis. method showed high sensitivity and accuracy. The method
detects the resolved signals of the purine H8 proton in ATP
The energy for many diverse cellular processes including signal and ADP, which when integrated yield directly the ATP decrease
transduction, transport, metabolism and muscle contraction is during ATPase activity (Fig. 1a). We verified feasibility and validated
derived by the hydrolysis of ATP.1 The characterization of such the assay on test samples with different spectrometers and then
processes relies on the understanding of the enzyme reactivity applied it to the characterization of the ATPase activity of actin. The
and is a fundamental step in the development of inhibitors of result is a generally applicable, facile method that directly detects
the enzymatic activity. Consequently a number of assays have ATP and GTP turnover that will find wide use in the characteriza-
been developed to monitor enzyme function and inhibitory tion of enzymatic activity.
activity. Methods employed for protein kinases often rely on the
detection of the phosphorylated substrate as a product and
utilize a radiolabeled phosphate donor (g-32P-ATP) or labelled
substrates and/or antibodies for substrate recognition and
detection.2 Other methods are available that measure the depletion
of ATP, or accumulation of ADP, and that are more generally
applicable to ATPases. These include colorimetric, luminescence
or fluorescence based assays for the detection of residual ATP,
produced ADP or inorganic phosphates.2–6
During the course of our research we often encounter the
necessity to assess the activity of a protein kinase (e.g., produced
recombinantly) or an ATPase. We therefore set out to develop an
NMR-based method that could be routinely used in the laboratory.
We chose to focus on the detection of ATP turnover because
it is applicable to both ATPases and kinases and to utilize 1H NMR
spectroscopy rather than the more classical 31P NMR.7–10 1H NMR is
a Department of Chemistry, Dartmouth College, Hanover 03755, USA.
E-mail: maria.pellegrini@dartmouth.edu, dale.f.mierke@dartmouth.edu
b Department of Biochemistry, Geisel School of Medicine, Dartmouth College,
Fig. 1 Quantitative analysis of ATP/ADP in control experiment. (a) ATP,
ADP structure: the H8 used for quantification is labeled. (b) NMR titration
experiment to quantify ATP% in control samples. (c) Data fitting and R2
analysis of control experiment.
Hanover 03755, USA
† Electronic supplementary information (ESI) available: Experimental details are
included in the supplementary. See DOI: 10.1039/c4cc04399e
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We first acquired mono-dimensional 1H NMR spectra of a had a minimal ATPase activity, while filament formation con-
series of control samples with defined ratios of ATP/ADP. The ditions increased ATPase activity, with a 50% ATP turnover after
purine H8 proton resonance is distinguishable for ATP and ADP approximately 1 hour (Fig. 2b-a, af). In the accelerated filament
at 8.47 and 8.48 ppm, respectively (Fig. 1b). The ATP percentage formation condition, the ATP turnover rate is further increased to a
for each sample was calculated from the integrated peaks. As 50% turnover time of less than 10 minutes (Fig. 2b-afp). Altogether
shown in Fig. 1, the experimentally determined ATP percentage these data suggest that 1H NMR is an accurate and efficient method
agrees very well with the theoretical values with an R2 of 0.995 for monitoring ATP turnover. Given the advances in NMR automa-
(Fig. 1c), demonstrating the accuracy of the method.
tion (e.g., a SampleJet sample changer, Bruker) all samples could be
Actin represents a family of polymerizable ATPases found in acquired with minimal operator intervention. The assay was imple-
all types of eukaryotic cells and many bacteria.11,12 ATP-bound mented in this instance in a non-homogenous format (single time
actin assembles into helical filaments, and incorporation into points required sample preparation), but depending on the concen-
the filament accelerates ATP hydrolysis 10 000-fold. Upon ATP tration of ATP utilized (which dictates signal/noise and therefore
hydrolysis and phosphate release, the resulting ADP-actin is data acquisition time) and the enzyme kinetics, the time-course
prone to depolymerization, at which point the depolymerized experiments could be executed in a continuous manner with
actin monomer can re-load with ATP and add to the filament acquisition of NMR spectra on the same sample at different time
again, creating a cycle of polymerization–depolymerization that points. The assay is also compatible with a wide range of ATP
will continue as long as ATP is present in solution.13 Since actin concentrations with the lowest tested in this experiment at approxi-
polymerization–depolymerization dynamics are tightly regulated mately 4 mM of ATP (importantly ATP concentrations higher than
by a variety of proteins, it is possible to modulate the turnover 1 mM pose no problems) The assay can also be used to monitor
rate with these factor proteins in vitro.
enzymatic reactions with AMP as substrate or product. In the actin
To illustrate the application of this method, we monitored samples there is a trace contamination of some kinase that can
the hydrolysis of ATP under three conditions: non-polymerizing further hydrolyze ADP into AMP (clearly observed in the overnight
(actin monomers/a); actin filament formation (actin and INF2- reaction, Fig. 2a). Importantly, the integration of ATP, ADP and AMP
FFC, inverted formin2, CAAX variant/af), and accelerated actin signals from this spectrum is equal to the initial amount of ATP.
filament formation (actin, profilin and INF2-FFC/afp).14 Time
Single point experiments can be easily utilized for assessing
points from three minutes to two hours were taken, as well as inhibitor activity, providing a medium throughput NMR-based
an overnight reaction of 19 hours (Fig. 2a). After acquiring screening assay with low risk of artifacts (with the limit that
spectra in automation, percentages of residual ATP at different inhibitor resonances should not overlap with both the ATP and the
time points were calculated in the same manner as the control. ADP H8 resonance). To demonstrate the application, we prepared a
The ATP percentage versus time plot indicates that actin alone small library of small molecules and one commercially available
actin inhibitor, latrunculin. The small-scale screening was carried
out on a Bruker 600 MHz spectrometer equipped with a 1.7 mm
cryoprobe, requiring only 35 ml of each sample and greatly reduced
the sample cost for the inhibitor screening. 1H spectra were collected
to analyze the percentage of residual ATP in the system after
30 minutes reaction in the accelerated actin filament formation
condition with or without small molecules (Fig. 2c). At a concen-
tration of 20 mM small molecule in each sample, latrunculin
achieved high level of inhibition with more than 80% ATP left after
30 minutes while in absence of inhibitor or with other random
molecules, the residual ATP was less than 20% (Fig. 2d). This
implementation of the screening method, with low sample demand,
was carried out in automation, utilizing a sample changer, and can
be potentially used in large scale screening of small molecule
libraries for inhibitors.
To expand the application of the method, we also tested the
possibility of monitoring GTP turnover. Similar to the ATP standard,
we prepared a series of GTP/GDP samples at know ratios, and used
the integration of their H8 peaks to determine the accuracy of
the experimental ratios (Fig. 3a and b). As expected, the calculated
ratios matched the theoretical ones well with an R2 of 0.989 (Fig. 3c),
demonstrating that the same method can be used to monitor
GTPase activity as well with similar accuracy.
Fig. 2 ATP hydrolysis kinetics by actin and inhibitor screening. (a) 1H NMR
spectra of ATP turnover by actin at different time points (a-actin alone,
af-actin and INF2-FFC, afp-actin, INF2-FFC and profilin). (b) Plot of residual
ATP percentage vs. reaction time. (c) 1H NMR spectra of ATP/ADP after
30 minutes reaction in afp conditions with/without small molecules
(0 – no small molecule; #1 – latrunculin; #2–8 – random small mole-
cules). (d) Residual ATP percentage in each sample.
We additionally measured the performance of the method with
NMR instruments at a lower magnetic field, using a walk-up
500 MHz spectrometer (Dartmouth’s Chemistry Department
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must be compatible with all of the enzymes) and possible inter-
ference of assay components. For example, in assays that include co-
factor proteins (e.g., formin and profilin in the case examined here)
and in the widely utilized application of inhibitor screening, the
effect of these additional components on the secondary (or
tertiary) enzyme activity cannot be ignored.21
Our results clearly demonstrate that using 1H NMR spectro-
scopy to measure ATPase activity is a sensitive and highly
automated approach. The method does not require antibodies,
secondary enzymes or additional reagents, affords a medium
throughput and can be employed under a variety of experimental
conditions. Because of the absence of additional reagents and
complex reactions, the assay is easily accessible to laboratories
that do not routinely engage with ATPase/kinase assays. The
range of ATP concentrations that can be utilized in the assay is
comparable to those employed in malachite green or LDH/PK
assays (micromolar to millimolar).22–24 These factors along with
the possible automation of data acquisition make this method
ideal for medium-throughput screening for inhibitors of ATP
and GTP dependent enzymes.
Fig. 3 Quantitative measurement of GTP/GDP in control experiment.
(a) GTP, GDP structure: the H8 used for quantification is labeled. (b) NMR
titration experiment to quantify GTP% in control samples. (c) Data fitting and
R2 analysis of control experiment.
Notes and references
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This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 12037--12039 | 12039