Báo cáo Y học: Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPcNH2 and GTPcF pdf

Synthesis, characterization and application of two nucleoside
triphosphate analogues, GTPcNH
2
and GTPcF
Michael Stumber
1
, Christian Herrmann
2
, Sabine Wohlgemuth
2
, Hans Robert Kalbitzer
1
, Werner Jahn
1
and Matthias Geyer
1,
*
1
Max-Planck-Institut fu
¨
r medizinische Forschung, Department of Biophysics, 69120 Heidelberg, Germany;
2
Max-Planck-Institut fu
¨
r molekulare Physiologie, Department of Structural Biology, 44227 Dortmund, Germany
Guanosine triphosphate nucleotide analogues such as
GppNHp (also named GMPPNP) or GTPcSarewidely
used to stabilize rapidly hydrolyzing protein-nucleotide
complexes and to investigate biochemical reaction path-
ways. Here we describe the chemical synthesis of guanosine
5¢-O-(c-amidotriphosphate) (GTPcNH

2
) and a new synthe-
sis of guanosine 5¢-O-(c-fluorotriphosphate) (GTPcF). The
two nucleotides were characterized using NMR spectrosco-
py and isothermal titration calorimetry. Chemical shift data
on
31
P,
19
Fand
1
H NMR resonances are tabulated. For
GTPcNH
2
the enthalpy of magnesium coordination is
DH° ¼ 3.9 kcalÆmol
)1
and the association constant K
a
is
0.82 m
M
)1
. The activation energy for GTPcNH
2
ÆMg
2+
complex formation is DH
à
¼ 7.8 ± 0.15 kcalÆmol

)1
,similar
to that for the natural substrate GTP. For GTPcF we ob-
tained a similar enthalpy of DH° ¼ 3.9 kcalÆmol
)1
while the
magnesium association constant is only K
a
¼ 0.2 m
M
)1
.The
application of both guanine nucleotide analogues to
the GTP-binding protein Ras was investigated. The rate of
hydrolysis of GTPcNH
2
bound to Ras protein lay between
the rates found for Ras-bound GTPcS and GppNHp, while
Ras-catalysed hydrolysis of GTPcF was almost as fast as for
GTP. The two compounds extend the variety of nucleotide
analogues and may prove useful in structural, kinetic and
cellular studies.
Keywords: nucleotides; nucleotide analogues; NMR spectro-
scopy; GTP hydrolysis; Ras.
Nucleotides are fundamental components in cellular meta-
bolism. Acting as substrates for nucleotide binding proteins,
they are the protagonists of a large variety of cellular
processes. Nucleotides can regulate enzymatic activity by
transitions between their mono-, di- and triphosphate
bound forms. These transitions often induce conformational

changes in the proteins, referred to as the ÔactiveÕ and
ÔinactiveÕ conformations. Perhaps the best known example is
the energy metabolism of adenosine nucleotides: hydrolysis
of ATP to ADP leads to functional molecular rearrange-
ments in the actomyosin mediated muscle contraction.
Guanine nucleotide-binding proteins on the other hand are
specialized in the control of intracellular communication
processes such as signal transduction (Ras and Rho
families) or protein and vesicle trafficking (Ran and Rab
families, respectively), which are combined with GTP-
hydrolysis (reviewed in [1–3]). Another aspect of nucleotide
mediated transformation is the transfer of the leaving
phosphoryl group (mostly the c-phosphate group) to
acceptors like water, amino-acid residues, or other nucleo-
tides. Often the association of a metal ion, usually magnes-
ium, with the phosphate groups of the nucleotide is crucial
for these events.
The study of nucleotide-binding proteins, their function,
structure and mechanism, often demands use of nonhy-
drolyzable or slowly hydrolyzable nucleotide analogues.
These modifications become necessary when stabilization of
a specific isoform of the protein is required. In cellular
assays the triphosphate analogues GTPcSandATPcSare
most commonly used, usually in order to generate the
constitutively active form of a protein. In structural biology,
long-term stability of the protein-nucleotide complex is
required in order to grow homogeneous crystals or to obtain
a single state of the protein. Here, the most commonly used
triphosphate analogues are GppNHp (also named
GMPPNP or GDPNP) and to a minor extent GppCH

2
p
(also named GMPPCP) and their respective adenosine
counterparts AppNHp and AppCH
2
p. Another application
of substrate analogues is the use of caged nucleotides to
characterize unstable protein intermediates by X-ray crys-
tallography [4]. Nucleotide modifications can also serve as
an approach to designing dominant negative forms of a
protein [5] or to solve the phase problem in crystallography
[6]. Even more specific is the application of aluminium
fluoride, beryllium fluoride or orthovanadate in the presence
Correspondence to M. Geyer, Max-Planck-Institut fu
¨
r medizinische
Forschung, Abteilung Biophysik, Jahnstraße 29,
D-69120 Heidelberg, Germany.
Fax: + 49 6221 486 437, Tel.: + 49 6221 486 396,
E-mail:
Abbreviations:GTPcNH
2
, guanosine 5¢-O-(c-amidotriphosphate);
GTPcF, guanosine 5¢-O-(c-fluorotriphosphate); GppNHp, guanosine
5¢-O-(b,c-imidotriphosphate); GppCH
2
p, guanosine 5¢-O-
(b,c-methylenetriphosphate); GTPcS, guanosine 5¢-O-(c-thiotriphos-
phate); ITC, isothermal titration calorimetry; DCC, dicyclohexylcar-
bodiimide; DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate;

THC, triethylammonium hydrogencarbonate.
*Present address: Max-Planck-Institut fu
¨
r molekulare Physiologie,
Department of Physical Biochemistry, 44227 Dortmund, Germany.
(Received 23 January 2002, revised 8 May 2002,
accepted 17 May 2002)
Eur. J. Biochem. 269, 3270–3278 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03003.x
of a nucleoside diphosphate. These compounds can form
stable analogues that mimic the transition state of the
terminal leaving group of the nucleotide within a protein-
nucleotide complex [7,8].
Mechanistic studies to analyse the enzymatic activity of a
nucleotide binding protein usually benefit from the avail-
ability of a broad range of different nucleotide phosphate
analogues. Here, advantage can be taken of the individual
characteristics of the nucleotide when applied to a protein.
Differences in metal ion binding properties as well as charge
distribution and hydrophobicity determine the specific
features of a nucleotide that provide insights into the
biological system. Also, nucleotide modifications such as
spin labeling make the protein-nucleotide complex access-
ible to spectroscopic techniques. Most prominent is the use
of fluorescent analogues (e.g. mant-GTP) for kinetic
measurements by fluorescence spectroscopy and
17
O-labe-
ling for EPR or NMR techniques.
Here we investigate two modified nucleoside triphos-
phates which are stable and show distinct characteristics:

guanosine 5¢-O-(c-amidotriphosphate) (GTPcNH
2
), for
which we describe the first synthesis, and guanosine 5¢-O-
(c-fluorotriphosphate) (GTPcF) [9], which we synthesized
by the method of Wittmann [10]. Both are shown in Fig. 1.
We characterized the stability and metal ion binding
properties of the two nucleotide analogues by NMR
spectroscopy and isothermal titration calorimetry. Both
nucleotides were bound to the small GTP-binding protein
Ras and the rates of hydrolysis were determined in
comparision to other nucleotide triphosphate derivatives.
Finally, the suitability for spectroscopic and structural
studies was tested by formation of the complex between
RasGTPcNH
2
and the Ras-binding domain of the Ras
effector protein c-Raf-1.
MATERIALS AND METHODS
General description of synthesis
High pressure liquid chromatography (HPLC) was done on
a Beckman ÔSystem GoldÒÕ. Nucleotides were analysed by
ion-pair chromatography on a reversed phase Super ODS
column, 50 · 4.6 mm (TOYOPEARLÒ) at a flow rate of
1.2 mLÆmin
)1
, using a linear gradient from 100% 10 m
M
tetrabutyl-ammonium bromide/10 m
M

sodium phosphate
buffer (pH 6.8) to 100% acetonitrile within 10 min. Detec-
tion was at 260 and 340 nm. The retention times given are
for orientation only.
GTP-triethylammonium salt was prepared by applying
GTP sodium salt to a Super Q column (TOYOPEARLÒ)
and elution with a gradient from 0 to 1
M
triethylammo-
nium hydrogencarbonate (THC). The eluate containing the
nucleotide (retention time in HPLC 4.65 min) was evapor-
ated under reduced pressure, redissolved in methanol, again
evaporated and dried over P
4
O
10
. Monoamido-phosphoric
acid, H
2
PO
3
NH
2
, was prepared as described [11].
Synthesis of GTPcNH
2
and GTPcF
To the solution of 0.8 g GTP triethylammonium salt in
5 mL dimethylsulfoxide were added 0.8 g DCC and 80 mg
pyridinium hydrochloride. After 20–24 h at room tempera-

ture the mixture was treated with about 5 mL concentrated
ammonia in water for 30 min. The solution was diluted with
60–70 mL water and, after filtration, applied to a Super Q
column (2.5 · 20 cm). The column was eluted at a rate of
5mLÆmin
)1
with a gradient from 0 to 1
M
THC within
120 min. Fractions containing the GTPcNH
2
(as checked
by UV absorption and HPLC, retention time 4.20 min)
were collected and evaporated. Any remaining THC was
removed by dissolving in methanol and repeated evapor-
ation under reduced pressure, yield of the pure GTPcNH
2
was 50–60%.
One gram of GTP triethylammonium salt was added to a
stirred solution of 2.5 mL tributylamine and 1.2 g
2,4-dinitrofluorobenzene in about 10 mL dimethylforma-
mide. After 6–8 h a clear solution was obtained. The
mixture was kept for 20–24 h at room temperature. The
crude product was precipitated with 100 mL acetone and
300 mL diethyl ether. The pellet was dissolved in water
(about 20 mL) and applied to a Super Q column
(18 · 2.6 cm). The column was eluted with a gradient of
0–1
M
THC within 2 h at a flow rate of 5 mLÆmin

)1
.
Fractions containing the reaction product (retention time of
4.47 min, no absorption at 340 nm) were collected and
evaporated as described for the GTP triethylammonium
salt. The product was dissolved in 20 mL methanol and
Fig. 1. Chemical structure of the nucleoside triphosphate analogues
synthesized. (A) Guanosine 5¢-O-(c-amidotriphosphate) (GTPcNH
2
)
and (B) guanosine 5¢-O-(c-fluorotriphosphate) (GTPcF). Displayed is
the
D
-riboside form of the respective nucleoside.
Ó FEBS 2002 Nucleotide analogues GTPcNH
2
and GTPcF(Eur. J. Biochem. 269) 3271
precipitated by addition of a solution of 250 mg NaClO
4
in a
few ml methanol to remove part of the colored by-products.
The pellet was dissolved in water (20 mL) and purified on a
Super Q column as described above, giving an almost
colorless substance (yield in the range 5–10%).
Preparation of NMR samples and NMR spectroscopy
31
Pand
19
F NMR spectra of free nucleotides were recorded
in aqueous solution of 90%/10% H

2
O/D
2
O. Typically the
lyophilized nucleotide was redissolved to a final concentra-
tion of 2–10 m
M
and 2500 lL of the sample volume was
placed in 10 mm NMR tubes (Wilmad). For titration
experiments various amounts of MgCl
2
were added from a
100-m
M
stock solution. Proton NMR experiments were
performed using 500 lL sample volume in 5 mm NMR
tubes (Wilmad).
31
P NMR spectra of C-terminal truncated
wildtype Ras protein (residues 1–167) complexed with
GTPcNH
2
ÆMg
2+
were recorded in 40 m
M
Tris/HCl, 5 m
M
MgCl
2

and 2 m
M
DTE at pH 7.4. Here, sample volumes of
2500 lLof1.0m
M
concentrated protein were measured
containing 10% D
2
O.
1
H,
19
Fand
31
P NMR experiments were performed on a
Bruker AMX-500 NMR spectrometer working at reson-
ance frequencies of 500 MHz, 470 MHz and 202 MHz,
respectively.
31
P spectra were referenced to 85% phosphoric
acid enclosed in a glass sphere which was immersed in the
sample and calibrated for various temperatures.
19
F spectra
were referenced to trifluoroacetic acid, based on the IUPAC
conventions for indirect referencing relative to internal DSS
[12]. Unless noted otherwise, phosphorus spectra were
recorded at 20 °C with a total spectral width of 60 p.p.m.
For one dimensional
31

P NMR spectra of free nucleotides,
64–512 free induction decays were summed after excitation
with a 65 degree pulse using a repetition time of 3–5 s. A
total of 32 K time domain data points were recorded and
transformed to 16 K real data points corresponding to a
digital resolution of 0.74 Hz point
)1
.The
31
P spin-spin
coupling constants of the nucleotide-Mg
2+
complexes were
determined from a nonfiltered 1D spectrum with a digital
resolution of 0.25 Hz per point after Fourier transforma-
tion.
All spectra were processed on a Silicon Graphics Indigo2
workstation using the software package UXNMR (Bruker,
Karlsruhe) for data processing and data evaluation. Phos-
phorus spectra used for exchange rate determination were
filtered by an exponential window function causing no
significant line broadening.
Determination of exchange rates
The Mg
2+
exchange rates of GTPcNH
2
were extracted
from a series of
31

P NMR exchange spectra. The spectra
were analyzed and compared to simulations based on the
mathematical treatment of the exchanging spin system
following Nageswara Rao [13]. The simulation of the
31
P
spectra was built on C
++
NMR library Ô
GAMMA
Õ [14],
modelingathreespinsystemwithanABC fi A¢B¢C¢
exchange. Chemical shift and J-couplings were determined
for NMR spectra of both ÔpureÕ states: without magnesium
complexation (state A) and with saturated magnesium
complexation (state B) (see Tables 1 and 2). Thus, the only
parameters to be adjusted were the relative populations of
the states A and B and the exchange rates k
1
for A fi B
and k
)1
for B fi A. As B ¼ 100%–A and k
)1
¼ k
1
*A/B
only two free parameters had to be fitted to the experimental
data. The simulations were performed on the complete
31

P
spectra (a-, b- and c-phosphorus nuclei), using chemical
shift values and J-coupling constants as listed in Tables 1
and 2.
Isothermal titration calorimetry
The interaction between a nucleotide and the magnesium
ion was investigated by means of ITC (ITC-MCS, Micro-
Cal, Inc.). Briefly, in such an apparatus the solutions are
thermostatted to the desired temperature, the nucleotide at
5.0 m
M
placed in a cell which is accurately temperature
controlled and the MgCl
2
solution at 50 m
M
in a syringe
dipping into the cell. The two solutions are mixed by
computer controlled stepwise injections (typically in inter-
vals of 4 min) from the syringe which serves at the same
time as a stirrer. The heat consumed due the endothermic
association process is measured by the detection of the
heating power which is necessary to keep the cell at constant
temperature [15]. All ITC experiments were performed at
25 °C. The data were analyzed using the manufacturer’s
software yielding the stoichiometry N, the binary equili-
brium association constant K
a
¼ [nucleotideÆMg
2+

]/
[nucleotide]/[Mg
2+
] and the enthalpy of association DH°,
the latter with the approximation that this parameter is
independent of the concentration. The change of entropy
DS° is calculated by the fundamental relationship
–RT lnK
a
¼ DH° – TDS°. The experimental error on DH°
Table 1. NMR chemical shifts of GTPcNH
2
,GTPcF, and GTP in
aqueous solution. Spectra were recorded in 90%/10% H
2
O/D
2
Oat
pH 7.4 and 25 °C.
31
Pand
19
F chemical shifts were referenced to 85%
phosphoric acid and trifluoroacetic acid, respectively, using the indirect
reference method with parameters adopted from IUPAC [12].
31
P chemical shift d (p.p.m.)
19
F chem.
Nucleotide abcshift d

GTPcNH
2
)11.46 )22.76 )1.12 –
GTPcNH
2
ÆMg
2+
)11.33 )21.50 )0.34 –
GTPcF )11.66 )23.49 )18.16 0.91
GTPcÆFMg
2+
)11.89 )23.19 )18.63 0.97
GTP )10.74 )21.22 )5.51 –
GTPÆMg
2+
)10.41 )19.01 )5.30 –
Table 2. J-Coupling constants of GTPcNH
2
and GTPcFinaqueous
solution.
J-coupling constants (Hz)
Nucleotide
2
J
PaPb
2
J
PbPc
1
J

PcF
GTPcNH
2
20.3 19.2 –
GTPcNH
2
ÆMg
2+
15.8 16.8 –
GTPcF 19.9 18.2 936.2
GTPcFÆMg
2+
15.4 12.7 934.0
GTP 19.8 19.8 –
GTPÆMg
2+
14.0 12.4 –
3272 M. Stumber et al. (Eur. J. Biochem. 269) Ó FEBS 2002
is 5% whereas the experimental error on K
a
is about 10–
20%. In addition, the stoichiometry factor N is obtained
from the fit to the data, where a value of 1 corresponds to
1 : 1 complex formation.
Protein preparation and guanine nucleotide exchange
In order to test the applicability of the two synthesized
triphosphate nucleotide analogues to nucleotide binding
proteins, the small GTP-binding protein Ras (residues
1–167) was synthesized in Escherichia coli and purified as
described [16]. Purified GDP, GTP, GTPcS and GppNHp

reagents were purchased from Sigma and GppCH
2
pwas
ordered from JenaBioScience. GDP, which binds very
tightly to Ras, was replaced with the respective GTP
analogue by the following procedures. For nucleotide
exchange GTPcNH
2
, GppNHp and GppCH
2
pwereeach
incubated at threefold molar excess with Ras in the presence
of 200 l
M
ammonium sulfate, 0.1 l
M
zinc chloride and 1 U
alkaline phosphatase per mg Ras overnight at 4 °C. In order
to load Ras with GTP, GTPcF, or GTPcS nucleotide-free
Ras was produced by incubation overnight at 4 °Cinthe
presence of 200 l
M
ammonium sulfate, 0.1 l
M
zinc chloride
and 0.2 U alkaline phosphatase per mg Ras. After size
exclusion chromatography, one of the nucleotides was then
added to the Ras protein. Excess nucleotide after either
procedure was removed (which is important in order to
obtain accurate single turnover hydrolysis rate constants).

The pooled Ras fractions were concentrated to 20 mgÆmL
)1
by centrifugal concentrators (Vivaspin 10 kDa cut-off,
VivaScience). The buffer used in all these procedures
contained 25 m
M
Tris/HCl at pH 7.4, 2.5 m
M
MgCl
2
,and
1m
M
DTE. The Ras catalysed nucleotide hydrolysis was
determined with HPLC by measuring the concentration of
protein-bound GTP or its triphosphate analogues and GDP
as described [17]. Intrinsic reaction rates were obtained
from the decay of the (triphosphate nucleotide)/(tri- and
diphosphate nucleotide) ratio with time, fitted to single-
exponential curves. The Ras-binding domain of human
c-Raf-1 (Raf-RBD, 81 residues) was expressed in E. coli
and purified as described recently [18].
RESULTS
NMR spectra, chemical shift data and J-coupling
constants of the two nucleotides
Proton, phosphorus and fluorine NMR measurements
confirmed the chemical structure and the high degree of
purification of the two synthesized triphosphate nucleotides.
As expected,
1

H NMR measurements of both GTPcNH
2
and GTPcF in aqueous solution at 20 °C, pH 7.4 showed
no difference to the natural substrate GTP [19] as the
guanine base is not affected by the modifications and as
the c-phosphate amide hydrogens are in fast exchange with
the solvent. In Fig. 2
31
P NMR spectra are shown for
GTPcNH
2
and GTPcF, and their respective metal ion
complexes with Mg
2+
.
For GTPcNH
2
the appearance of three discrete reson-
ance lines with similar intensity confirms the uniformity and
the conformational identity of the substrate. The observed
mean half width of, e.g. 4.7 Hz for the c-resonance line is
typical for a molecule of 523 Da mass at 20 °C in aqueous
solution. The resonance lines could be assigned by their
J-coupling constants and by comparison to unmodified
GTP. While the chemical shift of the a-phosphate group
changed only little upfield compared to GTP, the
b-phosphate was shifted upfield by about )1.5 p.p.m. and
the terminal c-phosphate shifted by almost 5 p.p.m. down-
field by the replacement of the hydroxy OH
–

with an amide
NH
2
. Complexation of GTPcNH
2
with Mg
2+
ledtoan
additional downfield shift of all phosphate groups, with the
b-phosphate changing most. This observation was similar
to the change in GTP when coordinated with magnesium,
but the absolute shift change was almost 1 p.p.m. smaller
(from 1.26 p.p.m. to 2.21 p.p.m.) than in the natural
substrate. The
2
J
PP
-coupling constants of GTPcNH
2
and
GTPcNH
2
Mg
2+
analogues showed smaller alterations
when compared to GTP. In both cases the b-phosphate
groups appeared as triplets as the coupling constants between
P
a
–P

b
and P
b
–P
c
were almost identical, while coordination
with magnesium again decreased the coupling constants.
In GTPcF four phosphorus lines appeared as the
coupling between the natural spin ½ nuclei
31
Pand
19
F
led to a splitting of the terminal phosphate resonance. This
direct coupling constant
1
J
PcF
was about 936 Hz and hardly
changed upon magnesium coordination (934 Hz), indica-
ting a strong interaction between the two nuclei. Chemical
shift changes of GTPcF compared to GTP were much more
distinct than for GTPcNH
2
. All three phosphate groups
shifted upfield; in the case of the c-phosphate the shift was
)12.6 p.p.m. By contrast, coordination to magnesium
caused only slight chemical shift changes, of which the
Fig. 2.
31

PNMRspectraofGTPcNH
2
(A) and GTPcF (B) (top) and
their respective magnesium ion complexes (bottom). Spectra were
recorded at pH 7.4 and 20 °C in aqueous solution.
Ó FEBS 2002 Nucleotide analogues GTPcNH
2
and GTPcF(Eur. J. Biochem. 269) 3273
largest was )0.5 p.p.m. for the c-phosphate. This might be
an effect of the low magnesium binding affinity, as will be
discussed later. A similar observation was made for the
19
F
NMR resonance line at position 0.91 p.p.m. which changed
only to 0.97 p.p.m. upon magnesium saturation. Finally,
the J-coupling values between the three phosphates again
tended to be very insensitive to modifications, and fell by
around 25% on complexation with magnesium. All chem-
ical shift data and J-coupling constants reported are
summarized in Tables 1 and 2.
Nucleotide stability
We next tested the stability of the GTPcNH
2
nucleotide
derivative. In 0.1
M
triethanolamine/HCl buffer at pH 7.6
the spontaneous hydrolysis of GTPcNH
2
at room tem-

perature was less than 1% in five days. In contrast, at
pH 4.5 in 0.1
M
potassium phosphate buffer the nucleotide
was hydrolysed to GDP with a half time of about 48 h.
Titration of GTPcNH
2
with HCl/NaOH monitored by
31
P
NMR spectroscopy showed no variation of the chemical
shifts of the three-fold negatively charged phosphate groups
from pH 3 to pH 11. At pH 2.8 the intrinsic hydrolysis
increased (so called acidic hydrolysis) and GTPcNH
2
3–
was
rapidly transformed to GDP
3–
+H
2
PO
4
–
+NH
4
+
by two
water molecules. The intermediate compound phosphor-
acid-amidate H

2
PO
3
NH
2
was not observed by NMR.
As a control, we titrated H
2
PO
3
NH
2
in the range from
pH 11 to pH 1.8. The
31
P chemical shifts for the three
different protonation states were found to H
2
PO
3
NH
2
at )6.90 p.p.m., [HPO
3
NH
2
]
–
at )2.65 p.p.m., and
[PO

3
NH
2
]
2–
at +7.97 p.p.m. The pK
a
values between these
three states were determined to pK
(0/1–)
¼ 3.02 ± 0.05 and
pK
(1–/2–)
¼ 8.46 ± 0.02 using a least square fit to 15
individual measured chemical shift values (data not shown).
Since the resonancelinesforthe a- and b-phosphate groups of
GDP at pH 2.8 were located at )10.73 and )10.20 p.p.m.,
respectively, a possible signal overlap between GTPcNH
2
,
GDP, HPO
3
NH
2
and H
3
PO
4
(P
i

) could be excluded. We
therefore assume that at low pH (pH < 3) GTPcNH
2
is
first transformed to ammonia and GTP, the latter being
subsequently hydrolysed to GDP and P
i
.
Magnesium binding and magnesium exchange rates
To analyse the metal ion binding properties of GTPcNH
2
we first performed a magnesium titration series and a
temperature series by NMR spectroscopy. Complete line-
shape analysis simulations of the complex formation of
GTPcNH
2
with Mg
2+
were performed on the entire
31
P
NMR spectra (a-, b- and c-phosphorus nuclei) and showed
a reasonably good agreement for all three resonance lines.
This is demonstrated in Fig. 3 where the part of the NMR
spectra and simulations that show the b-phosphate is
displayed. The b-resonance line underwent the biggest
resonance shift and was therefore most sensitive to changes
in the exchange rate, as the titration with magnesium from
null to complete saturation indicates (Fig. 3).
Next, we determined the binding energy of GTPcNH

2
to
magnesium by a complete lineshape analysis of a series of
NMR spectra. We adjusted the saturation of GTPcNH
2
with Mg
2+
to 45% and varied the temperature from 5 °Cto
65 °C in 13 steps of 5°. Five representative
31
P NMR spectra
of the b-resonance line and the corresponding simulations
are shown (Fig. 4). The fitted exchange rates in aqueous
solutions ranged from 900 to 9000 Hz with relative margins
from ± 22% at 5 °C to ± 8% at 30 °C. As the plot
against reciprocal temperature shows, the simulated
exchange rates k nicely fit to the Arrhenius equation
k ¼ k
0
exp(–DH
à
/RT) with R the gas constant and T the
absolute temperature (Fig. 5). Based on these values the
activation energy DH
à
for the GTPcNH
2
Mg
2+
complex

formation was determined to be 7.8 ± 0.15 kcalÆmol
)1
.
This result is similar to the activation energy for magnesium
binding of the natural substrate ATP which has been
determined to be 8.1 kcalÆmol
)1
[20].
Association of magnesium ions with different
nucleotides
In biological systems it is the complex between the
nucleotide and the magnesium ion which is bound to an
ATP or GTP binding enzyme rather than the nucleotide
only. Therefore, ITC was employed to quantify the
interaction between the nucleotides and the magnesium
ion (Fig. 6 and Table 3). As expected all nucleotides bound
one magnesium ion as indicated by the stoichiometry factor
N ¼ 1 (Table 3). Basically, for all complex formation
reactions an unfavorable enthalpy change was observed,
which was counteracted by a TDS° value two to three times
as large. In comparison to GTP the affinity for the
magnesium ion was lower for GTPcNH
2
and GTPcF.
For GTPcS the association constant was only two-fold
smaller whereas for GTPcNH
2
and GTPcF this constant
was significantly smaller, namely 34-fold and 140-fold,
respectively. Most probably this is due to the decreased

negative charge at the c-position in GTPcNH
2
and GTPcF
Fig. 3.
31
PNMRspectraofaMgCl
2
titration series added in increasing
amount to GTPcNH
2
. The resonance line of the b-phosphate in the
experimental measurements (left) and its corresponding simulation
(right) are shown. Note the shift and the intermediate broadening of
the resonance line. The amount of GTPcNH
2
Mg
2+
complexes relative
to free GTPcNH
2
nucleotide is indicated on the left. The determined
exchange rates based on the exchanging spin system simulation are
shown right. The spectra were measured at 20 °C and pH 7.4 in
aqueous solution.
3274 M. Stumber et al. (Eur. J. Biochem. 269) Ó FEBS 2002
where the protic hydroxy group is replaced by the amino
and fluoride groups, respectively. In contrast, the sulfur in
GTPcS may take on the role of the oxo-group. It should be
noted that the smaller affinities of GTPcNH
2

and GTPcF
are predominantly due to lower DS° values, possibly
reflecting the release of less water into bulk upon complex
formation.
Application of the nucleotides to the GTPase Ras
The suitability of the two nucleotide analogues for biolo-
gical macromolecules was finally tested using the small
GTP-binding protein Ras (reviewed in [21,22]). We success-
fully loaded the nucleotide analogues onto the 21 kDa
GTPase Ras using the alkaline phosphatase method, which
yielded a tightly bound protein-nucleotide complexes, as
found for RasÆGTP [23]. First the intrinsic GTPase rate of
wild-type H-Ras (1–167) complexed with magnesium ions
and various guanosine triphosphate nucleotide analogues
was determined by HPLC measurement (Table 4). At 37 °C
the intrinsic hydrolysis rate of Ras-bound fluorotriphos-
phate GTPcF was only twofold lower than for the natural
Fig. 5. Arrhenius plot of the simulated magnesium ion exchange rate
constants (k) vs. the reciprocal absolute temperature (1/T )for
GTPcNH
2
. The activation energy DH
à
for GTPcNH
2
ÆMg
2+
complex
formation is determined to 7.8 ± 0.15 kcalÆmol
)1

.
Fig. 6. Isothermal titration calorimetry of GTPcNH
2
with MgCl
2
. To a
solution of 5.0 m
M
GTPcNH
2
placed in the cell of the calorimeter a
solution of 50 m
M
MgCl
2
wasinjectedinstepsof6lL each (the first
step was 2 lL only). The increase in heating power was detected (upper
panel). The power pulses were integrated and plotted vs. the molar
ratio of injected MgCl
2
and nucleotide (lower panel). A fit to the
experimental data yields the stoichiometry factor N ¼ 0.96, the
association constant K
a
¼ 0.82 m
M
)1
and the enthalpy of association
DH° ¼ 3.9 kcalÆmol
)1

.
Table 3. Thermodynamic parameters for the association of magnesium
ions with different nucleotides obtained by isothermal titration calori-
metry. DS° is calculated according to the Gibbs-Helmholtz equation.
Nucleotide
N
(Nucl./Mg)
(mol/mol)
K
a
(m
M
)1
)
DH°
(kcalÆmol
)1
)
DS°
(calÆmol
)1
ÆK
)1
)
GDP 1.0 3.3 2.3 24
GTP 0.99 28 3.0 30
GTPcS 1.0 14 4.1 33
GTPcNH
2
0.96 0.82 3.9 26

GTPcF 0.84 0.20 3.9 24
Fig. 4.
31
P NMR spectra of a temperature series of GTPcNH
2
Mg
2+
.
The b-phosphate resonance line at )22.19 p.p.m. is shown in an
intermediate exchange state at 45% Mg
2+
saturation. Experimental
measurements (left) and simulated spectra (right) are displayed
showing temperature values and the simulated exchange rates,
respectively. Lyophilized GTPcNH
2
was dissolved to 2.1 m
M
con-
centration in aqueous solution and adjusted to pH 7.4 with HCl/
NaOH. MgCl
2
was added to 1 m
M
concentration. The precise
saturation was determined from the chemical shift position at 20 °C
(see Fig. 3 and Table 1).
Ó FEBS 2002 Nucleotide analogues GTPcNH
2
and GTPcF(Eur. J. Biochem. 269) 3275

substrate GTP while the rate for the thiotriphosphate
GTPcS was about 11-fold lower. Most stable with up to
190-fold lower hydrolysis rates were the two triphosphate
analogues with b,c-substitutions GppCH
2
p and GppNHp.
The Ras-catalysed hydrolysis rate of GTPcNH
2
finally lay
midway between the rates for GTPcSandGppNHp,witha
3-fold difference to both.
The more stable RasÆGTPcNH
2
ÆMg
2+
complex was
subsequently studied by
31
P NMR spectroscopy. A partic-
ular feature of the Ras protein is the flexibility of the effector
loop which can be detected in the triphosphate bound form
by a line splitting of the phosphorus resonances [24]. The
exchange is due to at least two distinct conformations which
can be observed also by heteronuclear NMR [25,26] or in
different crystal forms of Ras protein [27,28]. Flexibility in
the active center of G-proteins has been also observed for
RanGTP [29] and in different conformations of the switch
regions in the crystal structures of Rap2A complexed with
GTP, GDP and GTPcS [30]. As shown in Fig. 7 (bottom
spectrum) this feature was preserved for Ras bound to

GTPcNH
2
. At low temperature (5 °C) the b-phosphate
resonance was split into a less populated high field shifted
peak (b1,  27%) and a highly populated down field shifted
peak (b2,  73%). A temperature series from 2 °Cto30°C
revealed the coalescence of both lines at approximately
15 °C which is typical for a two-site exchange with a
transition from slow to fast exchange (data not shown). As
described for the intrinsic hydrolysis of GTPcNH
2
,theRas-
catalysed hydrolysis of bound GTPcNH
2
did not lead to the
observable formation of the compound H
2
PO
3
NH
2
in the
NMR spectra (which is expected at )2.7 p.p.m.). Instead,
the resonance signals for P
i
and Ras-bound GDP increased
during the time course of the experiment (Fig. 7, compare
bottom and top spectra) suggesting the formation of
ammonia and Ras-bound GTP before hydrolysis.
A concentration series with the effector protein Raf-RBD

at 5 °C added in increasing amount from 0.2 to 1 molar
ratio showed the progressive stabilization of one particular
conformation due to its high affinity for triphosphate bound
Ras (Fig. 7). Most remarkably, also the c-phosphate group
is perturbed by this interaction and shifted about
)0.8 p.p.m. upfield (Table 5). These data indicate the ability
of GTPcNH
2
to function as a triphosphate nucleotide
analogue with characteristic properties.
DISCUSSION
The data reported here demonstrate the synthesis of the two
guanosine triphosphate nucleotide analogues GTPcNH
2
and GTPcF, their biochemical characterization and appli-
cation to the GTP-binding protein Ras. GTPcNH
2
was
prepared by the method described by Knorre et al. [31] for
ATP derivatives. c-Amide derivatives of GTP were des-
cribed by Babkina et al. who used, e.g. the c-(4-azido)
anilide of GTP to substitute efficiently for GTP as a
photoaffinity label in the elongation factor protein EF-Tu
[32]. The GTPcF substrate analogue was first prepared by
Eckstein et al. [9], by the method of Haley & Yount [33],
and used to study its interaction with the GTP-binding site
of adenylyl cyclase [34]. We synthesized this substance by
Table 4. Intrinsic GTPase rate of wild-type H-Ras
(1)167)
Mg

2+
protein
at 37 °C bound to various guanosine triphosphate nucleotide analogues.
Hydrolysis rates were determined with HPLC by measuring the con-
centration of protein-bound tri- and diphosphate nucleotides. Buffer
conditions: 25 m
M
Tris/HCl at pH 7.4, 2.5 m
M
MgCl
2
and 1 m
M
DTE.
Nucleotide GTPase rate (10
)5
min
)1
)
RasGTP 2820
RasGTPcF 1427
RasGTPcS 252
RasGTPcNH
2
84.6
RasGppNHp 25.6
RasGppCH
2
p 15.0
Fig. 7.

31
P NMR spectra of protein bound Ras
Æ
GTPcNH
2
Æ
Mg
2+
and
concentration series with Raf-RBD. The ratio of Raf-RBD to Ras
varies from 0 (bottom spectrum) to 1 (top spectrum) as indicated on
the right. Spectra were recorded at pH 7.4 and 5 °Cin25m
M
Tris/
HClbuffer,2.5m
M
MgCl
2
and 1 m
M
dithioerythritol. Note the
splitting of the b-phosphate resonance into two states for
RasÆGTPcNH
2
ÆMg
2+
(bottom spectrum) and the stabilization of one
conformation upon complexation with Raf. Excess of free and bound
phosphate groups are labelled.
Table 5.

31
P chemical shifts of Ras
(1)167)
Æ
GTPcNH
2
Æ
Mg
2+
at pH 7.4,
5 °C. Spectra were recorded in 25 m
M
Tris/HCl buffer, 2.5 m
M
Mg
2+
and 1 m
M
DTE. The splitting of the a- and b-phosphate resonance
lines in protein bound triphosphate-nucleotides is a specific feature of
the Ras protein, indicating different conformations of the active center
[24].
31
P chemical shift (p.p.m.)
Proteinnucleotide a
(1)
a
(2)
b
(1)

b
(2)
c
RasGTPcNH
2
Mg
2+
– )11.80 )16.15 )16.85 1.90
RafRasGTPcNH
2
Mg
2+
– )11.76 – )16.81 1.07
RasGppNHpMg
2+
)11.15 )11.85 )2.69 )3.41 )0.32
3276 M. Stumber et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the simple method of Wittmann [10], which works very well
with adenine nucleotides. The presence of a guanine base
gives rise to the formation of yellow by-products, probably
due to the reaction of 2,4-dinitrofluorobenzene with the
amino group of GTP. Thus in this case the simplicity of the
method is at the expense of yield.
The kinetic parameters determined reveal the biochemi-
cal properties of the two nucleotide analogues. While the
activation energy of magnesium binding for GTPcNH
2
is
similar to that of the natural substrate [20,35], the
association constant K

a
for magnesium is significantly
smaller for both nucleotides analysed. Therefore, the more
stable c-amido triphosphate analogue may be particularly
useful for the study of the role of divalent cation binding,
e.g. to analyse a proposed reaction mechanism. For
example, the influence of the magnesium binding affinity
on the kinetics of the Ras guanine nucleotide exchange
factor Sos [36] can be studied with the nucleoside
diphosphate GDPcNH
2
derivative loaded onto Ras.
Additionally, an intermediate magnesium-free state may
be stabilized more easily with GTPcNH
2
or ATPcNH
2
analogues. The analogue ATPcNH
2
may provide new
insights into the equilibrium between different conforma-
tions of myosin [37]. Finally, specific labeling of the amide
group with
15
N isotopes may be useful for nitrogen
selective heteronuclear NOE experiments for the structural
analysis of the active center in solution. In combination
with specific labeling of single residues in the protein [38]
this may yield detailed insights into the dynamics of the
nucleotide binding site.

The GTPcF analogue may be particularly useful
because of the high sensitivity of
19
F NMR spectroscopy.
In a previous report the GTP binding site of the 110 kDa
protein tubulin was studied using the fluorotriphosphate
[39]. Here, fluorine relaxation rates were determined to
analyse the location of the divalent cation site relative to
the exchangable nucleotide. We are analysing the possi-
bility of two distinct nucleotide binding sites in the human
guanylate-binding protein 1 (hGBP1) [40] by titration of
GTPcF or GDPbF to the noncomplexed protein, assu-
ming different chemical environments for each putative
nucleotide binding site (data not shown). Finally, the
suitability of both nucleotide derivatives for the use in
solid state NMR spectroscopy should be tested in future
experiments.
For the slowly hydrolysing Ras protein the two
nucleotides described here close the 10-fold gap in intrinsic
hydrolysis rates between bound GTP and GTPcS, and
GTPcS and GppNHp (Table 4). This broad variety allows
an almost individual selection of hydrolysis stability from
protein bound GTP to protein bound GppCH
2
pforall
different purposes. The application onto the GTP-binding
protein Ras confirmed that the flexibility of the effector
loop of Ras is preserved (Fig. 7). For nucleotide binding
proteins the flexibility of the nucleotide binding site in its
active center is a vital prerequisite for the dynamic

function of the protein. This is particularly important for
mechanistic studies and the analysis of binding intermedi-
ates, as shown for the complex formation of Ras with the
effector protein Raf-RBD [24,41]. With these two deriv-
atives characterized we extend the variety of nucleotide
analogues available for kinetic, structural, and cellular
studies.
ACKNOWLEDGEMENTS
We thank John Wray and Roger S. Goody for discussions and Ulrich
Haeberlen, Alfred Wittinghofer and Kenneth C. Holmes for continu-
ous support. M.G. acknowledges support by the Peter und Traudl
Engelhorn Stiftung (Penzberg, Germany).
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