Báo cáo khoa học: Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPcS pot

Slow conformational dynamics of the guanine
nucleotide-binding protein Ras complexed with the GTP
analogue GTPcS
Michael Spoerner
1
, Andrea Nuehs
1
, Christian Herrmann
2
, Guido Steiner
1
and
Hans Robert Kalbitzer
1
1 Universita
¨
t Regensburg, Institut fu
¨
r Biophysik und physikalische Biochemie, Germany
2 Ruhr Universita
¨
t Bochum, Physikalische Chemie I, Germany
Guanine nucleotide-binding proteins of the Ras super-
family function as molecular switches, cycling between
a GDP-bound ‘off’ and a GTP-bound ‘on’ state. They
regulate a diverse array of signal transduction and
transport processes.
It has been shown using
31
P NMR spectroscopy
that Ras (rat sarcoma) protein occurs in two con-

formational states (state 1 and 2) when complexed
with the GTP analogues guanosine-5¢-(b,c-imido)tri-
phosphate (GppNHp) [1] or guanosine-5¢-(b,c-methy-
leno)triphosphate (GppCH
2
p) [2]. These two states
interconvert with rate constants in the millisecond
time scale. They are characterized by typical
31
P NMR chemical shifts, with shift differences up to
0.7 p.p.m. NMR structural studies have shown that
this dynamic equilibrium comprises two regions of
Keywords
conformational equilibria; GTP analog;
GTPcS; Ras
Correspondence
H. R. Kalbitzer, Institut fu
¨
r Biophysik und
physikalische Biochemie,
Universita
¨
tsstraße 31, Regensburg,
D-93040, Germany
Fax: +49 941 943 2479
Tel: +49 941 943 2595
E-mail: hans-robert.kalbitzer@biologie.
uni-regensburg.de
(Received 28 July 2006, revised 13 Novem-
ber 2006, accepted 8 January 2007)

doi:10.1111/j.1742-4658.2007.05681.x
The guanine nucleotide-binding protein Ras occurs in solution in two
different conformational states, state 1 and state 2 with an equilibrium
constant K
12
of 2.0, when the GTP analogue guanosine-5¢-(b,c-imido)tri-
phosphate or guanosine-5¢-(b,c-methyleno)triphosphate is bound to the
active centre. State 2 is assumed to represent a strong binding state for
effectors with a conformation similar to that found for Ras complexed to
effectors. In the other state (state 1), the switch regions of Ras are most
probably dynamically disordered. Ras variants that exist predominantly in
state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt)
bound to the GTP analogue guanosine-5¢-O-(3-thiotriphosphate) (GTPcS)
leads to
31
P NMR spectra that indicate the prevalence of only one con-
formational state with K
12
> 10. Titration with the Ras-binding domain of
Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding
state 2. In the GTPcS complex of the effector loop mutants Ras(T35S) and
Ras(T35A) two conformational states different to state 2 are detected,
which interconvert over a millisecond time scale. Binding studies with Raf-
RBD suggest that both mutants exist mainly in low-affinity states 1a and
1b. From line-shape analysis of the spectra measured at various tempera-
tures an activation energy DH
|
1a1b
of 61 kJÆmol
)1

and an activation entropy
DS
|
1a1b
of 65 JÆ K
)1
Æmol
)1
are derived. Isothermal titration calorimetry on
Ras bound to the different GTP-analogues shows that the effective affinity
K
A
for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 com-
pared to the wild-type with the strongest reduction observed for the GTPcS
complex.
Abbreviations
GppCH
2
p, guanosine-5¢-(b,c-methyleno)triphosphate; GppNHp, guanosine-5¢-(b,c-imido)triphosphate; GTPcS, guanosine-5¢-O-(3-
thiotriphosphate); ITC, isothermal titration calorimetry; Raf-RBD, Ras-binding domain of Raf-kinase; Ras, protein product of the proto
oncogene ras (rat sarcoma).
FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1419
the protein called switch I and switch II [1,3,4]. Solid-
state NMR shows that even in single crystals or crys-
tal powders of Ras(wt)•Mg
2+
•GppNHp the two
conformational states can be observed to be in dyna-
mic equilibrium at ambient temperatures [5,6].
A threonine residue located in the effector loop

(Thr35 in Ras) is conserved in all members of the
Ras superfamily and seems to play a pivotal role in
the conformational equilibrium. It is involved, via its
side-chain hydroxyl, in the coordination of the diva-
lent metal ion and, via its main-chain amide, in a
hydrogen bond with the c-phosphate of the nucleo-
tide when complexed to the effector [7,8]. The same
coordination pattern is most probably preserved in
state 2 of free Ras. Replacing this threonine in Ras
with an alanine or serine residue leads to a complete
shift of the equilibrium towards state 1 in solution,
when Ras is bound to the GTP analogues GppNHp
[9] or GppCH
2
p [2]. These Ras variants, previously
used as partial loss-of-function mutants in cell-based
assays, show a reduced affinity between Ras and
effector proteins without Thr35 being involved in
any interaction. X-Ray crystallography [9] on
Ras(T35S)•Mg
2+
•GppNHp and EPR investigations
[10] suggest that switch I and switch II exhibit high
mobility in state 1. Recently, X-ray structures of
M-Ras [11] and of the G60A mutant of human
H-Ras [12], both in the GppNHp-bound form, were
published. These Ras variants seem to exist in
conformational state 1, as shown using
31
P NMR

spectroscopy. In the X-ray structure the contacts of
Thr35 (Thr45 in M-Ras) with the metal ion and the
c-phosphate group do not exist.
31
P NMR data indi-
cate that state 2 corresponds to the conformation of
Ras found in complex with the effectors. State 1,
characteristic of the mutants Ras(T35S) and
Ras(T35A) in the GppNHp form, represents a weak-
binding state of the protein [9,13]. Upon addition of
the Ras effector Raf-kinase, the
31
P NMR lines of
Ras(T35S) but not Ras(T35A) shift to positions cor-
responding to the strong binding conformation of
the protein [9].
A conformational equilibrium in the interaction site
with effectors seems to be a general property of Ras
and other small GTPases [14]. The equilibrium is influ-
enced not only by specific mutations but also by the
nature of the GTP analogue bound (GppNHp or
GppCH
2
p). In this study we investigate the dynamic
behaviour of Ras in complex with guanosine-5¢-O-(3-
thiotriphosphate) (GTPcS), another commonly used
GTP analogue that is hydrolysed slowly to find more
evidence for the biological importance of the conform-
ational equilibria.
Results

Chemical shifts of the nucleotide analogue
GTPcS in the absence and in the presence of
magnesium ions
Chemical shift values for the phosphates and the thio-
phosphate group of the nucleotide depend strongly on
the degree of protonation of their oxygens. Further-
more, chemical shifts and pK values are influenced by
Mg
2+
binding to the protein–nucleotide complex. For
a better interpretation of the chemical shifts of the
protein-bound nucleotide analogue we first studied
GTPcS in the presence and absence of Mg
2+
ions
within a pH range of 2–13. The rate of exchange
between Mg
2+
and the nucleoside triphosphate is slow
enough to observe the resonances of the metal-free
form separately from the metal-complexed form at
lower temperatures. Therefore, experiments were per-
formed at 278 K to ensure that over the whole pH
range a significant contribution of metal-free nucleo-
tide, if existing, could be directly detected by addi-
tional resonance lines. At a magnesium concentration
of 3 mm the nucleotide is completely saturated with
the divalent ion in the pH range studied since further
increase of the Mg
2+

concentration does not influence
the observed chemical shifts (also see Experimental
procedures).
Figure 1 shows the titration curves for GTPcSin
the absence and presence of Mg
2+
. Separation of the
three phosphate signals by more than 60 p.p.m. is
rather large. Particularly in case of the c-phosphorus
(Fig. 1A,B) two pK values are necessary in order to
describe the observed dependence of chemical shifts in
the pH range studied. The corresponding pK values
and chemical shifts are summarized in Table 1 together
with the data for the analogues GppNHp and
GppCH
2
p [2]. As expected, the apparent pK values
decrease substantially in the presence of the metal ion.
By far the largest effect on the chemical shifts is found
for the b- and c-phosphate group, but a slight shift of
0.6 p.p.m. is also seen for the a-phosphorus resonance
in the Mg
2+
•GTPcS complex. In agreement with pre-
vious studies on ATP [15], our data suggest a mixture
of different metal complexes in solution with a high
population of complexes where the b- and c-phosphate
is involved, as shown previously for the GTP ana-
logues GppNHp and GppCH
2

p [2]. The pK
3
values in
GTPcS are much smaller than those reported for
GppNHp and GppCH
2
p. The value of pK
2
does not
depend much on the analogue when a relatively large
error is taken into consideration. pK
2
and pK
3
are
usually associated with the first and the second
Conformational dynamics of Ras bound to GTPcS M. Spoerner et al.
1420 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS
deprotonation step at the c-phosphate group of the
nucleotide for the transition from the threefold negat-
ively charged state to the fourfold negatively charged
state. In line with this suggestion the largest shifts are
observed for the c-phosphate group for the first
deprotonation step for the three analogues. However,
the second deprotonation step is associated with larger
changes in the b-phosphate shifts in GppNHp and
GppCH
2
p, indicating a more complex pH perturbation
of the electronic system in these analogues.

Fig. 1. Titration curves of free and Mg
2+
bound GTPcS. (A,C)
31
P chemical shift val-
ues of the a-, b- and c-phosphate groups
were determined on a 2.5 mL of a 1 m
M
GTPcS solution in 100 mM Tris, 95% H
2
O
and 5% D
2
O containing 0.1 mM 2,2-dimeth-
yl-2-silapentane-5-sulfonate for indirect refer-
encing. The pH was adjusted by adding HCl
or NaOH. Measurements were performed in
a 10-mm sample tube at 278 K. (B,D) Meas-
urements on the Mg
2+
complexes were per-
formed in the presence of 3 m
M MgCl
2
. The
dependence of chemical shifts on the pH
values was fitted to Eqn (7). The
31
P reso-
nances were assigned by selective

1
H- and
31
P-decoupling experiments.
Table 1. pH dependence of chemical shifts of different GTP analogues. Data were recorded at 278 K in solutions of 1 mM nucleotide in the
absence or presence of 3 m
M MgCl
2
in 95% H
2
O ⁄ 5% D
2
O. In a first approximation d
2
, d
3
, and d
4
correspond to the chemical shifts of two-,
three-, and fourfold negatively charged nucleotide. pK
2
and pK
3
are the corresponding pK
a
values of the three phosphates of the nucleotide.
d
2
values are given in parentheses the titration up to pH 1.5 does not allow the precise estimation of this value. For d
3

and d
4
the estimated
error is ± 0.05 p.p.m.
Nucleotide
Phosphate
group d
2
⁄ p.p.m. pK
2
d
3
⁄ p.p.m. pK
3
d
4
⁄ p.p.m.
GTPcS a ()11.3) ) 11.30 ) 11.04
b ()24.0) 2.8 ± 0.1 ) 24.0 5.78 ± 0.02 ) 23.06
c (40.8) 39.70 33.91
Mg
2+
•GTPcS a ()11.2) ) 11.27 ) 10.67
b ()24.2) 1.7 ± 0.5 ) 23.78 ) 20.51
c (41.6) 40.38 4.11 ± 0.02 36.85
GppCH
2
p
a
a ()10.86) ) 10.93 ) 10.82

b (7.14) 3.2 ± 0.15 8.74 8.96 ± 0.02 13.22
c (17.85) 14.63 6.57 ± 0.02 11.23
Mg•GppCH
2
p
a
a ()10.83) ) 10.47 ) 10.33
b (9.50) 2.3 ± 1.5 9.93 14.93
c (16.98) 14.29 11.46
GppNHp
a
a ()10.95) ) 10.80 8.79 ± 0.02 ) 10.55
b ()12.27) 3.4 ± 0.04 ) 10.91 ) 7.76
c (0.20) ) 1.64 ) 0.91
Mg•GppNHp
a
a ()11.17) ) 10.34 6.56 ± 0.02 ) 10.01
b ()9.36) 2.0 ± 0.8 ) 8.95 ) 5.46
c ()1.38) ) 2.16 ) 1.02
a
Data from Spoerner et al. [2].
M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS
FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1421
Conformational states of Ras complexed with
Mg
2+
•GTPcS
Figure 2 shows
31
P NMR spectra of Ras(wt) in com-

plex with the slowly hydrolysable GTP analogue
GTPcS at various temperatures. Assignment of the res-
onance lines was confirmed by a 2D
31
P–
31
P NOESY
experiment on Ras(wt)•Mg
2+
•GTPcS (data not
shown). Binding of GTPcS to the Ras protein leads to
rather large chemical shift changes. In contrast to the
observations made for the GTP analogues GppNHp
and GppCH
2
p [1,2] only one set of resonances can be
observed for the wild-type protein in the temperature
range 278–308 K (Fig. 2). This most probably means
that wild-type Ras occurs predominantly in one state
when GTPcS is bound. It is reasonable to assume that
a second structural state also exists and is character-
ized by different chemical shift values, as observed in
the GppNHp and GppCH
2
p complexes [1,2]. When
this second state has clearly different chemical shifts
compared with the first state then two scenarios are
consistent with the observed spectrum. If fast exchange
conditions prevail over the whole temperature range,
then only one averaged resonance signal per phosphate

group would be observed. If slow exchange conditions
prevail, a second conformational state, characterized
by clearly different chemical shifts, must have a rather
low population because no signals can be detected
above noise level. In this case, from the signal-to-noise
ratio the equilibrium constant for the two states can
be estimated to be > 10. Analysing the temperature
dependence of the line width, particularly of the
c-phosphorus resonance, slow exchange conditions are
more likely. At lower temperatures the line width
decreases with increasing temperature due to the
decrease of the rotational correlation time. At higher
temperatures the line width increases again (51 Hz at
298 K, 57 Hz at 303 K). Chemical shift also changes
within the temperature range of 278–308 K by
+0.26 p.p.m. At higher temperatures, the GTP ana-
logue hydrolyses, and resonances of Ras-bound GDP
are thus detected. In principle, one would expect to
observe thiophosphate and Ras-bound GDP as result
of GTPcS hydrolysis. In contrast, with all the meas-
urements performed in this study, inorganic phosphate
could be observed only using
31
P NMR. In addition,
H
2
S could be detected by its smell after a time. The
exact mechanism of thio phosphate decay could not be
clarified. It is dependent on the presence of Ras, but
may be also due to other protein impurities occurring

in low concentrations in the Ras preparations. In con-
trast to the situation observed for wild-type protein in
the complex of GTP cS with the mutant Ras(T35S) or
Ras(T35A), additional
31
P NMR lines are found at
low temperature (Fig. 3A). With increasing tempera-
ture, the lines initially become broader before coales-
cing again at higher temperature (Fig. 4A). From our
studies with GppNHp and GppCH
2
p we expect that
the effector interaction state 2 becomes destabilized by
replacing Thr35 with a serine or an alanine residue,
and therefore at least one of the new lines seen in the
mutant is likely to correspond to state 1. Because no
component of the two sets of resonances of Ras(T35S)
and Ras(T35A) has a chemical shift that corresponds
to that of Ras(wt) it is not clear whether the two sets
of resonance lines correspond to state 1 and state 2 or
if they represent two substates of state 1 (see below).
In the following, we call them state 1a and state 1b.
The equilibrium constant K
1a1b
¼ [1b] ⁄ [1a] between
these two states is 0.5. In the case of the serine mutant,
a weak third line of the c-phosphorus signal with a
similar chemical shift to the resonance of wild-type
Ras seems to exist (Fig. 3A); this is not visible in the
spectrum of the T35A mutant. The chemical shifts are

summarized in Table 2.
With knowledge of the resonance positions corres-
ponding to state 1a and 1b, we investigated whether
these states also exist in wild-type Ras bound to
GTPcS. Separation of the chemical shift values
between state 1b and state 2 of more than 4 p.p.m.
allowed us to perform a saturation-transfer experiment
with presaturation at frequencies around the signal
corresponding to state 1b. If exchange occurs over a
Fig. 2.
31
P NMR spectra of wild-type Ras complexed with
Mg
2+
•GTPcS at various temperatures. The samples contained
1m
M Ras(wt)•Mg
2+
•GTPcSin40mM Hepes ⁄ NaOH pH 7.4,
10 m
M MgCl
2
, 150 mM NaCl, 2 mM 1,4-dithioerythritol and 0.1 mM
2,2-dimethyl-2-silapentane-5-sulfonate in 5% D
2
O, 95% H
2
O,
respectively. The absolute temperature was controlled by immer-
sing a capillary with ethylene glycol and measuring the hydroxyl-

methylene shift difference [28].
Conformational dynamics of Ras bound to GTPcS M. Spoerner et al.
1422 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS
timescale < T
1
a decrease in the integral of the reson-
ance corresponding to state 2 should be observed, even
when state 1 is too sparsely populated to be detectable
directly. Some results are shown in Fig. 3B. A mini-
mum of the resonance integral of state 2 is obtained at
a presaturation frequency of 32.7 p.p.m., which corres-
AB
Fig. 3. Conformational equilibria of wild-type Ras and Ras mutants complexed with Mg
2+
•GTPcS. (A) The sample contained 1 mM
Ras(wt)•Mg
2+
•GTPcS (lower), 1.2 mM Ras(T35S)•Mg
2+
•GTPcS (middle), and 1 mM Ras(T35A)•Mg
2+
•GTPcS (upper) in 40 mM Hepes ⁄ NaOH
pH 7.4, 10 m
M MgCl
2
, 150 mM NaCl, 2 mM 1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfonate in 5% D
2
O, 95% H
2
O,

respectively. Data were recorded at 278 K. The assignment was determined by a
31
P–
31
P NOESY experiment on Ras(wt)•Mg
2+
•GTPcS.
31
P
resonances assigned to Ras–nucleotide complex in conformation of state 1a or state 1b are coloured in red, the resonances assigned to
state 2 are coloured green. (B)
31
P NMR saturation transfer experiment on Ras(wt)•Mg
2+
•GTPcS. The integrals of the resonance correspond-
ing to the c-thiophosphate group in state 2 of Ras(wt) are given in dependence of the frequency of presaturation d. For presaturation a weak
rectangular pulse of 1 s duration and a B
1
-field of 18 Hz were used. A Lorentzian function was fitted to the data. The integral of the c-phos-
phorus signal without presaturation is set to 100%.
Fig. 4. Experimental and simulated
31
P NMR data of Ras(T35S)•Mg
2+
•GTPcS at different temperatures. The sample contained 1.2 mM
Ras•Mg
2+
•GTPcSin40mM Hepes ⁄ NaOH pH 7.4, 10 mM MgCl
2
,2mM 1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfonate

in 5% D
2
O, 95% H
2
O. The absolute temperature was controlled by immersing a capillary with ethylene glycol and measuring the hydroxyl–
methylene shift difference [28]. (A) Experimental spectra; (B) simulated spectra. Experimental data were filtered by an exponential filter lead-
ing to an additional line broadening of 5 Hz. Total number of scans per spectrum were 1600–5400. The rate constant for the transition
state 1a to state 1b are indicated. Data were simulated as described in Experimental procedures. The transverse relaxation rates 1 ⁄ T
2
at
278 K (in the absence of exchange) obtained from the data analysis are 251 s
)1
for both state 1a and state 1b of the a-phosphate group of
bound GTPcS, 236 s
)1
and 204 s
)1
for the b-phosphate group of bound GTPcS in state 1a and state 1b, respectively, and 189 s
)1
for
state 1a and 1b of the bound c-thiophosphate group (values are given with an estimated error of ± 15 s
)1
).
M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS
FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1423
ponds to the frequency of state 1b detected for the two
Thr35 mutants. These results indicate the existence of
state 1b in wild-type Ras, but with a very sparse popu-
lation. A more detailed analysis including calculation
of exchange rates was not possible because of the lim-

ited signal-to-noise.
Dynamics of the conformational exchange
By analysing the temperature dependence of the
31
P NMR data from Ras(T35S)•Mg
2+
•GTPcS
(Fig. 4B) for the transition between substates 1a and 1b
the Gibb’s free activation energy DG
|
, the activation
enthalpy DH
|
and the activation entropy DS
|
can
be determined (Table 3) using a full-density matrix
analysis. The exchange rates obtained are somewhat
higher than that found between states 1 and 2 of
Ras(wt)•Mg
2+
•GppNHp or Ras(wt)•Mg
2+
•GppCH
2
p.
Whereas DG
|
of the exchange in Ras(T35S)•Mg
2+

•GTPcS is equal to that obtained for the other com-
plexes, both DH
|
, and DS
|
are somewhat lower. For the
other nucleotides studied, relaxation times T
2
at 278 K
for the a- and c-phosphate group were quite different
for the two conformational states 1 and 2. We did not
find such large differences between the corresponding
T
2
relaxation times for the conformational states 1a and
1b of Ras(T35S)•Mg
2+
•GTPcS.
Complex of Ras•Mg
2+
•GTPcS with the
Ras-binding domain of Raf-kinase
Addition of the Ras-binding domain of Raf-kinase
(Raf-RBD) to Ras(wt)•Mg
2+
•GTPcS leads to line
broadening of the resonances (Fig. 5, Table 2), but
only to very small changes in the chemical shifts
(|Dd| £ 0.16 p.p.m). This is in line with the assumption
that the wild-type protein occurs mainly in conforma-

tional state 2 when the GTP analogue GTPcSis
bound. Correspondingly, in Ras(T35S)•Mg
2+
•GTPcS,
lines preliminary assigned to states 1a and 1b decrease
in intensity when Raf-RBD is bound, whereas the
intensity of lines located close to those assigned in
wild-type Ras to state 2 increases (Fig. 5, Table 2).
The changes in chemical shift induced by Raf binding
are rather large in the mutant, suggesting that
none of the states visible in the spectrum of
Ras(T35S)•Mg
2+
•GTPcS corresponds to state 2 found
in the wild-type protein. Complex formation between
Raf-RBD and Ras(T35A)•Mg
2+
•GTPcS (Fig. 5,
Table 2) leads only to a line broadening of the two
lines of the c-phosphate group, and not to significant
changes in chemical shift or the relative populations of
the resonances. In particular, the relative intensity of
the downfield-shifted c-phosphorus resonance is not
increased in the presence of the effector as would be
expected if it corresponded to effector binding state 2.
Influence of the GTP analogue on the affinity
between Raf-RBD and Ras
The affinities of wild-type and (T35S)Ras complexed
with the different GTP analogues GppNHp, GppCH
2

p
and GTPcS to Raf-RBD were determined using isother-
mal titration calorimetry (ITC) at 298 K in a buffer
identical to that used in the NMR spectroscopy
experiments. Within the limits of error, the effective
Table 2.
31
P chemical shifts and conformational states of Ras complexed with different GTP analogues. Data were recorded at various tem-
peratures. Shifts were taken from spectra recorded at 278 K. The equilibrium constant K
12
between state 1 and 2 is calculated from inte-
grals of the c-thiophosphate resonances defined by K
12
¼ k
12
⁄ k
21
¼ [2]] ⁄ ([1a] + [1b]). State 2 is assigned to the conformation close to the
effector binding state. The error is < 0.03 p.p.m. for the chemical shifts and < 0.1 for the equilibrium constants. ND, not detected.
Ras-complex
a-phosphate b-phosphate c-phosphate
K
12
K
1a1b
d
1
(p.p.m.)
d
2

(p.p.m.)
d
1
(p.p.m.)
d
2
(p.p.m.)
d
1
(p.p.m.)
d
2
(p.p.m.)
Ras(wt)•Mg
2+
•GTPcS )11.30 )16.67 37.01 > 10 ND
b
Ras(T35S)•Mg
2+
•GTPcS )10.70 )17.96
a
)17.22
a
32.73
a
37.89
a
36.87 0.06 0.5
Ras(T35A)•Mg
2+

•GTPcS )10.80 )17.92
a
32.79
a
< 0.05 0.5
)17.19
a
37.91
a
Ras(wt)•Mg
2+
•GTPcS )11.19 )16.55 36.85 > 10 ND
b
+ Raf-RBD
Ras(T35S)•Mg
2+
•GTPcS )11.22 )16.52 36.54 > 10 ND
b
+ Raf-RBD
Ras(T35A)•Mg
2+
•GTPcS )10.50 )17.55 32.48
a
< 0.05 0.5
+ Raf-RBD 37.91
a
a
Chemical shifts in state 1a (lower) and 1b (upper).
b
Values could not be determined since signal cannot be detected.

Conformational dynamics of Ras bound to GTPcS M. Spoerner et al.
1424 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS
association constant K
A
between wild-type Ras and
Raf-RBD is not influenced by the type of bound ana-
logue (Table 4). However, in all cases, the contributions
of enthalpy and entropy to DG° differ between nucleo-
tide analogues. Although for the Thr35 mutant the error
ranges for the three nucleotide analogues overlap, a dif-
ference in affinities between Ras(T35S) bound to the
analogue GTPcS, where the oxygen between b- and c-
phosphate is still available, and GppCH
2
p may exist. A
significant decrease in K
A
, by a factor of $ 20, is seen,
independent of the analogue used when the wild-type
protein is compared with Ras(T35S). The decrease in
affinity is due to changes in DH° and DS°, which partly
compensate.
Discussion
The environment of the nucleotide bound to the
protein
NMR spectroscopy very sensitively reports changes in
the environment of a given atom by measuring a
change in its resonance frequency. Whenever chemical
shift changes are visible they indicate that there is a
change in the environment of the observed nucleus.

For phosphorus resonance spectroscopy on nucleo-
tides, it is known that two factors mainly determine
chemical shift changes, a conformational strain and
electric field effects polarizing the oxygen atoms of the
phosphate groups. In addition to these direct effects,
long-range effects may occur that are caused by a
structure-dependent change in the anisotropy of the
magnetic susceptibility. Here, ring current effects may
be the most dominant contribution.
We have previously studied the complexes of Ras
using the GTP analogues GppCH
2
p and GppNHp [2],
which differ in the position of the b–c-bridging oxygen
by replacing the naturally occurring oxygen either with
an apolar group or a hydrogen-bond donator. We
have now completed the picture using the slowly
hydrolysing GTP analogue GTPcS, in which the b–c-
bridging oxygen is not affected, but the physicochemi-
cal properties of the c-phosphate group are modified.
For a quantitative analysis of the chemical shift chan-
ges induced by protein binding it was necessary to
have reliable data for the system not perturbed by
Table 3. Exchange rates and thermodynamic parameters in different Ras–nucleotide complexes. The rate constants k
12
and k
21
(k
1a1b
and

k
1b1a
) were calculated by a line-shape analysis based on the density matrix formalism as described in Experimental procedures. The free acti-
vation energy DG
|
, the activation enthalpies DH
|
, and the activation entropies DS
|
, were calculated from the temperature dependence of the
exchange rates on the basis of the Eyring equation. The values for the transition between state 1 and state 2 k
12 and
k
21
are given. The
states are defined as in Table 1. DG
12
or DG
1a1b
is the difference in free enthalpy between state 2 (1b) and 1 (1a). T
2
times given are without
exchange contribution and were obtained from the line shape analysis. The estimated error is ± 0.3 ms.
Protein complex
Temp.
(K)
Exchange rate
constant (s
)1
)_

DG
j
1a1b
DH
j
1a1b
TDS
j
1a1b
DG
1a1b
k
1a1b
k
1b1a
(kJÆmol
)1
) (kJÆmol
)1
) (kJÆmol
)1
) (kJÆmol
)1
)
Ras(T35S)•Mg
2+
•GTPcS 278 70 137 41 ± 2 61 ± 1 18 ± 1 1.56 ± 0.15
a
288 170 330
298 430 810

k
12
k
21
DG
j
12
DH
j
12
TDS
j
12
DG
12
Ras(wt)•Mg
2+
•GppNHp
a
278 80 42 42 ± 5 70 ± 3 28 ± 2 ) 1.48 ± 0.15
288 250 135
298 700 387
Ras(wt)•Mg
2+
•GppCH
2
p
a
278 80 39 41 ± 5 63 ± 3 29 ± 2 ) 1.65 ± 0.15
288 260 131

298 740 391
Relaxation times T
2
(ms) of the resonances of
Protein-complex a-phosphate b-phosphate c-phosphate
(1) or (1a) (2) or (1b) (1) or (1a) (2) or (1b) (1) or (1a) (2) or (1b)
Ras(T35S)•Mg
2+
•GTPcS 278 4.0 4.0 4.2 4.9 5.3 5.3
Ras(wt)•Mg
2+
•GppNHp
a
278 5.8 3.9 4.8 4.8 4.1 7.1
Ras(wt) •Mg
2+
•GppCH
2
p
a
278 4.2 4.0 6.4 6.4 3.8 5.2
a
Data from Spoerner et al. [2]. Note that the values given differ somewhat from those given by Geyer et al. [1] because absolute tempera-
ture was controlled independently and the new assignment of the signals were considered.
M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS
FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1425
protein binding that we provide here. Although data
had been published previously for free GTPcS [16],
they were measured under different experimental
conditions and the referencing system (external stand-

ard) in particular is not sufficiently reliable for precise
comparisons.
When one compares the chemical shift changes Dd
in the free Mg
2+
–nucleotide complexes (Table 1) with
those induced by protein binding (Table 2) one may
obtain information on the change of the environment
of the phosphate groups in the different complexes. In
wild-type Ras in state 2, one finds Dd values of )0.26,
6.39 and 3.10 p.p.m., respectively for the a-, b- and
c-phosphate of GTPcS. The corresponding shift chan-
ges are )1.15, 7.51 and )2.41 p.p.m. for GppNHp and
)2.44, 6.32 and )3.03 p.p.m. for GppCH
2
p. The
a-phosphate groups in the three GTP analogues should
be least influenced by the modifications. In accordance
with this observation, in the absence of protein, their
response to a change in pH (acidity) is very small, only
an upfield shift of < 0.26 p.p.m. is observed when the
c-phosphate group is protonated by a decrease in pH.
After binding to the protein, for all three analogues an
upfield shift between of 0.26 and 2.44 p.p.m. is
observed, indicating that the environmental changes
are qualitatively similar but differ in detail.
Potential phosphate group interactions can be
derived from the published X-ray structures, although
one should be aware that they show differences in
effector loop details that may reflect the occurrence of

different conformational states in solution. Because
NMR data indicate that the interaction of Ras with
Raf-RBD stabilizes the effector loop in a well-
defined, state 2-like conformation, the X-ray structure
of the Ras-like mutant of Rap1A, called Raps
[Rap(E30D,K31E)], complexed with Mg
2+
•GppNHp
and Raf-RBD [7] can serve as a model.
The most important interactions derived from the
X-ray structure are depicted in Fig. 6. It is assumed to
represent state 2 of the protein. Interactions assumed
to be absent in state 2 and ⁄ or weakened (or abolished)
by the replacement of an oxygen atom with a sulfur
Fig. 5. 31p NMR spectra of wild-type Ras and Ras mutants bound
to Mg
2+
•GTPcS in complex with Raf-RBD. Initially the samples con-
tained 1.0 m
M Ras•Mg
2+
•GTPcS (lower), 1.2 mM Ras(T35S)•
Mg
2+
•GTPcS (middle) or 1.0 mM Ras(T35A)•Mg
2+
•GTPcS (upper) in
40 m
M Hepes ⁄ NaOH pH 7.4, 10 mM MgCl
2

, 150 mM NaCl, 2 mM
1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfo-
nate in 5% D
2
O, 95% H
2
O, respectively. A solution of 9.8 mM
Raf-RBD dissolved in the same buffer was added in increasing
amounts. The molar ratios of Raf-RBD ⁄ Ras are 1.5 for Ras(wt) and
2 in the mutant samples. Data were recorded at 278 K.
31
P reso-
nances assigned to Ras–nucleotide complex in conformation of
state 1a or state 1b are coloured red, the resonances assigned to
state 2 are coloured green.
Table 4. Affinities of Raf-RBD to Ras complexed with different GTP analogues. The association constant K
A
between Raf-RBD and Ras com-
plexed with different GTP analogues was determined using ITC. Measurements were performed at 298 K in 40 m
M Hepes ⁄ NaOH pH 7.4,
10 m
M MgCl
2
, 150 mM NaCl, 2 mM 1,4-dithioerythritol. Data were analysed using ORIGIN FOR ITC 2.9 assuming a 1 : 1 complex formation [28]
and DG° ¼ G
complex
) G
free
¼ -RTlnK
A

.
Raf-RBD complexed
with
K
A
(lM
)1
)
DG°
(kJÆmol
)1
)
DH°
(kJÆmol
)1
)
TDS°
(kJÆmol
)1
)
Ras(wt)•Mg
2+
•GppNHp 2.50 ± 0.4 )36.5 ± 0.6 )13.4 ± 1.5 23 ± 2.1
Ras(wt)•Mg
2+
•GppCH
2
p 2.50 ± 0.4 )36.5 ± 0.6 )18.4 ± 2.0 18 ± 2.6
Ras(wt)•Mg
2+

•GTPcS 2.44 ± 0.6 )36.4 ± 0.9 )7.5 ± 1.5 29 ± 2.4
Ras(T35S)•Mg
2+
•GppNHp 0.12 ± 0.04 )29.0 ± 0.06 )9.7 ± 1.0 19 ± 1.1
Ras(T35S)•Mg
2+
•GppCH
2
p 0.09 ± 0.04 )28.2 ± 0.06 )15.3 ± 1.5 13 ± 1.6
Ras(T35S)•Mg
2+
•GTPcS 0.18 ± 0.04 )30.0 ± 0.06 )13.6 ± 1.5 16 ± 1.6
Conformational dynamics of Ras bound to GTPcS M. Spoerner et al.
1426 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS
atom in the c-phosphate group are represented by bro-
ken lines.
Influence of the nucleotide bound on the Ras
conformational states
31
P NMR spectroscopy allows us to probe the con-
formational states of nucleotide-binding proteins, such
as Ras-related proteins, which lead to structural rear-
rangement in the active centre. In principle, whenever
chemical shift changes are visible they indicate that
there is a change of the environment of the phospho-
rus nuclei, although small changes in structure can
lead to large differences in chemical shifts and vice
versa. The main mechanisms leading to changes in
chemical shifts are conformational strain and electric
field effects polarizing the oxygen atoms of the

phosphate groups. In addition to these direct effects,
long-range effects may occur, caused by a structure-
dependent change in the anisotropy of the magnetic
susceptibility, with ring current effects making the
most dominant contribution.
Binding of the different GTP analogues to Ras leads
to large changes in chemical shift, namely a strong
upfield shift in the a-phosphate resonance and a strong
downfield shift in the b-phosphate resonance compared
with data from free Mg
2+
–nucleotide complexes
(Table 2). In complexes with GTPcS, a relatively small
upfield shift of 0.63 p.p.m. is observed for the a-phos-
phate resonance and a strong downfield shift of
3.84 p.p.m. is observed for b-phosphate resonance.
c-Phosphorus resonances do not show the typical shift
changes common to all analogues. Thus, qualitatively
the phosphorus of the a-phosphate group in the mag-
nesium complexes of GTP and its analogues is less
shielded when bound to the protein, whereas the
strong downfield shift in the resonance most probably
results from strong polarization of the phosphorus–
oxygen bonds in the b-phosphate group. Such bond
polarization in Ras•Mg
2+
•GppNHp has been dis-
cussed by Allin et al. [17], as an explanation of strong
infrared shifts seen in the P–O vibrational bands after
complexation. It should be mentioned that the degree

of shift differences in the chemical shift values cannot
be related in a simple way to the degree of conforma-
tional change causing this change.
Whereas wild-type Ras complexes with the GTP
analogues GppNHp or GppCH
2
p exist in a conform-
ational equilibrium between two main conformational
states 1 and 2, with a K
12
value of $ 2, the complex
with the analogue GTPcS obviously exists in predom-
inantly only one conformation. It shows the spectral
characteristics of state 2 as the effector binding state.
(a) The interaction with Ras-binding domains leads
AB
C
Fig. 6. Schematic representation of the coordination sphere of the phosphate groups and the thiophosphate of GTPcS in wild-type and
mutant Ras nucleotide complexes. G, guanosine. (A) Coordination that predominantly exists in wild-type protein containing Thr35. (B,C)
Other possible complexes with Ras(T35S) or Ras(T35A). Note, that not all contacts between the nucleotide and the protein are included.
Bonds that probably exist only in state 1 or are weakened or abolished in the thiophosphate group are represented by broken lines. The sul-
fur atom was assumed to be negatively charged as shown previously for free ATPcS [32]. However, in the protein bound nucleotide the
charge distribution is probably also influenced by the protein environment and could be thus different in different conformations.
M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS
FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1427
only to small chemical shift changes. (b) Weakening
or destruction of the naturally occurring hydrogen-
bond interaction of the side-chain hydroxyl group of
Thr35 with the metal ion, and of the main-chain
amide with the c-phosphate by mutations to serine or

alanine leads to large changes in chemical shift. (c)
These chemical shift changes can usually be reversed
in Ras(T35S) by Raf-binding because serine still con-
tains a side-chain hydroxyl, however this is not the
case in Ras(T35A). Geyer et al. [1] suggested that in
the GTP-bound form, Ras(wt) also exists predomin-
antly in one conformation. In terms of the conforma-
tional equilibria of Ras, GTPcS seems to be the
analogue which is more similar to physiological GTP
than both other commonly used analogues GppNHp
or GppCH
2
p.
Structural states of Ras(T35S) and Ras(T35A)
Mutation of Thr35 to serine or alanine leads to two
new phosphorus lines of the c-thiophosphate group
and the b-phosphate group, which both show charac-
teristics of state 1. The two states are in a dynamic
equilibrium as evident from their temperature depend-
ence. They are therefore assumed to represent sub-
states of state 1 and are called states 1a and 1b. The
alanine mutation makes coordination of the side chain
with the divalent ion typical for state 2 impossible and
can therefore only exist in state 1. In the serine
mutant, metal ion coordination is perturbed but still
possible. It shows, in addition to lines assigned to sub-
states 1a and 1b, a very weak line at the position of
the c-phosphate resonance in wild-type Ras, suggesting
that Ras(T35S) shows in equilibrium a sparse popula-
tion of state 2. As in the case of the complexes of

Ras(T35A) or Ras(T35S) with the two analogues
GppNHp and GppCH
2
p, the resonance of the a-phos-
phate is shifted downfield relatively to state 2, whereas
the b-phosphate resonance is shifted upfield and is split
into two. The c-phosphate resonance is also split into
two well-separated lines, but one is shifted downfield
and one upfield from the resonance positions obtained
with the wild-type protein.
As observed earlier for GppNHp and GppCH
2
p
complexes of Ras, and now for GTPcS, not only is the
hydroxyl group of Thr35 that interacts in the X-ray
structures with the metal ion important for stabiliza-
tion of state 2, but so too is its methyl group. This is
evident because in Ras(T35S) an hydroxyl group
remains available but state 2 is destabilized. Stabiliza-
tion of state 2 by the side-chain methyl group of
Thr35 does not seem to be due to a simple hydropho-
bic interaction, but rather to sterical restraints, because
it is located in a cavity formed by the side chains of
Ile36 and the charged ⁄ polar side chains of Asp38,
Asp57 and Thr58.
In GTPcS bound to Ras three different stereoiso-
mers of the thiophosphate group are possible (Fig. 6).
In principle, they can occur in state 1 and state 2 of
the protein, but the corresponding populations may
differ greatly. However, they are not equivalent ener-

getically because sulfur is coordinated more weakly to
magnesium ions than oxygen and is a weaker acceptor
of hydrogen bonds than oxygen. As a consequence,
GTPcS binds more weakly to Ras than does GTP
itself [18]. In state 2, the amide group of Thr35 is
probably involved in a hydrogen bond with one of the
nonbridging c-phosphate oxygen atoms and the diva-
lent ion with the other oxygen; the third oxygen is
probably involved in a hydrogen bond with the amide
of Gly60 and the interaction with the positively
charged side chain of Lys16. Energetically, a sterical
position such as that shown in Fig. 6A is strongly
favoured, in agreement with the experimental observa-
tion of a single phosphorus resonance for the c-phos-
phate (Fig. 6A). In the mutant proteins, state 1 is
strongly preferred because the side-chain interaction of
Thr35 with the Mg
2+
ion is perturbed (T35S) or
impossible (T35A). It has been suggested previously [2]
that weakening of metal ion coordination most prob-
ably leads to a concerted breaking of the hydrogen
bond between the amide group of amino acid 35 and
the c-phosphate group.
Indeed, M-Ras [11] and H-Ras(G60A) [12] in the
GppNHp form show
31
P NMR spectra typical of
state 1 and recently published X-ray structures show
that the amide group of Thr35 is distant from the

c-phosphate group. Ford et al. [12] proposed a third
conformational state for human wild-type H-Ras
because their spectrum contained three
31
P resonances
corresponding to the a- and c-phosphate (note that a
new resonance assignment published by Spoerner et al .
[2] was not known to Ford et al. [12]). However,
because the third state could not be observed in our
experiments, and the chemical shifts are very close to
those observed for H-Ras•Mg
2+
•GDP, they should
most probably be assigned to the a- and b-resonances
of Ras-bound GDP.
When a hydrogen bond exists between the amide
group of amino acid 35 and the c-phosphate in the
mutant proteins, in the GTPcS-complex the free
energy differences DG° and thus the equilibrium popu-
lations of the three stereoisomers are changed (Fig. 6).
In the stereoisomer that most probably dominates in
wild-type Ras (Fig. 6A), coordination of the c-phos-
phate group with the metal ion and the interaction
Conformational dynamics of Ras bound to GTPcS M. Spoerner et al.
1428 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS
with Gly60 and Lys16 is still possible, in the two other
cases either the metal ion coordination is weakened
when the sulfur is oriented towards the metal ion
(Fig. 6B) or the hydrogen-bond interaction with Gly60
is weakened (Fig. 6C). It seems plausible that a wild-

type-like arrangement is the energetically most
favoured; meaning that state 1a should be assigned to
this stereoisomer. For the resonances assigned to
state 1b it is more difficult to derive a structural hypo-
thesis. However, the chemical shifts of the c-phosphate
resonance in state 1b are close to those observed in
metal-free GTPcS (Tables 1 and 2) suggesting that it
represents the arrangement seen in Fig. 6B, with
coordination with the metal ion abolished.
Dynamics and energetics of the conformational
transitions
The DG
|
values for the transition between state 1 and
2 in complexes of wild-type protein with GppNHp and
GppCH
2
p are 42 and 41 kJÆmol
)1
, respectively [1,2].
For the complex between wild-type protein and
GTPcS the activation energy cannot be determined by
NMR because only state 2 is visible. However, it is
reasonable to assume that the activation energy is sim-
ilar. For the transition between states 1a and 1b we
determined a DG
|
value of 41 kJÆmol
)1
. Thus the acti-

vation energies are identical within the limits of error.
This may be due to chance or may suggest that a sim-
ilar transition state is involved in the transition
between states 1 and 2 and between states 1a and 1b.
Dynamic equilibria in Ras complexed with GTP and
different GTP analogues have been described previ-
ously [4,19,20] in
15
N-enriched Ras. It was shown that
a number of amide resonances are not visible in 2D
heteronuclear NMR spectra, most probably because of
exchange broadening. In the complex between
Ras(1–171) and GppNHp the amide resonances of 22
nonproline residues are not visible, whereas in the
complex with GTPcS or GTP $ 20 additional reso-
nances can be detected. Some of these resonances are
broader in the GTPcS complex than in the GTP com-
plex [4].
It is clear that any protein exists in multiple con-
formational states (now often called excited states)
with different populations. Ito et al. [4] called this phe-
nomenon regional polysterism. Different probes are
also differentially sensitive to different conformational
states. The two main conformational states 1 and 2
coexist with almost equal populations in the GppNHp
complex, and exchange between theses two states,
which probably involves structural changes in loop L1,
switch 1 and switch 2, can qualitatively explain the
excessive line broadening of the resonances of residues
located in these regions. Additional local conforma-

tional changes may strengthen this effect. GTP and
GTPcS exist predominantly in state 2 meaning that
the line broadening associated with the transition will
be smaller. The equilibrium between different stereo-
isomers around the thiophosphate group may contrib-
ute to the increased line width seen in some resonances
in the GTPcS complex.
Affinity of Ras-binding domains of Raf-kinase to
Ras complexed with different GTP analogues
ITC measurements show that under our experimental
conditions the affinities between Ras(wt) and the
tightly binding Raf-RBD are not influenced much by
the type of bound GTP analogue. The association con-
stant is identical within the limits of error for all three
GTP analogues (Table 4). At first this seems surprising
because NMR spectroscopy shows that the bound ana-
logue clearly influences the equilibrium between the
two conformational states. However, NMR spectrosco-
py indicates that, after binding of the RBD, Ras most
probably exists in its correct structural state. Because
the data analysis used assume a two-state model and
the free enthalpy difference between state 2 in the free
and complexed form can be assumed to be very small,
only the conformational equilibrium with state 1 could
influence the total enthalpy change measured by ITC.
However, in the GppNHp or GppCH
2
p complexes of
the wild-type protein, the difference in DG
12

between
the two states is $ 2kJÆmol
)1
(Table 3) and thus much
smaller than DG° involved in effector binding, which is
of the order of 36 kJ Æmol
)1
(Table 4). In addition, only
the relatively small fraction of the protein occurring in
state 1 in GTPcS would contribute to a nucleotide-spe-
cific variation in DG° and thus would be scaled down
proportionally to K
12
)1
.
In the T35S-mutant the population of free Ras is
shifted to state 1, but the binding of Raf-RBD restores
the correct conformation (similar to state 2). The wild-
type and mutant proteins differ mainly in the methyl
group of threonine which is missing in Ras(T35S).
From the NMR point of view, Ras(wt) and the serine
mutant seem to exist in the same conformation when
bound to effectors. This is not true for the complexes
of Raf-RBD with Ras(T35A) where the interaction
with the RBD cannot restore the correct conformation
[9]. For the T35S-mutant the dissociation constant
increases by about one order of magnitude with the
largest increase seen for GppCH
2
p. DG° increases by

7.5, 8.3, and 6.5 kJÆmol
)1
for GppNHp, GppCH
2
p and
GTPcS, respectively. The small differences may reflect
M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS
FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1429
the energy difference between states 1 and 2 in this
mutant, which cannot be derived from our NMR data.
Relatively small, but in some cases significant, differ-
ences in DH° and TDS° values are seen in the three
nucleotide complexes. Qualitatively, the differences
may be rationalized with the help of the NMR results
as follows. In a dynamic equilibrium Ras in complex
with GppNHp or GppCH
2
p has a mobile effector loop
which is fixed upon RBD binding. Therefore, the
change in the configurational entropy (as part of the
total entropy) is smaller than in the GTPcS complex,
where the effector loop is suggested to be oriented in
the correct position already.
The nucleotide analogue bound to Ras influences
the equilibrium between states 1 and 2. Replacing the
oxygen bridging the b- and c-phosphate group in
GTPcS with an imido or methylene group shifts the
population of state 2 to state 1. Although it was
shown that P–O–P bonds have very open bond angels
[21], which should lead to delocalization of the electron

density into the neighbouring atoms, the bridging oxy-
gen may still be a weak hydrogen bond acceptor. That
is an interaction involving a hydrogen bond donator
and ⁄ or a group with positive partial charge could be a
reason for stabilization of state 2 in the GTPcS com-
plex that is abolished by replacement of the bridging
oxygen. Taking a closer look at the crystal structure of
Ras in the GTP-bound state [22], only the main-chain
NH of Gly13 and ⁄ or the amino group of Lys16 and
the bridging P
b
–O–P
c
oxygen seem to be able to con-
tact each other by forming a hydrogen bond. The
interacting groups are also close enough when
GppNHp is bound, as derived from X-ray structure
[23], although in this case a strong hydrogen bond is
not to be expected.
Conclusions
Ras bound to triphosphate nucleosides exists in (at
least) two conformational states which can be identi-
fied using
31
P NMR spectroscopy. One of these states
(state 2) represents the high-affinity binding state for
effectors; the second state (state 1) represents a differ-
ent state of the protein with much reduced affinity to
effectors. The equilibrium between the states can be
shifted by using different GTP analogues or by specific

mutations of Ras. A hydrogen bond of the amide
group of Gly13 and ⁄ or the amino group of Lys16 with
the b–c-phosphorus-bridging oxygen may be one fac-
tor responsible for stabilization of state 2 in the GTP
complex. Thus, Ras(wt)•Mg
2+
•GTPcS exists predom-
inantly in state 2. Other factors stabilizing state 2 are
clearly the interactions of the amide and side-chain
hydroxyl groups of Thr35 with the c-phosphate group
and metal ion, respectively. Ras variants existing in
state 1 show two substates states 1a and 1b. The trans-
ition velocity between these two states and thus the
energy of the transition state is similar to that found
for transition between states 1 and 2 of Ras bound to
the analogues GppCH
2
p or GppHNp. The activation
barrier may reflect a transient breakage of the bond
between the metal ion and the c-phosphate.
Experimental procedures
Protein purification
Wild-type and Thr35 mutants of human H-Ras(1–189) were
expressed in Escherichia coli strain CK600K with ptac vector
plasmids and purified as described previously [18]. Nucleo-
tide exchange to GppNHp, GppCH
2
p or GTPcS was done
using alkaline phosphatase treatment in the presence of a
twofold excess of the GTP analogue as described at John

et al. [24]. Free nucleotides and phosphates were removed
by gel filtration. The final purity of the protein was > 95%
as judged from the SDS ⁄ PAGE. The Ras-binding domain
of human cRaf-1 (Raf-RBD, amino acids 51–131) was
expressed in E. coli and purified as described previously [25].
Sample preparation
Typically 1 mm Ras•Mg
2+
•GTPcS was dissolved in 40 mm
Hepes ⁄ NaOH pH 7.4, 10 mm MgCl
2
, 150 mm NaCl, 2 mm
1,4-dithioerythritol and 0.1 mm 2,2-dimethyl-2-silapentane-
5-sulfonate in 5% D
2
O, 95% H
2
O. For binding studies a
solution of 5 or 7 mm Raf-RBD contained in the same buf-
fer was added in appropriate amounts to the samples.
NMR spectroscopy
31
P NMR spectra were recorded with an Avance-500 NMR
spectrometer (Bruker Biospin, Karlsruhe, Germany) oper-
ating at a
31
P frequency of 202 MHz. Measurements were
performed in a 10 mm probe using 8 mm Shigemi (Tokyo,
Japan) sample tubes at various temperatures. Seventy-degree
pulses were used together with a total repetition time of 7 s.

Protons were decoupled during data acquisition by a GARP
sequence [26] with strength of the B
1
-field of 980 Hz. For
referencing a X-value of 0.4048073561 reported by Maurer
and Kalbitzer [27] was used, which corresponds to 85%
external phosphoric acid contained in a spherical bulb. The
assignment of the phosphate resonances was established
by a
31
P–
31
P NOESY experiment on 1.2 mm solution of
Ras(wt)•Mg
2+
•GTPcS at 283 K with a mixing time of 1.5 s
and a total repetition time of 14 s. Saturation experiments
were performed at 278 K using a B
1
-field of 18 Hz for a per-
iod of 1 s for presaturation.
Conformational dynamics of Ras bound to GTPcS M. Spoerner et al.
1430 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS
Sample temperature was checked by using the line separ-
ation (methylene-hydroxyl) of external ethylene glycol [28].
Thus, the absolute accuracy of the temperatures given in
here is better than ± 0.5 K.
Calculation of the exchange rates and the
thermodynamic parameters
Exchange rates were calculated by a line shape analysis

using full-density matrix formalism [29] based on a pro-
gram described by Geyer et al. [1] and modified later. Vis-
cosity-dependent changes of the rotational correlation time
s
rot
were corrected as described by Spoerner et al. [2].
Care was taken that the spin systems were largely in ther-
modynamic equilibrium before each scan in the experiments
by using a repetition time of 7 s together with 70 ° pulses.
Before fitting the data the noise level was decreased by mul-
tiplying the FID with an exponential filter leading to an
additional line broadening of 5 Hz. The difference in the
free activation energy DG
|
, the activation enthalpy DH
|
, and
the activation entropy DS
|
, were obtained by fitting the
temperature dependence of the exchange rates s
ex
to the
Eyring equation with
1=s
ex
¼ k
1
þ k
À1

ð1Þ
For the fit of the data the two-bond phosphorus–phosphorus
coupling constants were taken from proton decoupled spec-
tra of GTPcS measured at 278 K in the same buffer used for
the experiments with Ras. For free GTPcS the absolute
value of
2
J
ab
and
2
J
bc
are 19.7 Hz and 29.1 Hz, respectively.
pH dependence of chemical shifts
The pH dependence of chemical shifts was measured in
samples containing 2.5 mL solution containing 1 mm nuc-
leotide, 0.1 mm 2,2-dimethyl-2-silapentane-5-sulfonate in
5% D
2
O, 95% H
2
O. To estimate the optimal magnesium
concentration it was varied in the range from 0 to 40 mm.
Whereas at pH 2 no plateau in the chemical shifts could be
obtained within this range of MgCl
2
concentration, at
3mm MgCl
2

a plateau in the magnesium induced chemical
shifts was observed for the GTPcS at pH 7 and pH 9.
Therefore this concentration was used for the study of the
nucleotide–metal complexes. The pH of the solutions was
adjusted by adding HCl or NaOH and was determined with
a calibrated glass electrode.
The dependence of the chemical shift d on the pH is
often described by a modified Henderson–Hasselbalch
equation using microscopic equilibrium constants K
ji
and
chemical shift contributions d
ji
for the description of the
equilibrium between the protonated and the deprotonated
form of a functional group. Even in small molecules such
as GTP the description can become very complex because
different forms with the same total charge exist in the
equilibrium. For a molecule with N sites 2
N
different states
and thus 2
N ) 1
independent equilibrium constants and
chemical shifts are required for a full description. The prob-
lem can be simplified by defining macroscopic equilibrium
constants K
i
and chemical shift contributions d
i

to N +1
dissociation steps. As long as fast exchange conditions pre-
vail the d
i
can be calculated from the microscopic d
ij
and
the concentrations p
ij
of the M components of a dissoci-
ation step i by
d
i
¼
1
P
M
j¼1
p
ji
X
M
j¼i
p
ji
d
ji
ð2Þ
and is independent of the ligand L (H
+

) concentration,
because the relative concentrations in the different states
(ji) with the same number of bound ligands are constant
(defined by the respective microscopic equilibrium con-
stants). The concentration of the molecule p
i
in a given
macroscopic dissociation state i is then
p
i
¼
X
M
j¼i
p
ji
ð3Þ
With the macroscopic equilibrium constants K
i
K
i
¼
p
i
L
p
iÀ1
ð4Þ
one obtains
p

i
p
0
¼ P
i
j¼1
K
j
L
ð5Þ
The average chemical shift is then
d ¼
d
0
þ
P
N
i¼1
d
i
P
i
j¼1
K
j
L
1 þ
P
N
i¼1

P
i
j¼1
K
j
L
ð6Þ
With the definition of the pH the chemical shift is then
given as a function of pH by
d ¼
d
0
þ
P
N
i¼1
d
i
10
ipHÀ
P
i
j¼1
pK
j
1 þ
P
N
i¼1
10

ipHÀ
P
i
j¼1
pK
j
ð7Þ
A more detailed derivation of Eqn (7) is found in the
PhD thesis by Freund [30].
Isothermal Titration Calorimetry (ITC)
Measurements were performed with a MicroCal MCS ITC
apparatus at 298 K. Ras(wt) and Ras(T35S) bound to
Mg
2+
•GppNHp, Mg
2+
•GppCH
2
porMg
2+
•GTPcS and
M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS
FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1431
also Raf-RBD were dissolved in identical buffer (40 mm
Hepes ⁄ NaOH pH 7.4, 10 mm MgCl
2
, 150 mm NaCl, 2 mm
1,4-dithioerythritol). A 60 or 80 lm solution of Ras protein
in the cell was titrated with 0.6, 1.0 or 1.5 mm solution of
Raf-RBD. Data were analysed using origin for itc 2.9

(MicroCal Software Incorporation, Northampton, MA,
USA) assuming a 1 : 1 complex formation [31].
Acknowledgements
This work was supported by the Volkswagenstiftung
and the DFG.
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