AbstractThe intrinsic fluorescence properties of the oncogene protein p21N-ras, p21H-ras and one of its transforming mutants, p21N-ras (Va1112), have been investigated. A mutant containing a single tryptophan at position 28 in p21H-ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect

Drake, Alex

Hancock, John F.

Kuroda, Reiko

Neidle, Stephen

Skelly, Jane V.

Suter, David A.

Elsevier - Publisher Connector 

Volume 262, number 1, 127-130 FEBS 08229 March 1990 
Conformational effects of nucleotide exchange in ras p21 proteins as 
studied by fluorescence spectroscopy 
Jane V. Skelly, David A. Suter, Reiko Kuroda, Stephen Neidle, John F. Hancock+ and Alex Drake” 
CRC Biomolecular Structure Unit, Institute of Cancer Research, Sutton, Surrey Sh42 5NG, +Aca&mic Department of Haematology, 
Royal Free Hospital, London NW3 2QG and “Department of Chemistry, Birkbeck College, 20, Gordon Street, London WClH OQB, 
England 
Received 15 December 1989 
The intrinsic fluorescence properties of the oncogene protein p21Nmm, ~21”“” and one of its transforming mutants, ~21~~~ (Va112), have been investi- 
gated. A mutant containing a single tryptophan at position 28 in ~21”~” (Trp28) has been specifically engineered to provide a probe of protein 
conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease 
in the intensity of the fluorescence mission spectrum due to tyrosine occurred on GDP/GTP nucleotide xchange in the native and mutant proteins. 
Selective xcitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the 
wavelength of maximum emission on nucleotide xchange. A reduction in the residual Mg2+ ion concentration enhanced this effect. 
p21 ras protein; Guanine nucleotide binding; Tryptophan fluorescence 
1. INTRODUCTION 
The p21 products of the 3 functional ras genes, c.Ha, 
c.Ki and N-ras bind GTP/GTP, possess intrinsic 
GTPase activity [l-5] and display some sequence 
homology with the guanine nucleotide regulatory pro- 
teins (G proteins) [6-91. This has led to speculation that 
the ~21s act as signal transducers modulated by GTP 
hydrolysis and GDP/GTP exchange [lo]. Point muta- 
tions at codons 12, 13, 59, 61 or 63 in ras genes have 
been shown to result in the production of oncoproteins 
capable of transforming established cell lines [l l-131. 
Many of these mutated proteins have altered GTPase 
activities and the amino acids involved occur in a highly 
conserved nucleotide binding domain [4,5,14-171. The 
p21*=* GTP complex represents the active conforma- 
tion and the GDP bound form, the inactive one. When 
stimulated by a postulated detector molecule in the 
membrane, the protein then becomes activated, 
possibly through a conformational change required to 
accommodate the phosphate. The altered conforma- 
tion may be translated into an intracellular signal by in- 
teraction with one or more postulated effector 
molecules (target proteins). Recently, a cytoplasmic 
protein GAP (GTPase activating protein) has been im- 
plicated as an effector protein [19]. It is believed that 
this protein interacts with ras proteins in the proposed 
effector binding region of the molecule, residues 
Correspondence address: J.V. Skelly, CRC Biomolecular Structure 
Unit, Institute of Cancer Research, Sutton, Surrey SM2 5NG, 
England 
Published by Elsevier Science Publishers B. V. (Biomedical Division) 
32-40, and stimulates the GTPase activity of normal 
p2 1 but not its transforming mutants [ 19,201. However, 
mutations in the effector domain have no effect on the 
intrinsic nucleotide exchange or GTPase activities of 
p21 [20]. The biological active state of p21 is regulated 
by the rate of hydrolysis of GTP. Transforming muta- 
tions occurring in the guanine dinucleotide beta- 
phosphate binding loop alter the protein conformation 
resulting in a stabilised active form and a reduction in 
the rate of GTP hydrolysis. It is speculated that GAP 
may serve to orientate this phosphate binding loop in 
the proposed ‘catalytic’ domain into a more accessible 
conformation for the GTPase enzyme [ 171. The theory 
implies that the conformational change would be 
significant and therefore, would be likely to be 
translated throughout the molecule. This paper reports 
on attempts to ascertain whether structural differences 
between p21 *GDP, and p21 .GTP complexes can be 
detected by fluorescence, a technique that is highly sen- 
sitive to changes in protein secondary structure. 
The intrinsic fluorescence of p21 is dominated by 9 
tyrosine residues in p2 1 N-ras, and 10 in p21 H-ras. A single 
tryptophan residue can act as a useful intrinsic probe of 
structure in steady-state fluorescence measurements by 
providing information on the local environment of the 
fluorophore. A tryptophan residue was specifically 
engineered to replace phenylalanine at position 28, 
which was predicted to lie close to the dinucleotide 
binding domain. This substitution was favoured as it 
preserves the essential hydrophobic character of the 
side chain and has minimal effect on the biological ac- 
tivity of the protein. Recent X-ray crystallographic 
00145793/90/$3.50 0 1990 Federation of European Biochemical Societies 127 
Volume 262. number 1 FEBSLETTERS March 1990 
evidence has shown that the phenyl side chain lies 
almost perpendicular to the guanine base [17]. 
Circular dichroism spectra were recorded in the far 
UV region of the spectrum (250-200 nm) to detect 
possible structural differences between the mutants and 
the effects of guanine nucleotide exchange. 
2. MATERIALS AND METHODS 
The construction of E. coli strains containing normal and mutant 
N-ras and Ha-ras expression plasmids is described elsewhere [20]. 
Harvested cells were lysed by sonication and the cell debris was 
pelleted by centrifugation. Nucleic acids were precipitated by addi- 
tion of polyethylene imine (1% v/v). The proteins were purified by 
ion exchange on a DEAE Sephacel column equilibrated with buffer 
A (10 mM Tris-HCI, pH 7.5, 50 mM NaCl, 10 mM MgClr, 5 mM 
dithiothreitol). The protein was eluted with a salt gradient (50 to 
500 mM NaCl) and fractions assayed for p21 by an [‘HIGTP binding 
assay [2]. p21-containing fractions were pooled and precipitated with 
75% saturated ammonium sulphate. The precipitate was resuspended 
in buffer B (10 mM Tris-HCl, pH 7.5, 100 mM NaCI, 10 mM MgClz, 
5 mM dithiothreitol) and applied to a G-75 Sephadex column. 
p21-containing fractions were pooled and stored as an ammonium 
sulphate precipitate at 4°C. Prior to usage the protein was dialysed 
against several changes of buffer A. 
GTP binding assays were carried out at 37°C. [‘H]GTP (2 ,uM) was 
incubated with p21 .GDP (0.5 PM) in a total reaction volume of 40 ~1 
containing 10 mM Tris-HCl, pH 7.5, containing 50 mM NaCI, 
10 mM MgClr. Samples were removed at 20-min intervals and quen- 
ched with cold buffer (1 ml). After filtration through Millipore 
HAWP membranes, the [‘HIGTP-bound p21 was determined by 
scintillation counting. 
Circular dichroism spectra were recorded on a Jasco J4OCS spec- 
tropolarimeter. Silica optical cells of path lengths 0.05-0.1 cm were 
used. The chart record was in degrees of optical polarisation (8) x 
MRW (mean residue weight). All spectra were baseline corrected. In 
each case the control spectrum of the guanine nucleotide in buffer 
was subtracted. The spectra were recorded at intervals of 30 and 
60 min. 
Fluorescence measurements were recorded on a Perkin Elmer 
Luminescence Spectrofluorimeter LS-5. The observed excitation and 
emission spectra were corrected for variation in monochromator effi- 
ciency and photomultiplier sensitivity and expressed in arbitrary 
units. The appropriate buffer contributions were subtracted and the 
spectra were corrected for absorbance due to the guanine nucleotide. 
Quinine sulphate in 1 N HzS04 was used as a standard to calibrate 
the instrument. Optical band widths of 2.5 and 4 nm were used for 
excitation and emission, respectively. A 2 x 10 mm cuvette was used. 
All fluorescence measurements were recorded at room temperature 
(25°C). p21 was incubated with up to Cfold excess nucleotide (molar 
ratio) at 37°C prior to the measurements. 
Protein concentration was determined using the Bradford reagent 
1221. 
3. RESULTS AND DISCUSSION 
The circular dichroism spectra of ~21~“~~, p21HmraS, 
P21 N-ras (Val12) and p21HdraS (Trp28) are essentially 
superimposable. The data are consistent with similar 
high alpha-helix and beta-sheet contents in all 4 
proteins. 
Incubation of a two-fold molar excess of GTP with 
normal p21 at 37°C resulted in a significant reduction 
in the negative ellipticity after 30 min. After a total in- 
terval of 60 min the signal had further decreased and a 
128 
shift in the wavelength minimum from 218 to 215 nm 
was recorded. A similar experiment with GDP also 
resulted in a significant decrease in the signal. 
However, in this case, there was no observed shift in 
wavelength (fig. 1). A control experiment in which pro- 
tein was allowed to remain in the cell compartment at 
37°C for 6 h without additional GDP/GTP showed 
that there was some loss of signal over this period. This 
could be attributed to partial loss of protein due to 
denaturation. GTP/GDP exchange rates were deter- 
mined by filter binding as described in section 2. The 
rate of exchange is in agreement with results previously 
reported [23] and was found to be consistent with the 
rate at which the spectral shift is observed. A reduction 
in signal intensity which occurs in all preparations may 
be attributed to partial denaturation of the protein. 
These results support those of Pingoud et al. [24] who 
report small differences in the far UV circular 
dichroism spectra between p21 in the GTP and GDP 
forms but contradict later results suggesting that dif- 
ferences are not detectable in this region of the CD 
spectrum [25]. In a similar experiment using a limited 
amount of p21NmraS with a point mutant at Val12, the 
change observed for the normal ras protein was also 
detected. 
Upon excitation at 278 nm, the intrinsic fluorescence 
emission spectrum of normal p21N-‘aS and p21H-TaS is 
dominated by tyrosines as there are no tryptophans pre- 
sent. The spectra recorded after incubation of p21H-‘“” 
and p21 (Va112) with GTP at 37°C for 30 min shows a 
decrease in intensity (fig.2) whilst the wavelength of 
maximum emission at 306 nm (Acm) is unchanged. This 
is characteristic of tyrosine fluorescence. A 10% 
decrease in intensity was obtained in a control experi- 
ment which was incubated at 37°C in buffer A only. 
Adjusting the pH of p21 to 2.0, at which the protein is 
inactive, produces an increase in fluorescence intensity. 
Decreased tyrosine fluorescence in the native state fre- 
quently originates from H-bonding of the tyrosyl 
groups. In p21H-TaS (Trp28) the single tryptophan was 
selectively excited at 295 nm. An emission spectrum 
with a maximum at 342 nm was observed (fig.3). Prein- 
cubation with GTP for 30 min resulted in a reduction 
of approximately 20% in fluorescence intensity and a 
shift in the emission maximum to 338 nm. A control 
experiment in which protein was incubated at 37°C 
showed an approximate 10% increase in fluorescence 
intensity but no shift in wavelength maximum. The 
presence of a 4-fold molar excess of the non- 
hydrolysable analogue of GTP, guanosine- ’ -O- 
(3-thiotriphospate) (GTPyS) with p21H-IaS (Trp28) 
causes a similar effect on fluorescence intensity relative 
to GDP. It is thus evident that the substitution at 
phenylalanine 28 does not impair GTP/GDP exchange. 
A time course study of these changes correlates with the 
rate of exchange of GTP with ~21. GDP and the rate 
of association of the non-hydrolysable analogue 
Volume 262, number 1 FEBS LETTERS March 1990 
2io 200 250 
Wavelength (nm) 
Fig.1. Circular dichroism spectra of p21H-‘@. (a) ~21~~“~ (20 PM) in 
buffer A. (b) ~21~~~ (20pM) incubated in buffer A + GDP 
(1OO~M) for 30 min. (c) p21nns (20rM) incubated in buffer A + 
GTP (100 PM) for 30 min. (d) ~21~~“~ (20 PM) incubated in buffer 
A + GTP (1OOrM) for 60 min. 
GTPyS, and therefore, for p21H-“’ (Trp28), 
fluorescence can be used to assess quantitative binding 
of guanine nucleotides and their analogues (data not 
shown). In the presence of residual Mg2+ and GTP, 
p21 *GDP exists as a stable complex with a half-life of 
approximately 20 min [23]. It is reported that the af- 
finity of p21 for GTP and GDP are approximately the 
same at residual concentrations of 5 mM Mg2+ whereas 
at 0.5 pM Mg2+ p21 has a IO-fold greater affinity for 
GTP [21,23]. To bias the binding in favour of GTP and 
thereby reduce the incubation periods, the nucleotide 
exchange experiments were carried out in the presence 
of 10 mM EDTA. It was calculated that this would 
reduce the Mg2+ to below 0.5 ,uM. The presence of 
C’ 
y \ 
280 310 360 do 440 480 5 
Wavelength 
Fig.2. Emission spectra of p21H-“’ excited at 278 mu (a) p21H+‘6 
(0.5 PM) in buffer A. (b) ~21~‘” (0.5/rM) incubated’at 37°C for 
30 min in buffer A. (c) ~21~~“’ (0.5 rM) + GTP 2 M incubated at 
37°C in buffer A. 
Wavelength bm) 
Fig.3. Emission spectra of ~21~‘“~ excited at 295 nm. (a) 
p21H‘=*(Trp28) (0.5 +M) in buffer A. (b) p21H”as(Trp18) (0.5 FM) 
incubated for 30 min in buffer A + 2 FM GDP. (c) p21H‘ms(Trp28) 
(0.5 /IM) incubated for 30 min in buffer A + 2pM GTP. (d) 
p21Hm”‘(Trp28) (0.5 FM) in buffer A + 10 mM EDTA. 
EDTA alone was shown to have no effect on the 
fluorescence spectrum of p21NmraS and p21H-*“. 
However, excitation of p21HWra” (Trp28) at 295 nm in 
10 mM EDTA resulted in a significant increase in 
fluorescence due to the single tryptophan residue. This 
is presumably caused by displacement of a dinucleotide 
from the guanine nucleotide binding site due to chela- 
tion of Mg2+ which is essential for effective nucleotide 
binding, thereby permitting Trp28 to become more ac- 
cessible to solvent. The fluorescence was instantly 
quenched by the subsequent addition of GDP and/or 
GTP. The slight differences between the fluorescence 
spectra of p21 H-‘aS(Trp28) - GDP and p21H-‘aS(Trp28). 
GTP may reflect conformational differences between 
them. Similar experiments to confirm these results are 
being carried out using preparations of nucleotide free 
protein. 
0 
These preliminary results suggest hat the GDP and 
GTP ~21s are conformation~ly distinct in both the 
normal and transforming mutant Va112. The distinct 
forms can be detected both by circular dichroism and 
intrinsic fluorescence spectroscopy. The genetically- 
engineered mutation at Phe28 will provide an impor- 
tant probe in further studies of structural and dynamic 
aspects of the mechanism of nucleotide exchange and 
hydrolysis which are currently in progress. The fact 
that substitution of this phenyl~~ine residue by tryp- 
tophan does not impair GDP/GTP binding suggests 
that it is not directly involved in nucleotide binding. In 
conclusion, the results of this study are consistent with 
the current hypothesis for the mechanism of action of 
p21 which suggests that the protein can adopt two con- 
formations. The implication of this awaits a full com- 
parison of the X-ray structures of the GDP- and 
GTP-bound forms. 
129 
Volume 262, number 1 FEBS LETTERS March 1990 
Acknowledgements: We are grateful to Mike Tilby, Department of 
Medicine, Institute of Cancer Research for providing facilities for 
fluorescence spectroscopy and Alan Hall, Chester Beatty Laboratory 
for providing the E. coli strains. We also gratefully acknowledge 
Christ Marshall for many helpful discussions. This work was sup- 
ported by the Cancer Research Campaign. 
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Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy 

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Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy

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