Identification of a Putative Sordarin Binding Site in
Candida albicans Elongation Factor 2 by Photoaffinity
Labeling*
Juan Manuel
Domínguez
and
J. Julio
Martín
From the Research Department, GlaxoSmithKline S. A. PTM,
C/Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
Received for publication, May 9, 2001, and in revised form, June 4, 2001
 |
ABSTRACT |
Candida albicans EF-2 binds sordarin
to a single class of binding sites with Kd = 1.26 µM. Equimolar mixtures of EF-2 and ribosomes, in the
presence of a non-hydrolyzable GTP analog, reveal two classes of high
affinity sordarin binding sites with Kd = 0.7 and
41.5 nM, probably due to the existence of two ribosome
populations. Photoaffinity labeling of C. albicans EF-2 in
the absence of ribosomes has been performed with
[14C]GM258383, a photoactivatable sordarin derivative.
Labeling is saturable and can be considered specific, because it can be
prevented with another sordarin analog. The fragment
Gln224-Lys232 has been identified as
the modified peptide within the EF-2 sequence, Lys228 being
the residue to which the photoprobe was linked. This fragment is
included within the G"-subdomain of EF-2. These results are discussed
in the light of the high sordarin specificity toward fungal systems.
| |
INTRODUCTION |
The natural product sordarin and its semisynthetic derivatives
constitute a selective class of inhibitors of protein synthesis in
fungi. Their potential as systemic antifungal agents has been evidenced
by their broad spectrum and in vivo therapeutic efficiency (1, 2; see Ref. 3 for a recent review). Among all the processes included in protein synthesis, ribosomal translocation is the step
impaired by sordarin (4). This molecule binds to
EF-21 itself, although such
binding is greatly favored by the presence of ribosomes (5). On the
other hand, mutations leading to sordarin resistance have been found
both in EF-2 and in the yeast ribosomal protein rpP0 (6, 7). Hence, the
EF-2·ribosome complex is proposed as the functional target of
sordarin antifungals.
Translocation has been thoroughly studied in prokaryotes and a good
picture of the global mechanism at molecular level is starting to
emerge. Key aspects in such progress have been the elucidation of
ribosome structure at good resolution (8) and detailed crystallographic
studies of EF-G, the prokaryotic homolog of EF-2 (9, 10). Also,
visualization of the ribosome·EF-G complex by cryo-electron
microscopy (11), as well as other functional studies (12, 13), have led
to a better understanding of the process (see Ref. 14 for a review).
EF-G, the protein that promotes ribosomal translocation in prokaryotes,
is a tadpole-like molecule organized into five domains. The globular
domain I is responsible for GTP binding and hydrolysis. It contains
several structural motifs characteristic of the G-protein superfamily
(indeed, it is referred to as "G-domain") plus an extra insert
called the G'-subdomain. The role of the latter remains unknown,
although it has been suggested to act as a nucleotide-exchange factor
(9). Interaction of the G-domain with the ribosome triggers GTP
hydrolysis, and the energy released is transformed into mechanical movement. Eventually, domain IV (a fibrous domain at the other end of
the molecule) moves away, stretching the EF-G shape and literally pushing the newly formed peptidyl-tRNA from the A to the P
ribosomal site (15). It is still unclear how energy is transformed into
movement, although it seems to be related to rearrangements within both
EF-G and the ribosome that allow the complex to act as a molecular
ratchet (15, 16).
The eukaryotic system has not been so deeply studied. However, it seems
to be more sophisticated, as demonstrated by the larger number of
proteins constituting the eukaryotic ribosome and by the ability to
regulate EF-2 function by specific kinases (17) and ADP-ribosylation
(18). Nevertheless, the general features of the process are assumed to
be the same. In coherence with this, there is a substantial degree of
homology between bacterial EF-G and eukaryotic EF-2. The more relevant
differences are the greater length of EF-2 and the regulatory
mechanisms depicted above. Likewise, it is remarkable that the
G'-subdomain has been replaced in EF-2 by another insert termed the
G"-subdomain, which is 15-30 residues longer, with a position that is
displaced beyond in the sequence, and shows no homology with the
prokaryotic G'-subdomain (19). On the other hand, EF-2 is a highly
conserved protein within the entire eukaryotic kingdom. This fact makes
especially striking the existence of EF-2 inhibitors such as sordarin,
which exclusively impair fungal and not bacterial, mammalian, or plant
protein synthesis machinery with the added capacity to discriminate
between closely related fungal species (6, 20, 21).
Identification of the residues involved in sordarin binding to EF-2
might help to explain such selectivity. For this reason, we have
performed photoaffinity labeling studies of EF-2 from the pathogenic
fungus Candida albicans, using a radiolabeled sordarin derivative.
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EXPERIMENTAL PROCEDURES |
Elongation factor 2 and salt-washed ribosomes were isolated from
C. albicans 2005E as previously described (5). EF-2
concentration was determined spectrophotometrically using
E280 = 67310 M
1·cm1 as deduced from its
amino acid sequence (Swiss-Prot O13430). Ribosome concentration was
also calculated spectrophotometrically assuming one
A260 unit corresponds to 18 pmol of ribosomes
(22). [14C]GM258383 (1.8 GBq/mmol) and
[3H]sordarin (180 GBq/mmol) were prepared by the Isotope
Chemistry Group at GlaxoSmithKline Medicines Research Center
(Stevenage, United Kingdom). Non-radiolabeled sordarin derivatives were
prepared by the Medicinal Chemistry Unit at GlaxoSmithKline S.A. (Tres Cantos, Spain). Sephadex G-25 (PD-10 columns) was from Amersham Pharmacia Biotech (Uppsala, Sweden). All other chemicals were from
Sigma Chemical Co.
Binding Assays--
Binding of [3H]sordarin to
EF-2 alone or to equimolar mixtures of EF-2 and ribosomes was studied
by equilibrium dialysis using microvolumetric dialysis capsules (Cellu
Sep, San Antonio, TX). The two chambers of each capsule were separated
by dialysis membrane with a cutoff of 6 kDa. One chamber was filled
with 150 µl of [3H]sordarin at appropriate
concentration in 25 mM Hepes-KOH, pH 7.4, 85 mM
potassium acetate, 4 mM magnesium acetate, and 1.5 mM DL-dithiothreitol. The other chamber was
filled with the same solution containing either 3.6 µM
EF-2 or 333 nM EF-2, 333 nM ribosomes, and 33.3 µM Gpp(NH)p. Samples were incubated overnight at 30 °C
under rotary shaking. Finally, duplicate 50-µl aliquots were
withdrawn from each chamber, and their radioactivity was measured. Free
sordarin was calculated from radioactivity values of the first capsule
chamber, whereas bound sordarin was calculated by subtracting the
latter from each corresponding value of the second capsule chamber.
Photolabeling of EF-2--
The reaction was performed in
Eppendorf tubes containing 50 µl of 20 µM EF-2 in 30 mM Tris-HCl, pH 7.5, 10 mM KCl, 200 µM EDTA, 20% (v/v) glycerol, 10 mM
-mercaptoethanol and the appropriate amount of
[14C]GM258383. After preincubating for 15 min at 25 °C
in the dark, samples were placed on ice and irradiated at 254 nm with
an UV lamp (2000 microwatts/cm2). Further sample processing
was dependent on the aim of the experiment. For quantification
purposes, samples were denatured by adding 150 µl of 8 M
guanidine chloride followed by 5 min of heating at 80 °C. Then, free
drug was removed by gel filtration through Sephadex G-25, and the
extension of covalent labeling was determined by liquid scintillation
counting. For other purposes samples were processed as opportunely described.
Identification of the Photolabeled Residue--
Photolabeling of
EF-2 was performed as described above using 100 µM
[14C]GM258383 and 10-min UV-irradiation. Gel filtration
on Sephadex G-25 was then used to remove drug excess and to exchange
buffer to 100 mM ammonium acetate, pH 5.0. Further
reduction and alkylation with 4-vinylpyridine, trypsin digestion, and
reverse-phase HPLC were performed according to established methods
(23). Amino acid sequences were determined with an Applied Biosystems
pulse-liquid sequencer model 477A connected on-line to a reverse-phase
HPLC unit for identification of the stepwise released
phenylthiohydantoin-amino acids. Mass determinations were performed
with a Bruker Biflex III MALDI time of flight mass spectrometer. These
analysis were done by Eurosequence b.v. (Groningen, The Netherlands).
| |
RESULTS AND DISCUSSION |
GM258383, the photoactivatable aryl azide sordarin derivative used
in the present work (see structure in Fig.
1), inhibits protein synthesis in
C. albicans cell-free systems (IC50 = 2 µM, determined as in Ref. 20). This biological activity
makes GM258383 appropriate for studies intended to identify sordarin
binding site. Ideally, this photoprobe was intended to label the
ribosome·EF-2 complex, because this is considered the functional
target of sordarin. However, the spectral properties of GM258383
(maximum absorption at 260 nm and no absorption at wavelengths longer
than 300 nm) are almost identical to those of ribosomes; consequently,
photoactivation of the probe is quenched by the inner filter effect of
ribosomes. Indeed, all efforts to photolabel mixtures of ribosomes and
EF-2 were unsuccessful (data not shown). Therefore, labeling
experiments were carried out with EF-2 alone.
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Fig. 1.
Chemical structure of sordarin, GM193663, and
GM258383. The asterisk denotes 3H
(sordarin) or 14C (GM258383) positions in the radioactive
derivatives.
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To characterize the binding of sordarin to its target we have performed
equilibrium dialysis experiments with [3H]sordarin and
either EF-2 alone or equimolar mixtures of EF-2 and ribosomes in the
presence of a non-hydrolyzable GTP analog, i.e. Gpp(NH)p
(Fig. 2). Sordarin binding to EF-2 alone
showed saturation, the Scatchard plot corresponding to that of one
single class of binding sites (Fig. 2A). Fitting the
experimental points to a single hyperbola yielded Kd = 1.26 µM. This indicates that sordarin binds to EF-2 in
a specific manner to a defined binding site within the EF-2 molecule
and hence supports performing photoaffinity labeling with EF-2 alone.
This affinity, similar to that described for rat liver EF-2 and its
natural substrate GTP (Kd = 3.0 µM)
(24), seems to be high enough to reduce the risk of nonspecific
labeling (25, 26). On the other hand, the presence of ribosomes unveils
two classes of binding sites with higher affinity, as deduced from the
Scatchard plot (Fig. 2B). Kd values for
these two sites (calculated from data fitting to a double hyperbola)
were 0.7 and 41.5 nM. These values are far apart with
respect to the value with EF-2 alone and might be indicative of the
presence of two populations of ribosomes (pre- and post-translocated)
in agreement with our previous observations (4). The affinity increase
may result from conformational changes within EF-2 upon interaction
with the ribosome rather than from the creation of a new site in EF-2, because the latter seems unlikely in view of the already notable affinity shown by EF-2 alone. The possibility of a combined binding site in the interface between ribosome and EF-2 cannot be ruled out
either. Interestingly, it has been recently shown that the sordarin
derivative GM193663 increases the reactivity of 26 S rRNA in
Saccharomyces cerevisiae ribosomes, most notably affecting to the sarcin-ricin loop, which becomes more exposed (27).
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Fig. 2.
Equilibrium dialysis measurement of
[3H]sordarin binding to EF-2 alone (A)
or to equimolar mixtures of ribosomes and EF-2
(B). Experiments were performed as described
under "Experimental Procedures." Data were also displayed as
Scatchard plots (insets) and then accordingly fitted to a
single (A) or a double (B) hyperbola from which
Kd values were calculated. Dotted lines
(B, inset) show individual binding to each class
of site according to the parameters deduced from the double hyperbola.
Results presented here are representative of three experiments.
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The use of aryl azides (such as GM258383) in photoaffinity labeling is
widespread. Nevertheless, the long lifetime of the reactive
intermediate has been deemed a serious caveat that may lead to
nonspecific labeling (28). Fig.
3A shows EF-2 photolabeling with [14C]GM258383 at different irradiation times.
Labeling followed pseudo-first order kinetics with
kobs of 0.215 min1 and was
quantitatively completed after 10 min. When EF-2 was irradiated in the
presence of increasing concentrations of [14C]GM258383, a
hyperbolic saturation curve was obtained (Fig. 3B), with
maximum incorporation at 0.39 mol/mol EF-2. The most convincing result
to support the specificity of photolabeling with
[14C]GM258383 is presented in Fig. 3C, where
it is demonstrated that sordarin derivative GM193663 prevented
photolabeling in a dose-dependent manner, so that
incorporation of the photoprobe was completely precluded in the
presence of high concentrations of GM193663 (Fig. 4, lane 3). On the other hand,
when the reaction mixture was not irradiated at 254 nm no labeling was
detected by SDS-polyacrylamide gel electrophoresis and fluorography
(Fig. 4, lane 4). In all, it is proved that GM258383 is
covalently bound to a EF-2 sordarin binding site as a result of a
photoactivatable process.
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Fig. 3.
Characterization of EF-2 photolabeling.
A, photolabeling kinetics. B, effect of
photoprobe concentration. C, photolabeling prevention with
GM193663. The experiments were performed as described under
"Experimental Procedures" using the indicated amounts of
[14C]GM258383 (100 µM in A and
C) and the denoted irradiation times (10 min in B
and C).
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Fig. 4.
Fluorography of samples from EF-2
photolabeling. 20 µM EF-2 was incubated with 100 µM [14C]GM258383 and either not exposed to
UV light (lane 4) or UV-irradiated for 10 min in the
presence (lane 3) or absence (lane 2) of 10 mM GM193663. Samples were then diluted 20-fold with
denaturing electrophoresis sample buffer, and 5 µl was loaded on a
10% acrylamide gel. After SDS-polyacrylamide gel electrophoresis was
run the gel was dried and subjected to fluorography. Lanes 1 and 5, molecular weight markers.
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To identify the EF-2 residue modified by [14C]GM258383,
photolabeled EF-2 was digested with trypsin and the resulting peptide mixture was resolved by reverse-phase HPLC (Fig.
5). Five fractions (named
"A" to "E") showed significant
radioactive levels, which accounted for more than 60% of the total.
Besides, there is a spurious trail of radioactivity at the end of the
chromatogram, which is not associated with UV peaks at 214, 254, 280, or 297 nm; hence, it might be attributed to traces of free decomposed radioligand. Fraction A was further purified by HPLC (Fig.
6) and yielded two radioactive peaks,
"A1" and "A2." Sequence analysis of these
two peaks, by Edman degradation, rendered uFANxYSK (peak A1;
where "u" denotes no unambiguous assignment and
"x" denotes no detection) and FANxYSKK (peak
A2). MALDI-MS of peak A1 rendered a signal at
m/z = 1437 (MH+), which could be
attributable to the mass of the peptide QFANKYSK (986 Da) plus an added
mass of 450 Da corresponding exactly to the expected mass increase due
to the linked photoprobe (Fig. 7).
Likewise, MALDI-MS of peak A2 gave a signal at
m/z = 1437 (MH+), attributable
to the mass of FANKYSKK (986 Da) plus the photoprobe. Such sequences
correspond to the fragment Gln224-Lys232
within the C. albicans EF-2 sequence, Lys228
being the modified residue. Modification of this lysine residue, which
explains that trypsin did not cleave at this position and that the
residue was not identified after Edman degradation, is the result of
one of the mechanisms described for the photochemistry of aryl azides.
As depicted in Fig. 7, it proceeds via triplet nitrene, leading to
abstraction of a hydrogen radical from the protein and further
combination of the newly formed radicals to yield the covalent bond.
The hydrogen radical is preferably abstracted from a carbon atom
adjacent to heteroatoms of the amino acid side chain, as is the case of
C
in Lys (26).
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Fig. 5.
Separation of trypsin-generated
peptides. 6 nmol of EF-2 photolabeled with
[14C]GM258383 was digested with trypsin as described
under "Experimental Procedures." The resulting peptide mixture was
resolved by reverse-phase HPLC on a Nucleosil 10C18 column (150 × 2.1 mm) using a linear gradient of 0-100% acetonitrile in 0.04%
(v/v) trifluoroacetic acid during 120 min at 0.35 ml/min. Fractions
were collected for different intervals, and one-tenth of their volume
was used for radioactivity counting by liquid scintillation.
A, UV profile of the column eluate; arrows denote
positions of the peaks that were selected according to the content of
radioactivity. B, Radioactivity expressed as
total cpm in each of the collected fractions.
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Fig. 6.
Purification of fraction A. Fraction A
from the first HPLC run (Fig. 5) was subjected to re-chromatography on
the same column using a linear gradient of 0-90% acetonitrile in
0.05% (w/v) ammonium acetate, pH 6.0, during 120 min at 0.35 ml/min.
Fractions were collected for different intervals and one-third of their
volume was used for radioactivity counting by liquid scintillation.
A, UV profile of the column eluate; arrows denote
positions of the peaks that were selected according to the content of
radioactivity. B, radioactivity expressed as total cpm in
each of the collected fractions.
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Fig. 7.
Reactions involved in EF-2 photolabeling with
[14C]GM258383. Reactions are inferred from the
experimental results of this paper, according to the photochemistry of
aryl azides described in Ref. 26.
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The same procedure was followed with fraction B. Further
chromatography yielded a single radioactive peak (Fig.
8). No sequence could be obtained from
Edman degradation of this peak. However, MALDI-MS revealed one signal
at m/z = 1419 (MH+) attributable
to the mass of qFANKYSK (968 Da; where "q" is
pyroglutamic acid) plus the mass of the photoprobe (450 Da).
Transformation of Gln into Glu and subsequent lactam formation blocked
the N terminus of the peptide, thus preventing Edman degradation.
Finally, when the rest of the radioactive peaks were analyzed in the
same form, either no radioactivity was detected in the
re-chromatography, or no sequence nor MS results were obtained. This
suggests that these peaks might correspond to modified peptides present
in scarce amounts.
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Fig. 8.
Purification of fraction B. Fraction B
from the first HPLC run (Fig. 5) was subjected to re-chromatography on
the same column using a linear gradient of 0-90% acetonitrile in
0.05% (w/v) ammonium acetate, pH 6.0, during 120 min at 0.35 ml/min.
Fractions were collected for different intervals, and one-third of
their volume was used for radioactivity counting by liquid
scintillation. A, UV profile of the column eluate; the
arrow denotes the position of the peak that was selected
according to the content of radioactivity. B, radioactivity
expressed as total cpm in each of the collected fractions.
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The modified Lys228 is located in the G-domain (also called
domain I) of EF-2, more precisely at the beginning of the insert called
G"-subdomain. The analog insert in bacterial EF-G (called the
G'-subdomain) is located before the G5 motif, between helix DG and strand 6G (see Ref.9). However, in
eukaryotic EF-2, the insert is longer and placed after the G5 motif.
Because this G"-subdomain is involved in sordarin binding, such
differences between G"- and G'-subdomains can play a role in
determining the innocuousness of sordarins on bacterial protein synthesis.
Fig. 9 shows the alignment of
the G"-subdomains from all EF-2 sequences presently available. It is
noteworthy that such a subdomain is longer in EF-2 from higher animals
than in the rest of eukaryotic species due to a 13-residue fragment
located at the beginning of the subdomain. More remarkable is the
existence of two putative Walker motifs (29), with Walker-A being
present only in this group of species. Recently, Gonzalo et
al. (30) identified in rat liver EF-2 a second nucleotide binding
site specific for ATP, which may probably involve these additional Walker motifs. Although the underlying physiological function is not
known, these observations show that, despite the high homology, significant differences exist in G"-subdomains among species. Thus, the
high selectivity of sordarin toward one such set of species (the fungi)
appears less surprising.
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Fig. 9.
Sequence alignment of G"-subdomains of EF-2
from different sources. Alignment was performed using ClustalW
software (36) with default gap penalties. The peptide identified by
photoaffinity labeling with [14C]GM258383 is highlighted
in a black box. Regions identified previously (30) as
possible Walker A and B motifs are shaded.
Asterisks denote conserved residues within all the
sequences. FUN, fungi (C.alb, C. albicans; S.cer, S. cerevisiae;
C.psi, Candida parapsilosis; C.tro,
Candida tropicalis; C.gla, Candida
glabrata; D.dis, Dictyostelium discoideum;
C.neo, Cryptococcus neoformans; C.lus,
Clavispora lusitaniae). ANI, animals
(H.sap, Homo sapiens (human); R.nor,
Rattus norvegicus (rat); C.gri, Cricetulus
griseus (Chinese hamster); G.gal, Gallus
gallus (chicken); D.mel, Drosophila
melanogaster (fruit fly); C.ele, Caenorhabditis
elegans). PRO, protists (L.maj,
Leishmania major; P.fal, Plasmodium
falciparum; C.par, Cryptosporidium parvum;
B.hom, Blastocystis hominis; E.his,
Entamoeba histolytica; T.cru, Trypanosoma
cruzi; G.int, Giardia intestinalis);
PLA, plants (B.vul, Beta vulgaris;
C.kes, Chlorella kessleri).
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The G"-subdomain is thought to directly interact with the sarcin-ricin
loop of 26 S rRNA in fungi. The peptide identified in this work is very
close to Trp218, an essential residue in the interaction
with the loop (31). Putting together these observations with the
above-mentioned effect of GM193663 on S. cerevisiae 26 S
rRNA (27), the hypothesis of a combined binding site between ribosome
and EF-2 becomes more plausible. This in turn opens the possibility
that differences among ribosomes also contribute significantly to
sordarin specificity.
In a previous paper, a theoretical three-dimensional model for S. cerevisiae EF-2 was presented (32). The model was built from
homology mapping of EF-2 onto the crystal structure of
Thermus thermophilus EF-G. In this model a sordarin binding
site was proposed based on the position of the mutations conferring
resistance to sordarin: 9 of 14 mutated residues (32, 33) mapped
closely on domain III, defining a possible binding pocket. Accordingly, Shastry et al. (21) have recently shown in this domain a
block of eight amino acids (corresponding to residues 517-524 in
S. cerevisiae EF-2) that may define a sordarin specificity
region in natural fungal species. We have not made use of this
three-dimensional model, because the photolabeled peptide is included
in the G"-subdomain, which is not present in the template EF-G
molecule, and hence the accuracy of the model may not suffice for our
purposes. Nevertheless, it is obvious that the putative binding region
deduced from our studies is far from the pocket predicted from these
resistant mutations. Recent studies strongly suggest that domain III is closely related to the G-domain both structurally and functionally. It
seems to influence the GTP binding center (34) and to participate in
the transmission of conformational rearrangements from the G-domain to
domain IV after GTP hydrolysis (35). Therefore, these mutations in EF-2
may help to overcome the effect of the drug by affecting EF-2 function
rather than sordarin binding to EF-2 alone, thus precluding the
transition to a high affinity sordarin binding complex upon interaction
with the ribosome. On the other hand, there are five more
sordarin-resistant mutations out of domain III, two of them located
near the photolabeled peptide (6).
In summary, the results presented in this paper may contribute to the
sordarin mode of action. In any case, studies designed to afford a more
detailed definition of the molecular effects of this drug at ribosomal
level are still needed.
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ACKNOWLEDGEMENTS |
We thank Dr. H. J. Bak and Dr. W. J. Weijer (Eurosequence b.v.) for their excellent work, and M. Nieto,
R. Sarabia, and A. Monjo for their assistance.
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FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.: 34-91-807-0301;
Fax: 34-91-807-0595; E-mail: juan_m_dominguez@gsk.com.
Published, JBC Papers in Press, June 11, 2001, DOI 10.1074/jbc.M104183200
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ABBREVIATIONS |
The abbreviations used are:
EF-2, elongation
factor 2;
EF-G, elongation factor G;
Gpp(NH)p, :
-imidoguanosine
5'-triphosphate;
MALDI, matrix-assisted laser desorption/ionization;
MS, mass spectroscopy;
HPLC, high performance liquid
chromatography.
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