Binding of the Chemotaxis Response Regulator CheY to the Isolated, Intact Switch Complex of the Bacterial Flagellar Motor
LACK OF COOPERATIVITY*
Yael Sagi
,
Shahid Khan
and
Michael Eisenbach
¶
From the
Department of Biological Chemistry, The
Weizmann Institute of Science, 76100 Rehovot, Israel and the
Molecular Biology Consortium, Chicago, Illinois
60612
Received for publication, March 28, 2003
, and in revised form, May 6, 2003.
 |
ABSTRACT
|
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In bacteria, the chemotactic signal is greatly amplified between the
chemotaxis receptors and the flagellar motor. In Escherichia coli,
part of this amplification occurs at the flagellar switch. However, it is not
known whether the amplification results from cooperativity of CheY binding to
the switch or from a post-binding step. To address this question, we purified
the intact switch complex (constituting the switch proteins FliG, FliM, and
FliN and the scaffolding protein FliF) in quantities sufficient for
biochemical work and used it to investigate whether the binding of CheY to the
switch complex is cooperative. As a negative control, we used complexes of
switchless basal bodies, formed from the proteins FliF and FliG and similarly
isolated. Using double-labeling centrifugation assays for binding, we found
that CheY binds to the isolated, intact switch complex in a
phosphorylation-dependent manner. We observed no significant
phosphorylation-dependent binding to the negative control of the switchless
basal body. The dissociation constant for the binding between the switch
complex and phosphorylated CheY (CheY
P) was 4.0 ± 1.1
µM, well in line with the published range of CheY
P
concentrations to which the flagellar motor is responsive. Furthermore, the
binding was not cooperative (Hill coefficient
1). This lack of
CheY
P-switch complex binding cooperativity, taken together with earlier
in vivo studies suggesting that the dependence of the rotational
state of the motor on the fraction of occupied sites at the switch is
sigmoidal and very steep (Bren, A., and Eisenbach, M. (2001) J. Mol.
Biol. 312, 699709), indicates that the chemotactic signal is
amplified within the switch, subsequent to the CheY
P binding.
 |
INTRODUCTION
|
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The motility of bacteria such as Escherichia coli and
Salmonella enterica serovar Typhimurium derives from the rotation of
their flagella (for reviews, see Refs.
1 and
2). Each flagellum is driven by
a bidirectional rotary motor embedded in the cytoplasmic membrane. The
direction of flagellar rotation determines the swimming behavior of the cells,
enabling bacteria to approach beneficial environments and escape hostile ones.
Thus, the essence of bacterial chemotaxis is modulation of the direction of
flagellar rotation (3). This
modulation is carried out by a switch that responds to signals received from
the chemotaxis receptors (for reviews, see Refs.
46).
The switch extends from the base of the flagellar motor into the cytoplasm. It
is composed of three proteins, FliG (
35 molecules per switch
(7,
8)), FliM (
35 molecules
per switch (7,
8)), and FliN (
100
molecules per switch (9)), and
is mounted on the MS ring of the flagellar motor, formed from the protein FliF
(1012).
The signals from the chemotaxis receptors to the switch are transduced by the
response regulator CheY. The activity of this protein is modulated by
phosphorylation. When phosphorylated in response to chemotactic stimulation,
the protein is detached from the histidine kinase CheA, which is a part of the
receptor supramolecular complex, and acquires an elevated affinity for the
switch protein FliM
(1316).
The outcome is an increased probability of shifting the direction of flagellar
rotation from the default direction, counterclockwise, to clockwise (for
recent reviews, see Refs. 17
and 18).
Bacteria such as E. coli and Salmonella sense stimuli
over a wide concentration range and, despite the wide range, do so with very
high sensitivity, suggesting high amplification of the chemotactic signal
(19,
20). One of the major
questions in bacterial chemotaxis is which of the signal transduction steps
amplify the chemotactic signal. One amplification step likely occurs at the
receptor level, the amplification being provided by propagation of the
excitation signal from the stimulated receptor molecule to neighboring
receptor molecules within the receptor cluster
(2124;
for a review, see Ref. 17). In
view of recent findings that switching from counterclockwise to clockwise upon
an increase in the intracellular level of
CheY
P1 is highly
cooperative (a Hill coefficient of
10
(25)), another amplification
step probably occurs at the switch level (for a review, see Ref.
26). However, it is not known
whether this high amplification reflects cooperativity of CheY
P binding
to FliM (4,
25,
27,
28) or amplification of
post-binding events at the switch
(28). This question cannot be
addressed by direct in vitro assays of CheY binding to purified FliM
because it is not known whether purified FliM in solution truly represents the
native protein within the switch complex and because the inter-FliM
interactions, which normally occur within the switch
(29), probably do not occur
with soluble FliM molecules.
Recently, Lux et al.
(30) overexpressed, in a
non-flagellated strain, all three switch proteins together with FliF and
purified the resulting membrane-associated structure. These structures have
now been identified on the basis of immunochemical
(30), morphological
(31), and in vivo
CheY-binding criteria (32) as
intact functional switch complexes mounted on the transmembrane MS ring. These
advances open up the possibility of detailed mechanistic analysis of the
cooperative motor response upon binding CheY. Here, we have used this
preparation to characterize the phosphorylation-dependent binding of CheY to
the intact switch complex in vitro.
 |
EXPERIMENTAL PROCEDURES
|
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Preparation of Switch ComplexFor the preparation of switch
complexes, we overproduced FliG and FliF from the overproducing plasmid
pKOT107 and overproduced FliM and FliN from the overproducing plasmid pKLR2 in
the nonflagellated E. coli strain BL21 (
DE3)
(33). The overproduced switch
complex, found at the cytoplasmic membrane, was purified according to the
procedure of Francis et al.
(34) for isolating extended
basal bodies with several modifications. Cells (21 liters) of strain BL21
(
DE3) containing the plasmids pKOT107 and pKLR2 were grown in Luria
broth supplemented with ampicillin (100 µg/ml) and chloramphenicol (30
µg/ml). The overproduction of the proteins was induced at OD600
= 0.4 by isopropyl-1-thio-
-D-galactopyranoside (1
mM). The cells were grown for an additional 3 h and harvested by
centrifugation. The pellet was resuspended in 250 ml of ice-cold sucrose
solution (0.5 M sucrose, 0.1 M Tris-HCl, pH 7.9), and
then lysozyme (final concentration, 1 mg/ml, from a freshly prepared stock
solution) and EDTA (final concentration, 10 mM) were added. The
mixture was gently stirred at 4 °C for about 11 h. The resulting
spheroplasts were lysed by two-step addition of Triton X-100: first to a final
concentration of 1% in the presence of MgSO4 (10 mM);
then, following gentle stirring at 4 °C for 3 h, to a final concentration
of 2% in the presence of MgSO4 (10 mM). The suspension
was further gently stirred at 4 °C for 0.5 h. EDTA (20 mM) was
added, and the suspension was stirred for 60 min. Unlysed cells and inclusion
bodies were removed by centrifugation at 23,000 x g for 30 min,
and the pH of the supernatant was raised with NaOH to pH 10 to disintegrate
the outer membrane structures. The lysate was spun at 123,000 x
g for 60 min, and the pellet was resuspended in a solution containing
Tris-HCl (10 mM, pH 7.9), KCl (100 mM), sucrose (10%
w/v), and Triton X-100 (0.1%). The suspension was recentrifuged at 23,000
x g for 30 min, and then the supernatant was spun at 200,000
x g for 40 min. The pellet was resuspended in a solution
containing Tris-HCl (50 mM, pH 7.9) and Triton X-100 (0.1%). To get
rid of residual inclusion bodies that potentially remained in the suspension,
the suspension was centrifuged again at 23,000 x g for 10 min.
The purified switch complex was stored at 4 °C with Complete protease
inhibitor (Roche Applied Science).
Preparation of Switchless Basal BodyFor the preparation of
a switchless basal body, we used the same procedure used for the preparation
of the switch complex, with the exception that the cells contained only one
plasmid, the plasmid pKOT107 for the overproduction of FliG and FliF
alone.
Labeling of CheYInitially we radiolabeled CheY in
vitro by methylating the
-amine of its lysine residues and the N
terminus with formaldehyde and NaB[3H]4, as described
previously (35). The
experiments shown in Fig. 2
were carried out with CheY labeled in this way. However, subsequently we found
that high levels of labeling (higher than those used in
Fig. 2) inhibited the ability
of CheY to undergo in vitro phosphorylation by the phosphodonor
acetyl phosphate. We, therefore, switched to in vivo labeling, which
does not involve chemical modification of CheY. E. coli strain M15
(36), carrying the plasmid
pQE12-CheY-His-tag for the overproduction of His6-CheY (received
from A. Wolfe, Loyola University), was grown to OD590 = 0.5 in H1
minimal medium of Kaiser and Hogness
(37) supplemented with
histidine, methionine, and threonine (1 mM each), as well as with
leucine (200 µM), thiamin (5 µg/ml), ampicillin (100
µg/ml), and glucose (0.3%, w/v). The overproduction of CheY was induced at
OD590 = 0.5 by
isopropyl-1-thio-
-D-galactopyranoside (0.5 mM).
For labeling, isopropyl-1-thio-
-D-galactopyranoside was
supplemented with [14C]leucine (final concentration, 0.17
µCi/ml). The cells were grown for an additional 4 h, harvested by
centrifugation, and sonicated. Non-soluble material (inclusion bodies and cell
debris) was removed by centrifugation at 123,000 x g for 30
min. The His6-tagged CheY protein was purified from the supernatant
by Ni-NTA affinity chromatography (Qiagen) according to the manufacturer's
instructions, with some modifications. We followed protocol 11 (batch
purification of His6-tagged proteins under native conditions) with
the exception that, following the collection of the flow-through, the washing
of the lysate-Ni-NTA column was with 8090 ml of washing buffer (until
no protein was eluted) rather than 4 ml, and the elution of CheY from the
Ni-NTA column was with 2030 ml of elution buffer instead of 2 ml. We
concentrated CheY by ultrafiltration through a 10 kDa cut-off membrane in an
Amicon chamber (model 52). CheY was stored at 80 °C. We verified
that the His6-tag at the C terminus of CheY did not interfere with
the function of the protein according to two criteria: (a) we
expressed His6-tagged CheY in the non-chemotactic strain AW546
eda+ cheY201 (received from J. Adler) and found
that His6-tagged CheY restored chemotactic responsiveness to this
strain, judged by its ability to form typical chemotactic rings on a semisolid
agar (0.3% agar) plate containing Tryptone
(38); and (b) we
measured the ability of His6-tagged CheY to undergo acetyl
phosphate-mediated in vitro phosphorylation by measuring the changes
in the fluorescence of Trp58, known to be strongly reduced upon
CheY phosphorylation (39). The
fluorescence of CheY was determined using a fluorimeter with excitation and
emission wavelengths set at 295 and 345 nm, respectively. Tag-free CheY or
His6-tagged CheY was diluted to a final concentration of 10
µM into 50 mM Tris-HCl buffer (pH 7.9) containing 5
mM MgSO4, and its fluorescence was recorded.
Subsequently, acetyl phosphate (final concentration, 20 mM) was
added, and the fluorescence was again recorded. Both His6-tagged
CheY and tag-free CheY were similarly quenched (by
60%) upon the addition
of acetyl phosphate.

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FIG. 2. Specific phosphorylation-dependent binding of CheY to the switch
complex. The switch complex and switchless basal body were prepared and
assayed for CheY binding as described under "Experimental
Procedures," using [3H]CheY (7000 dpm/pmol) and
[14C]glucose (final concentration, 3500 dpm/µl). The results,
presented as the ratio between the quotient
[3H]CheY/[14C]glucose in the pellet subsequent to
centrifugation and the quotient [3H]CheY/[14C]glucose in
the suspension before centrifugation, are the mean ± S. D. of
duplicates in a representative experiment. A, comparison between
binding of CheY to the switch complex and to the switchless basal body. The
amount of switch complex or switchless basal body in each case was 59 µg of
total protein. B, examination of the potential contribution of
FliM-containing inclusion bodies to the observed CheY binding. The amount of
the switch complex was 148 µg of total protein. See text for details.
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Binding of CheY to the Switch Complex and to the Switchless Basal
BodyThe switch complex or the switchless basal body, at the amount
indicated in the figure legends, was incubated with [3H]CheY
(20007000 dpm/pmol) and [14C]glucose (final concentration,
3500 dpm/µl) or [14C]CheY (His6-tagged; 1317
dpm/pmol) and [3H]glycine (600 dpm/pmol) in a solution of Tris-HCl
(50 mM; pH 7.9), MgSO4 (5 mM), and where
indicated, bovine serum albumin (10 mg/ml). For phosphorylating conditions,
acetyl phosphate (25 mM) was added to the reaction mixture. After a
5-min incubation at room temperature (24 °C), the switch complex or the
switchless basal body was pelleted by centrifugation at 200,000 x
g for 10 min at 4 °C. The pellet was resuspended in 100 µl of
Tris-HCl (50 mM, pH 7.9). Aliquots of the reaction mixture prior to
centrifugation and of the supernatant and pellet subsequent to centrifugation
were counted for 14C and 3H by a
counter
(Tri-Carb liquid scintillation analyzers).
 |
RESULTS
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Isolation of the Switch Complex for Biochemical StudiesTo
produce an intact switch complex in quantities sufficient for biochemical
work, we overproduced the three switch proteins (FliG, FliM, and FliN) along
with FliF from two plasmids as done by Lux et al.
(30) and then purified the
overproduced switch complex from the cytoplasmic membrane as described under
"Experimental Procedures." In electron micrographs, the purified
switch complex (Fig. 1)
appeared similar to that obtained by Lux et al.
(30). As a negative control,
we used the host BL21 cells, lacking the overproducing plasmids. These cells,
following the same isolation procedure, did not yield the structures shown in
Fig. 1.

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FIG. 1. Electron micrograph of an isolated switch complex preparation. Low
magnification view of negatively stained structures obtained upon coexpression
of the FliF, FliG, FliM, and FliN proteins. Switch complexes are indicated by
the black arrows.
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Demonstration of Specific CheY
P Binding to the Switch
ComplexTo measure the binding between CheY and the purified switch
complex, we equilibrated the complex with [3H]CheY and then
separated switch-bound CheY from free CheY by centrifugation. To be able to
measure the volume of medium entrapped within the pellet, we also included in
the medium an inert radiolabeled compound, [14C]glucose. The
3H/14C ratio in the pellet was higher in the presence of
the phosphodonor acetyl phosphate than in its absence
(Fig. 2A), suggesting
the occurrence of phosphorylation-dependent binding of CheY to the switch
complex. Because CheY binds to FliM, we used, as a negative control, a
switchless basal body. We isolated it from cells overexpressing FliF and FliG
only, using the same isolation procedure as for the intact switch complex.
Unlike the case of the intact switch complex, we did not observe a
phosphorylation-dependent increase in CheY binding to the switchless basal
body (Fig. 2A). These
results suggest that the phosphorylation-dependent binding to the intact
switch complex is specific and that the isolated switch complex is functional
with respect to CheY binding.
A potential source of concern in the above experiments was that traces of
FliM-containing inclusion bodies might have remained in the preparation of the
switch complex and might have contributed to the observed binding of
CheY
P. We verified that this was not the case by the following measures:
(a) we did not observe a white fluffy precipitate, typical of
inclusion bodies, on top of the precipitate of the switch complex;
(b) before precipitating the switch complex at 200,000 x
g, we got rid of FliM-containing inclusion bodies by spinning the
preparation at 23,000 x g, which is above the force required
for their full precipitation (12,500 x g
(40)); and (c) we
carried out control experiments in which we equilibrated the switch complex
with [3H]CheY and then separated switch-bound CheY from free CheY
by centrifugation either at 23,000 x g or at 200,000 x
g. We observed only a phosphorylation-dependent increase in the
3H/14C ratio in the pellet at 200,000 x g
(Fig. 2B), suggesting
that the preparation of isolated switch complex did not contain inclusion
bodies and that the observed binding of CheY
P was to the switch complex
alone.
Quantification of the CheY-Switch BindingTo quantify the
binding between CheY and the switch complex, we titrated the complex with
increasing amounts of radiolabeled CheY under phosphorylating and
nonphosphorylating conditions (presence and absence of acetyl phosphate,
respectively). As a control for nonspecific binding, we used the switchless
basal body at equal total-protein concentrations. Under nonphosphorylating
conditions, CheY bound to the switch complex only slightly better than to the
negative control of switchless basal body
(Fig. 3). Under phosphorylating
conditions, CheY selectively bound to the switch complex. To quantify the
binding, we had to estimate the total amount of FliM within the switch
complex. We did this by two independent means: according to the number of CheY
molecules bound to the switch complex at saturation and according to a Western
blot analysis of the switch complex with anti-FliM antibodies
(Fig. 4). Both methods yielded
a similar estimate of the FliM amount, 0.95 and 1.1 nmol FliM/mg of total
protein, respectively. These values were measured with the same preparation of
switch complex used for the results shown in Figs.
3 and
4. The average ratio between
these estimations for three different preparations of the switch complex was
0.9 ± 0.2 (mean ± S.D.), suggesting that the stoichiometry
between CheY and FliM is 1:1. Quantitative analysis of the data of this
representative experiment revealed a dissociation constant of 3.6 and 120
µM for phosphorylated and nonphosphorylated CheY, respectively
(Fig. 3, insets).
Correcting the CheY
P concentration in the experiment according to the
estimate that CheY is 85.5% phosphorylated in vitro under these
conditions (41), we calculated
that the dissociation constant for the CheY
P-switch complex is 2.9
µM. A Hill fit to the binding data yielded a Hill coefficient of
1.0 (r2 = 0.99), suggesting that, within the uncertainties
of the experimental error, the binding of CheY
P to the isolated switch
complex is not cooperative. This apparent lack of cooperativity was observed
in each of the three batches of isolated switch complex assayed for
cooperativity (average Hill coefficient = 1.3 ± 0.2 (± S.D.)).
The dissociation constant varied in the range of 2.95.6
µM between different batches of the isolated switch complex,
yielding a mean of 4 ± 1 µM (± S.D.).

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FIG. 3. Concentration-dependent CheY binding to the switch complex. The
binding assay was carried out in the presence of bovine serum albumin (10
mg/ml) with switch complex or switchless basal body (115 µg of total
protein in each case), [14C]CheY (His6-tagged; 17
dpm/pmol), and [3H]glycine (600 dpm/pmol) as described under
"Experimental Procedures." , switchless basal body. ,
switchless basal body in the presence of acetyl phosphate. , intact
switch complex. , intact switch complex in the presence of acetyl
phosphate. Left inset, double reciprocal plot of non-phosphorylated
CheY; Kd = 120 µM. Right
inset, double reciprocal plot of CheY in the presence of acetyl
phosphate; Kd = 3.6 µM. The term
r stands for the number of moles of CheY bound to 1 mol of the switch
complex. We calculated it from the number of CheY molecules found to be bound
(after subtraction of nonspecific binding to the switchless basal body) to a
single molecule of FliM and then multiplying it by 35, the estimated number of
FliM molecules within a single switch complex
(7,
8). The free CheY concentration
was calculated by subtracting the concentration of CheY measured to be bound
to the switch from the total CheY concentration.
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FIG. 4. FliM quantification by Western blots. Aliquots of purified switch
complex and of purified FliM (15 ng/µl) were mixed with sodium dodecyl
sulfate sample buffer, boiled for 10 min, resolved on sodium dodecyl
sulfate-12% (w/v) polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes as described previously
(46). The blots were probed
with anti-FliM antibody (a gift from S. I. Aizawa) and peroxidase-conjugated
anti-rabbit antibody (Sigma). Films were scanned with an imaging densitometer
(model GS-690; Bio-Rad). The intensities of the bands were quantified by
Multi-Analyst software (Bio-Rad). Calculation of the amount of FliM in each
preparation of the purified switch complex was made on the basis of a
calibration curve prepared according to the band intensities of increasing
concentrations of FliM.
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 |
DISCUSSION
|
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In this study we isolated, in quantities sufficient for biochemical work,
the flagellar switch complex of E. coli and demonstrated that it is
functional, at least with respect to CheY binding. We found that this binding
(Kd = 4 ± 1 µM) is
phosphorylation-dependent, is specific, has a CheY:FliM stoichiometry of 1:1,
and is not cooperative. The significance of these findings is discussed
below.
Earlier in vivo studies found a steep dependence of the
probability of clockwise rotation on the intracellular concentration of active
CheY (25,
27,
42,
43). Carrying out the
measurements in single cells, Cluzel et al.
(25) found that the increase
was over a narrow range of CheY
P concentrations and highly cooperative
(Hill coefficient
10). These observations suggested that the switch acts
as some sort of amplifier, translating small changes in the concentration of
CheY
P into large changes in clockwise probability. However, these
observations could not distinguish between amplification caused by
cooperativity of CheY binding to the switch
(4,
25,
27,
28) and amplification evolved
within the switch (28).
Earlier studies, which used in vitro binding assays with purified
soluble proteins (or peptides thereof), could not address this question either
(13,
16,
44,
45). A distinction between
both possibilities, although indirect, was later made by Bren and Eisenbach
(46), who used FliM proteins
almost locked in their clockwise or counterclockwise states as representatives
of CheY-bound and CheY-free FliM, respectively. By expressing these FliM
proteins to different levels, they found that the dependence of the clockwise
probability on the relative level of FliM in the clockwise state is very
steep. Because that study bypassed the CheY
P-FliM binding step, it
suggested that at least part of the signal amplification occurs at a
post-binding step. In the current study, we found no cooperativity of
CheY
P binding to the switch, endorsing the notion that the chemotactic
signal is amplified within the switch rather than at the preceding binding
step of CheY
P to the switch.
While preparing this manuscript for publication, a study by Sourjik and
Berg (47) was published in
which resonance energy transfer was used for in vivo measurements of
CheY binding to the switch. Sourjik and Berg
(47) found essentially no
cooperativity of binding and a dissociation constant of
3.7
µM, very similar to the value measured by us in this study. The
similarity between the in vivo results of Sourjik and Berg
(47) and the in vitro
results with purified intact switch complex obtained in our current study
suggests that the isolated switch complex is functional and reliably
represents the function of the switch within the cell. Furthermore, our
measured dissociation constant for CheY
P (4 ± 1 µM)
is well in line with the range of CheY
P concentrations over which the
motor changes its bias from counterclockwise to clockwise (26
µM) (25). As
might be expected, it is lower than the dissociation constant measured for the
binding of an N terminus-containing peptide of FliM
(45) to CheY
P (
27
µM) (16).
The current study, taken together with the studies of Sourjik and Berg
(47), Cluzel et al.
(25), and Bren and Eisenbach
(46), strongly argues that the
chemotactic signal is amplified within the switch, subsequent to the
CheY
P binding. Such amplification suggests that the switching process can
be described as an allosteric transition between the rotational states of the
switch, with CheY
P as an allosteric activator. A model for an allosteric
transition of switching was recently proposed by Duke et al.
(48).
 |
FOOTNOTES
|
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* This study was supported by Grant 2000037 from the United States-Israel
Binational Science Foundation, Jerusalem, Israel. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶
Incumbent of the Jack and Simon Djanogly Professorial Chair in Biochemistry.
To whom correspondence should be addressed: Dept. of Biological Chemistry, The
Weizmann Institute of Science, P. O. Box 26, 76100 Rehovot, Israel. Tel.:
972-8-934-3923; Fax: 972-8-934-4112; E-mail:
m.eisenbach{at}weizmann.ac.il.
1 The abbreviations used are: CheY
P, phosphorylated CheY; Ni-NTA,
nickel-nitrilotriacetic acid. 
 |
ACKNOWLEDGMENTS
|
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We thank Dr. S. R. Caplan for insightful discussions and critical reading
of the manuscript.
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