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(Received for publication, April 28,
1995; and in revised form, August 2, 1995) From the
Earlier studies have suggested that CheZ, the phosphatase of the
signaling protein CheY in bacterial chemotaxis, may be in an oligomeric
state both when bound to phosphorylated CheY (CheY
Bacteria such as Escherichia coli or Salmonella
typhimurium use chemotaxis to navigate toward favorable
environments and retreat from non-favorable ones(1) . The
sensory information from the receptors is integrated by a cytoplasmic
signal transduction network of chemotaxis proteins (see for review, (2, 3, 4, 5) ) and transmitted to
the flagella by the signaling molecule CheY (6, 7) .
CheY interacts with the switch-motor complex at the base of the
flagellum (6, 7, 8, 9, 10) and
changes the direction of rotation from the default direction,
counterclockwise(9, 10, 11, 12, 13, 14, 15, 16, 17) to
clockwise(12, 15, 17) , and thereby causes
the cell to reorient(18) . The clockwise causing activity of
CheY is regulated by phosphorylation(19) . The phosphorylation
level is determined by the kinase CheA and the phosphatase
CheZ(20, 21, 22) . The activity of the
kinase, CheA, is modulated by chemotactic stimuli via the membrane
chemotaxis receptors and the chemotaxis protein
CheW(23, 24, 25) . On the other hand,
regulation of CheZ activity by chemotactic stimuli has not been
demonstrated. In a previous study we found that the binding of CheZ
to phosphorylated CheY (CheY
Figure 1:
Autoradiogram of DMS-mediated
cross-linking of CheZ. The cross-linking was carried out as described
under ``Experimental Procedures.''
[
A mixture of EDC and
NHS mediates the cross-linking of amines to carboxyl
groups(38) . CheZ exposed to this mixture formed a single
defined band at 46 kDa (Fig. 2A, lane 2). In the
presence of non-phosphorylated CheY, most of CheZ remained as a dimer (lane 3). A sharp change in the oligomeric state of CheZ was
observed when CheY was added together with AcP. Under these conditions
all the CheZ formed a >94 kDa cross-linking product (lane
4), which appeared on a 10% SDS-PAGE as a 90-200-kDa smear
and a distinct band at 145 kDa (Fig. 2B). As in the
case of DMS, AcP in the absence of CheY did not form the >94-kDa
complex (Fig. 3A, lane 3). These results confirm that
an oligomeric complex of CheZ is formed in the presence of CheY
Figure 2:
Autoradiogram of EDC-NHS-mediated
cross-linking of CheZ. The cross-linking was carried out for 1 h as
described under ``Experimental Procedures.''
[
Figure 3:
The dependence of CheZ oligomerization on
CheY
To confirm that the cross-linking products observed in this study
resulted from specific interactions, we repeated all the above
mentioned experiments in the presence of SDS (0.5%). (SDS does not
interfere with cross-linking mediated by random
collisions(39) .) Only the monomeric form of CheZ was observed;
the formation of all the cross-linking products was prevented by SDS
(data not shown), indicating that the interactions were specific. The EDC-NHS-mediated cross-linking of CheZ (Fig. 2) was much
more efficient than the DMS-mediated cross-linking (Fig. 1).
This may be the consequence of the small number of lysine residues and
the high number of aspartate and glutamate residues in CheZ (6 residues versus 37, respectively(40) ). For this reason the
subsequent cross-linking studies were carried out with EDC-NHS. If
the phosphorylation-dependent oligomerization of CheZ is
physiologically significant, it should depend on the concentration of
CheY
Figure 4:
The
amount of CheY associated with the oligomer.
[
Figure 5:
Size estimation of CheZ by zonal
centrifugation on a sucrose gradient. The positions of the molecular
size marker proteins are indicated by arrows.
To enable the measurement of the
rotational diffusion of CheZ by fluorescence depolarization, we had
first to fluorescently label CheZ. For this purpose we replaced
phenylalanine 214 of CheZ with cysteine by using a mismatch primer and
PCR, as described under ``Experimental Procedures.'' The
mutated protein, CheZ214FC, retained normal CheY phosphatase activity (Fig. 6) and therefore could serve as a model for wild-type
CheZ. CheZ214FC contains a single cysteine residue and therefore could
be labeled at a specific site by fluorescein-5-maleimide. The
anisotropy, which reflects the rotational diffusion and therefore also
the size of the fluorescein-labeled
CheZ214FC(43, 44) , was dependent on the concentration
of added CheY (Fig. 7A). In the absence of AcP, the
anisotropy increased only moderately with the concentration of CheY and
was not saturated within the concentration range of the experiment
(0-45 µM CheY). This moderate change in anisotropy
was probably the result of the low-level binding of non-phosphorylated
CheY to CheZ(26) . In the presence of AcP, the anisotropy was
increased over 2-fold, indicating a large decrease in the rotational
diffusion of CheZ upon CheY phosphorylation. The increase in anisotropy
was saturated already at 4 µM CheY. The anisotropy change
was rapid; it was completed prior to the first measurable time point
(
Figure 6:
Phosphatase activity of CheZ214FC. The
fraction of CheY
Figure 7:
Fluorescence depolarization studies of
fluorescein-labeled CheZ214FC. The experiment was carried out in the
absence (
In this study we have shown that CheZ is a dimer which
further oligomerizes upon interaction with CheY The different approaches used
in this study to probe the molecular size of CheZ have yielded
conflicting results. When run on a size-exclusion column, CheZ appears
as a tetramer ( (27) and this study). In the cross-linking
experiments, CheZ appeared as a dimeric protein (Fig. 1Fig. 2Fig. 3). In zonal centrifugation, the
estimated size of CheZ was between a monomer and a dimer (Fig. 5). Since it is well known, both experimentally (45, 46, 47) and theoretically(48) ,
that the molecular size of non-spherical proteins can be overestimated
severalfolds by size-exclusion
chromatography(45, 46, 47, 48) , and
that the size can be underestimated in zonal
centrifugation(47) , it is reasonable to assume that CheZ is a
non-spherical dimer. The major finding of this study is that, in the
presence of CheY In the fluorescence depolarization experiments, the
anisotropy of fluorescein-labeled CheZ was increased to a large extent
in the presence of CheY It is
well known in a variety of systems, including bacterial signal
transduction systems(50, 51) , that oligomerization
regulates protein activity. Accordingly, it is conceivable that the
oligomerization may either activate or inhibit the phosphatase activity
of CheZ. In the first possibility, a burst of CheY phosphorylation will
activate CheZ and will thereby promote faster deactivation of CheY.
Fast deactivation of CheY will prevent non-beneficial too long periods
of tumbling. If the other possibility is correct and oligomerization
inhibits the phosphatase activity of CheZ, the oligomerization may
serve as an amplification step in which phosphorylation of CheY leads
to oligomerization of CheZ, inhibition of its phosphatase activity,
and, consequently, further increase in the level of CheY
This paper is dedicated
to Julius Adler on the occasion of his sixty-fifth birthday.
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 1226-1231
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
THE SIGNAL PROTEIN OF BACTERIAL CHEMOTAXIS (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
P) (Blat, Y.,
and Eisenbach, M.(1994) Biochemistry 33, 902-906) or
free (Stock, A., and Stock, J. B.(1987) J. Bacteriol. 169,
3301-3311). The purpose of the current study was to determine the
oligomeric state of free CheZ and to investigate whether it changes
upon binding to CheY
P. By using either one of two different sets
of cross-linking agents, free CheZ was found to be a dimer. The
formation of the dimer was specific, as it was prevented by SDS which
does not interfere with cross-linking mediated by random collisions.
The dimeric form of CheZ was confirmed by sedimentation analysis, a
cross-linking-free technique. In the presence of CheY
P (but not in
the presence of non-phosphorylated CheY), a high molecular size
cross-linked complex (90-200 kDa) was formed, in which the
CheZ:CheY ratio was 2:1. The size of the oligomeric complex was
estimated by fluorescence depolarization to be 4-5-fold larger
than the dimer, suggesting that its size is in the order of 200 kDa.
These results indicate that CheZ oligomerizes upon interaction with
CheY
P. This phosphorylation-dependent oligomerization may be a
mechanism for regulating CheZ activity.
P) is 2 orders of magnitude higher
than to non-phosphorylated CheY, and that several molecules of CheZ can
bind to a single CheY
P molecule(26) . Earlier observations
indicated that CheZ can be in two oligomeric forms, 115 and >500
kDa, as estimated by size-exclusion chromatography (27) (the
molecular size of the monomer is 23.9 kDa(27) ). The
observations of both studies taken together suggest that the oligomeric
state of CheZ is modulatable. Here we examine this possibility and
demonstrate that CheZ is a dimer which oligomerizes upon interaction
with CheY
P. The possibility that this phosphorylation-dependent
oligomerization is a regulation mechanism for CheZ activity is
investigated in a subsequent work (28) .
Bacterial Strains and Plasmids
The
overexpression of nonlabeled and radiolabeled E. coli CheZ
(CheZ
), (
)and of radiolabeled E. coli CheY (CheY) was carried out in strain RP437, wild type
for chemotaxis(29) , carrying the plasmid pRL22(30) .
Nonlabeled CheY was overexpressed in strain EW30 (26) which contains a deletion from cheA to cheZ and carries the plasmid pRL22(
PvuII). For the
overexpression of S. typhimurium CheY (CheY
) we
constructed the plasmid pEWY5 as follows. cheY was amplified
from total DNA of strain ST1 (31) by polymerase chain reaction
(PCR) using the primers 5`-CCGAATTCATGGCGGATAAAGAGC-3` and
5`-CCGGATCCTCAGATGCCCAGTTTCTCAAAG-3`, which contained added EcoRI and BamHI sites, respectively, at their 5` end.
The amplified fragment was digested by EcoRI and BamHI, and ligated with pBTac1 (Boeringer Mannheim)
predigested by EcoRI and BamHI. The resultant
plasmid, pEWY5, overexpressed CheY under the control of tac promoter. The plasmid pEWC1, used for the overexpression of S.
typhimurium CheZ214FC, was constructed similarly to the
construction of pEWY5 except that the primers
5`-CCGAATTCATGATGCAACCATCTATCAAGCC-3` and
5`-CCGGATCCTTAACAGCCAAGACTGTCCAGCAGGTC-3` were used for the PCR.
CheY and CheZ214FC were overexpressed in RP1091
(RP437(cheA-cheZ)(14) ] and RP3098
[RP437(flhC-flhA)(32) ), respectively.Protein Radiolabeling
Radiolabeling of CheY and CheZ was carried out by inducing the expression
of CheY and CheZ in the presence of L-[
C]leucine as described
previously(26) .Protein Purification
The purification of
CheY (nonlabeled) and CheZ (nonlabeled and
radiolabeled) was carried out on a Cibacron column followed by a G-50
(for CheY) or Sepharose CL-6B (for CheZ) column as described
previously(26) . Radiolabeled CheY was purified as
the non-labeled CheY, only that smaller-scale columns (4 ml
of Cibacron and 65 
1 cm G-50) were used. CheY was
purified as CheY(26) , except that the bacteria
were grown on Luria broth and induced with 0.66 mM
isopropyl-
-D-thiogalactopyranoside. CheZ214FC was
purified as follows. RP1091 cells containing pEWC1 were grown at 35
°C in 1.5 liter of Luria broth containing 100 µg/ml ampicillin.
At OD = 0.4, overexpression was induced by the
addition of 1 mM
isopropyl-
-D-thiogalactopyranoside. After 4 h, the cells
were harvested by centrifugation, washed once by buffer A (20 mM HEPES, pH 7.4, 2 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride), resuspended in 35 ml of buffer A,
and sonicated. Membranes and unlysed cells were removed by
centrifugation at 165,000 g for 60 min. The lysate was
loaded onto a 40-ml Sepharose CL-6B column pre-equilibrated with buffer
A. The column was washed with 100 ml of buffer A, followed by 170 ml of
buffer A containing 275 mM NaCl. CheZ was eluted from the
column by a 250-ml linear gradient of 275-450 mM NaCl in
buffer A. The CheZ-containing fractions were pooled and concentrated by
ultrafiltration through a 5-kDa cut-off membrane, using an Amicon
chamber (model 52). The concentrated fractions were loaded onto a 20-ml
hydroxylapatite column, pre-equilibrated with buffer B (10 mM NaP
, pH 7.0, 2 mM dithiothreitol, and 0.2
mM phenylmethylsulfonyl fluoride). The column was washed with
60 ml of buffer B, and CheZ was eluted with a 200-ml linear gradient of
buffer B containing 10-200 mM NaP. The
CheZ-containing fractions were concentrated to a volume of 2 ml and
loaded onto a Sephadex G-150 column (50 1.5 cm),
pre-equilibrated with a solution of 50 mM Tris-HCl, pH 7.9,
200 mM NaCl, 2 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride. The CheZ-containing fractions were
concentrated and stored in 10% glycerol at -80 °C.Cross-linking
The reaction mixture (20 µl) for
cross-linking contained NaCl (90 mM), KCl (10 mM),
MgCl
(5 mM), Tris-HCl (50 mM, pH 8.5 for
cross-linking by dimethylsuberimidate (DMS) or 35 mM, pH 7.9,
for cross-linking by 1-ethyl-3-(dimethylaminopropyl)carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS)), and
CheZ (1.3 µM). CheY (20
µM), and acetyl phosphate (AcP, 18 mM) were also
added as appropriate. The reaction was initiated by the addition of DMS
(3 mg/ml) or EDC followed by NHS (66 and 13 mM, respectively),
and allowed to proceed for 3 h (with DMS) or 40-60 min (with EDC
plus NHS) at room temperature (22-25 °C). The reaction was
terminated by the addition of 5 µl of sodium dodecyl sulfate (SDS)
sample buffer (5 concentrated). The samples were boiled for 10
min, and 22-µl aliquots were used for 15% SDS-polyacrylamide gel
electrophoresis (PAGE). After electrophoresis, the gel was stained,
dried, and autoradiographed. Quantitative analysis of the radioactivity
associated with the different cross-linking products was carried out
with a PhosphoImager (Fujix, Bas 1000).CheZ Labeling with Fluorescein
CheZ214FC (100
µM, 200 µl) was incubated with 0.5 mM fluorescein-5-maleimide (Molecular Probes) in Tris-HCl (50
mM, pH 7.9) for 30 min at room temperature (22-25
°C). After incubation, CheZ was separated from the unreacted
fluorescein by application onto a 0.8-ml G-50 mini-column and briefly
spinning at 480 g. The eluted labeled CheZ was
dialyzed against Tris-HCl (50 mM, pH 7.9) containing 1.5
mM dithiothreitol. The labeled CheZ was stored at -80
°C.Size Estimation of CheZ by Zonal
Centrifugation
CheZ, CheY (14 kDa), CheW
(18 kDa), and ovalbumin (43 kDa) samples (150 µl of 2 mg/ml) were
loaded onto a 5-20% isokinetic sucrose gradient (5 ml) in
Tris-HCl (50 mM, pH 7.9), KCl (100 mM), and
MgCl (5 mM). The samples were spun at 290,000
g for 24 h (20 °C) in a Beckman SW50.1 rotor.
After centrifugation, 130-µl fractions were collected from the top
of the gradient and analyzed for protein concentration by the Bradford
technique(33) . The size of CheZ was estimated by
plotting a calibration line of (molecular size) against
the distance traveled by the protein peak(34) .
Size Estimation by Size-exclusion
Chromatography
CheZ alone or a mixture of CheZ (20
µM), CheY (100 µM), and AcP (18 mM)
was run through a size-exclusion HPLC Bio-Sil TSK-250 column (7.5
600 mm; Bio-Rad; 23 °C), utilizing HP 1040 A diode array
chromatography system, and Waters HPLC system, composed of two pumps
(model 510) and an automatic controller. Prior to separation, the
column was pre-equilibrated with a solution of Tris-HCl (50
mM, pH 7.9), KCl (100 mM), MgCl (5
mM), and AcP (18 mM). The flow rate was 1 ml/min.Fluorescence Depolarization of CheZ
Fluorescence
depolarization studies with fluorescein-labeled CheZ214FC were carried
out with a Perkin Elmer LS 50 B luminescence spectrometer equipped with
a fluorescence polarization accessory. The excitation and emission
wavelengths were 490 and 520 nm, respectively (5-nm slit width).Phosphatase Activity of CheZ
The phosphatase
activity of CheZ was assayed by monitoring the steady-state level of
CheY phosphorylation in the presence of
[P]acetyl phosphate (AcP, synthesized as
described(35) ) and varying concentrations of CheZ. CheY (20
µM) was incubated in Tris-HCl (50 mM, pH 7.9) and
MgCl
(5 mM) with [P]AcP (20
mM, 200-400 cpm/pmol) and varying concentrations of
CheZ. The reaction (20 µl) was quenched after 10 min of incubation
at room temperature (25 °C) by 100 µl of 10% ice-cold
trichloroacetic acid and 20 µg of ovalbumin. (We verified that
trichloroacetic acid does not hydrolyze CheY˜P under our
experimental conditions.) The level of CheY phosphorylation was
determined as described earlier(26) .
CheY Phosphorylation Leads to Formation of a High
Molecular Size CheZ-CheY Complex
In order to examine the
possibility of phosphorylation-dependent oligomerization of CheZ, we
monitored the oligomeric state of CheZ by cross-linking in the presence
or absence of CheYP. When cross-linked by DMS which cross-links
amines(36) , some of the CheZ molecules appeared to form two
bands migrating on SDS-PAGE as 43- and 49-kDa proteins (Fig. 1, lane 2). These bands probably correspond to CheZ dimers (the
monomeric size of CheZ is 23.9 kDa). The two distinct bands of the
dimer possibly reflect two different positions of DMS links between the
CheZ monomers. The addition of CheY (14 kDa) did not cause any change
in the cross-linking products observed (lane 4). However, the
addition of CheY together with the phosphodonor AcP (37) caused
the appearance of two additional cross-linking products of 38 and
>94 kDa (lane 5). (A more accurate determination of the
size of >94 kDa product is made below.) AcP in the absence of CheY
had no effect (lane 3). The 38-kDa band probably corresponds
to a 1:1 CheY-CheZ complex which reflects the increase in binding of
CheZ to CheY upon phosphorylation(26) . The >94 kDa band may
be an oligomeric complex of CheZ which possibly contains also CheY (see
below). In order to verify that the CheZ dimer and the oligomeric
complex are not cross-linking artifacts, we used an additional
cross-linker and cross-linking-free techniques.
C]CheZ (3.2 nCi) was used for each reaction.
The top band in each of the lanes belongs to nonspecific
aggregates unable to enter the separating
gel.
P.
C]CheZ (3.2 nCi) was used for each reaction. A, 15% polyacrylamide gel. B, as lane 4, but
on 10% polyacrylamide gel.
P. The cross-linking was carried out as in Fig. 2A, except for the modifications indicated below.
Unless mentioned otherwise, the CheY and CheY57DE concentrations were
20 µM. A, cross-linking of
[
C]CheZ in the presence of varying
concentrations of CheY. B, quantification of the dependence of
the amount of oligomer formed on the concentration of CheY. The
relative intensity of each oligomer band was measured by a
PhosphoImager and calculated relatively to the intensity of the band
formed in the presence of 20 µM CheY. The results are the
mean of two experiments, one of which is the experiment shown in panel
A. C, cross-linking of [C]CheZ under
conditions which prevent the phosphorylation of
CheY.
P. As shown in Fig. 3, A and B, the
amount of the oligomer was indeed dependent on the concentration of
CheY added in the presence of access AcP. Furthermore, CheY57DE which
cannot be phosphorylated due to the substitution of Glu for the
phosphorylation site, Asp
(26, 41) , did
not promote the oligomer formation even in the presence of AcP (Fig. 3C, lane 7). Similarly, depletion of
Mg
(necessary for CheY phosphorylation(42) )
also prevented the formation of the oligomer (lane 5).
The Amount of CheY in the Oligomer
In order to
estimate the relative content, if any, of CheY in the oligomer, we
carried out the cross-linking experiment with radiolabeled CheY or
radiolabeled CheZ in parallel (Fig. 4).
[C]CheY cross-linked in the presence of CheZ and
AcP (Fig. 4, lane 4) indeed formed a band migrating at
the same position as the CheZ oligomer (lane 1). This band was
not formed in the absence of CheZ and AcP (lane 3; the faint
high molecular size band seen in this lane represents aggregates too
large to enter the gel). This result indicates that the CheZ oligomer
contains also some CheY cross-linked to it. We quantified the amount of
CheY cross-linked to the CheZ oligomer by a PhosphoImager. The result
was 0.48 ± 0.18 (mean ± S.D., three independent
determinations) molecules of CheY per monomer of CheZ, suggesting a
CheZ:CheY ratio of 2:1.
C]CheZ (3.2 nCi, specific activity 126 Ci/mol)
or [C]CheY (20 nCi, specific activity 50 Ci/mol)
(the radiolabeled protein in each lane is indicated by an asterisk) were cross-linked by EDC-NHS as in Fig. 2A, only that the cross-linking was carried out
for 40 min.
Estimation of the Oligomeric Status of CheZ in the
Absence of CheY
Our observation
that, in the absence of CheYP by Sedimentation Analysis
P, CheZ appears as a dimer in
cross-linking experiments, is in apparent conflict with Stock and
Stock's (27) observation, reproduced by us, that CheZ
behaves as a tetramer when run on an HPLC size-exclusion column. In
order to solve this discrepancy we estimated the size of CheZ by zonal
centrifugation on a sucrose gradient. As shown in Fig. 5, the
CheZ peak appeared between the peaks of CheW (18 kDa) and ovalbumin (43
kDa) at a location which, according to the formula described under
``Experimental Procedures,'' is the site of a 35 ± 4
kDa (mean ± S.D., two determinations) globular protein. This
estimation is closer to the cross-linking results, which indicated that
CheZ is a dimer (47.8 kDa), than to the HPLC results which indicated a
tetramer. The difference between the size estimations of the different
approaches suggests that CheZ is non-globular (see
``Discussion'').
Estimation of the Oligomeric Status of CheZ in the
Presence of CheY
To confirm
the oligomerization of CheZ in the presence of CheYP by Fluorescence Depolarization
P, we initially
attempted to use size-exclusion chromatography. Running a mixture of
CheZ, CheY, and AcP through the HPLC column yielded only two peaks,
eluted at retention times identical to those obtained when CheZ and
CheY were run separately. This could mean either that, in contrast to
the cross-linking results, the CheY
P-induced oligomerization does
not occur at all, or that the oligomer is unstable and readily
dissociates when CheY and/or AcP are separated from CheZ on the column.
To distinguish between these possibilities, we used fluorescence
depolarization, which, unlike size-exclusion chromatography and
sedimentation analysis, allows interaction between CheZ and CheY
P
throughout the experiment.
30 s). In order to estimate the magnitude of the size change, the
anisotropy of CheZ was measured at varying viscosity values in the
presence or absence of CheY
P. The data, presented in the form of a
Perrin plot (43, 44) , are shown in Fig. 7B. In the Perrin plot the slopes of the curves
are inversely related to the molecular dimension and therefore to the
molecular size(43, 44) . The slope was 4.8-fold
smaller in the presence of CheY
P than in its absence, suggesting
that upon CheY phosphorylation CheZ formed a complex which is
approximately 4-5-fold larger in volume than the CheZ dimer. The
fact that we did not detect CheY
P-induced oligomerization of CheZ
in size-exclusion chromatography, but did observe it using fluorescence
depolarization, suggests that the oligomer is unstable and dissociates
in the absence of CheY
P.
P out of total CheY (0.26 ± 0.06) in the
absence of CheZ was considered as 100%. The results are the mean
± S.D. of three independent experiments.
, wild type;
, CheZ214FC.
) or presence () of 18 mM AcP. A,
the effect of CheY on the fluorescence anisotropy (A =
(I - I
)/(I
+ 2I
)) (44) of CheZ. The measured
solution contained fluorescein-labeled CheZ214FC (0.2 µM),
Tris-HCl (50 mM, pH 7.9), MgCl
(5 mM) and
increasing concentrations of CheY. B, Perrin plot
of the fluorescence anisotropy of fluorescein-labeled CheZ214FC (1.0
µM in the same buffer conditions as in A). When
present, the concentration of CheY was 20 µM.
The medium viscosity was increased by sucrose up to 4.6 cPoise. In this
viscosity range the change in fluorescence anisotropy reflects mainly
the rotation of the whole CheZ-fluorescein conjugate, and to a much
lesser extent the free rotation of the probe (fluorescein). The latter
rotation is not expected to be affected by the presence of CheY and
AcP. Therefore, to a first approximation, the slopes of these lines,
can be taken as the inverse of the respective molecular volumes (43) .
P. The significance
of these findings is discussed below.
P, there is further oligomerization of the CheZ
dimer. However, it is not possible to determine, on the basis of the
results, whether CheY
P is an integral part of the oligomer at a
CheZ:CheY
P ratio of 2:1, or whether it is just bound to an
oligomer comprising CheZ alone. The results of the fluorescence
depolarization implied that the oligomer is about 4-5 times
larger than the CheZ dimer formed in the absence of CheY
P. This
suggests that the size of the oligomer is in the order of 200 kDa. This
size is about at the top of the size range observed in the
cross-linking experiments (Fig. 2B). The size
estimation of the oligomer from the fluorescence depolarization is only
a first degree approximation, because it was based on the assumption
that both the oligomer and the CheZ dimer are
spherical(43, 44) , an assumption which, according to
our own results, is probably incorrect. Determination of the exact size
or shape is beyond the resolution of the applied methods. A better
estimation of the size of the oligomer is rather difficult by currently
available methods because, at this stage, there is no obvious way to
separate the oligomer from CheY
P and maintain it in a stable form.
For example, techniques in which the shape contribution can be
estimated (e.g. light scattering) cannot distinguish between
the oligomer and CheY
P in the mixture. Nevertheless, these
difficulties in size and shape estimation do not affect the conclusions
reached in this study, as neither the exact size nor the shape of the
oligomer are necessary for the conclusions. It should be noted that the
CheZ oligomer, observed in this study, is different from the CheZ
homopolymer (27) and the CheZ-CheA
multimeric
complex(49) , observed earlier, in the sense that the oligomer
of this study is not stable and it readily dissociates in the absence
of CheYP.
P (Fig. 7A). This
observation could, in principle, be attributed either to a significant
increase in the molecular volume of CheZ, or to a large conformational
change at the vicinity of the fluorescein moiety that restricts its
free rotation. The following observations strongly suggest that a
significant increase in the molecular volume of CheZ, i.e. CheZ oligomerization, is the mechanism responsible for the
anisotropy change. (i) To a first approximation, the Perrin plot (Fig. 7B) is composed of two distinct rotations: a fast
rotation of the probe, and a slow rotation of the whole protein. To
determine the rotational freedom of the probe, we extrapolated the
straight line of the Perrin plot to 1/anisotropy = 0. Since the
straight line shown in the figure represents the rotation of the
protein, the extrapolated value represents the hypothetical case in
which the protein rotation is frozen but the probe rotates freely. As
shown in Fig. 7B, both the dimeric and the oligomeric
forms of CheZ fall in the same anisotropy range (1/anisotropy values of
4.0 and 4.8 for the dimer and oligomer, respectively). This indicates
that the rotation of the probe itself is not significantly affected by
the oligomerization, and therefore that the anisotropy change is not
the result of a change in the probe rotation. (ii) An increase in
anisotropy could, in principle, be due to a decrease in the lifetime of
the excited state of the probe, reflected in a reduced efficiency of
the fluorescence. However, the large oligomerization-dependent change
in anisotropy was accompanied by only a minor reduction (7%) in the
fluorescence efficiency. The lack of substantial changes in the
fluorescence efficiency and in the motional freedom of the probe, is
supported by the observation that both the 214F
C substitution (Fig. 6) and the conjugation of this cysteine with fluorescein
maleimide (
)did not affect the activity of CheZ.P. The
results described in the subsequent paper (28) suggest that the
first possibility is the correct one.
), E. coli CheZ; AcP, acetyl phosphate; CheY, E.
coli CheY; CheY, S. typhimurium CheY;
CheZ, S. typhimurium CheZ; DMS,
dimethylsuberimidate; EDC, 1-ethyl-3-(dimethylaminopropyl)carbodiimide
hydrochloride; NHS, N-hydroxysuccinimide; PAGE, polyacrylamide
gel electrophoresis; PCR, polymerase chain reaction; HPLC, high
performance liquid chromatography.)
We thank Prof. Meir Shinitzky for his encouragement,
helpful discussions, and critical reading of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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