From the
Departments of Pathology and Microbiology
and
Biochemistry, School of Medical Sciences,
University of Bristol, Bristol BS8 1TD, United Kingdom
Received for publication, December 5, 2002 , and in revised form, May 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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The native EPEC Tir molecule resolves at a higher molecular mass (78
kDa) on SDS-PAGE than is predicted from its sequence (
57 kDa), suggesting
that the protein retains some form of residual structure in SDS
(7). Anomalous SDS gel shifts
may therefore act as a probe for Tir conformation. Upon translocation into
host cells EPEC Tir undergoes a significant increase in its apparent molecular
mass (7885 kDa). This has been shown to be due to phosphorylation, and
proceeds through sequential 5-kDa (designated T') and 2-kDa (designated
T') shifts (6,
7,
11). More recently, Tir has
been shown to be an in vitro and in vivo substrate for the
cAMP-dependent protein kinase (PKA) with the sequential in vitro
phosphorylation of Ser-434 and Ser-463 (within the C-terminal domain)
mimicking those shifts displayed by Tir isolated from infected mammalian cells
(11). Tir is also
phosphorylated on a single tyrosine residue (Tyr-474), enabling the direct
recruitment of the Nck adaptor molecule and the actin nucleation machinery
(12,
13). However, tyrosine
phosphorylation does not contribute to shifts in apparent molecular mass on
SDS gels (6). Although the
mechanism by which non-tyrosine phosphorylation affects Tir function has not
been elucidated, it has been postulated that it may induce changes in Tir
structure either to aid additional kinase modification and/or promote Tir
insertion into the host plasma membrane from a cytoplasmic location
(2,
9). In this study biophysical
methods have been used to characterize the effects of PKA-mediated
phosphorylation on the properties of Tir in order to address the physiological
relevance of such modifications. The results presented indicate a mechanism by
which PKA modification of Tir may play a role in its function within host
cells.
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EXPERIMENTAL PROCEDURES |
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Protein PurificationBL21-DE3
(15) E. coli were
transformed with pET27b encoding either TirHSVHis or C-TirHSVHis. Protein
expression was induced by addition of
isopropyl-1-thio--D-galactopyranoside (1 mM) to
cell cultures grown at 37 °C to an OD600 nm of 0.6 and
incubation for an additional 3 h. Cells pellets were resuspended in
sonication/binding buffer (20 mM imidazole, 50 mM HEPES,
150 mM NaCl, 2 mM
-mercaptoethanol, pH 7.5) and
lysed using a Vibra-Cell probe sonicator, 60% duty cycle (Sonics &
Materials, Inc.). Soluble proteins were separated from cell debris and
insoluble proteins by centrifugation (18,000 rpm, 30 min) and fast protein
liquid chromatography (FPLC) was used to pass the supernatant through a nickel
affinity chromatography column (5-ml Hi TrapTM nickel-chelating column;
Amersham Biosciences) equilibrated in binding buffer. Bound TirHSVHis or
C-TirHSVHis proteins were eluted with an imidazole gradient (0.051
M), and the protein-containing fractions identified via SDS-PAGE.
These fractions were combined and dialyzed overnight against a buffer
containing 20 mM Tris, 50 mM NaCl, pH 7.5 and
subsequently concentrated in a Vivaspin 20-ml concentrator (5000 molecular
weight cutoff, Sartorius). Protein concentration (OD280 nm) was
estimated with the extinction coefficient values for Tir and C-Tir determined
using Expasy web software ProtParam.
Native Polyacrylamide Gel ElectrophoresisResolution of monomeric and dimeric protein was achieved on native gels containing 10% polyacrylamide (37.5:1 bis-acrylamide/acrylamide), 400 mM Tris, pH 8.8 in the separating gel and 3% polyacrylamide and 120 mM Tris, pH 6.8 in the stacking gel. Samples were diluted in sample application buffer (400 mM Tris, pH 8.8, 20% glycerol, and 0.1% bromphenol blue) prior to loading onto gels. Gel electrophoresis was performed for 2 h at 150 V and in the presence of electrophoresis buffer containing 0.2 M Tris, 0.1 M glycine. Protein bands were visualized by staining the gels with Coomassie Blue in 10% (v/v) glacial acetic acid and 50% (v/v) ethanol followed by destaining in a 10% (v/v) glacial acetic acid/5% (v/v) ethanol solution.
Two-dimensional Gel ElectrophoresisC-Tir protein samples (10 µM in 20 mM Tris, pH 7.5) were diluted in IEF buffer (8 M urea, 2% ampholyte pH 310 (Amersham Biosciences), 0.5% (v/v) Tween-20, 10 mM dithiothreitol, 2% CHAPS) and used to rehydrate 17 cm IPG strips (pH 47, BioRad). These strip were focused for 24 h using a pHaser isoelectric focusing system (V-h 50,000, maximum voltage 5000, duration 24 h, holding voltage 124, maximum current 50 µA, 17 °C) and equilibrated in IPG equilibration buffers I and II (Genomic Solutions) prior to the second dimension on 10% SDS-PAGE. Gels were stained and destained as described for native gel electrophoresis.
In Vitro PKA PhosphorylationPhosphorylation of Tir and C-Tir proteins were carried out at 30 °C in PKA buffer (New England Biolabs) supplemented with 100 µM ATP (Roche Applied Science). Phosphorylation reactions were carried out with low concentrations of PKA (0.05 units per ng of protein equivalent to 1:100, PKA/Tir, or 1:300, PKA/C-Tir, mol/mol, respectively) or at a 100-fold higher concentration. Phosphorylated proteins were purified and concentrated on a 1-ml Hi TrapTM nickel-chelating column, eluting in half the volume applied to column, and dialyzed overnight with a buffer containing 20 mM Tris, 50 mM NaCl, pH 7.5.
Circular Dichroism SpectroscopyCircular dichroism spectra were obtained at room temperature using a Jobin-Yvon CD6 spectropolarimeter. Spectra of native and phosphorylated samples (1:100, PKA/Tir, or 1:300, PKA/C-Tir, mol/mol, respectively) having protein concentrations in the range of 0.050.2 mg/ml were measured in 2-mm pathlength quartz cuvettes. Spectra at high protein concentration (0.51 mg/ml) were measured in 0.1-mm quartz cuvettes. All spectra are averages of between 5 and 11 scans with relevant protein-free buffer spectra subtracted and were plotted without smoothing using SigmaPlot (SPSS Inc.). Deconvolution of CD spectra into component secondary structural contributions was done using the CDPro suite of programs (16), which enable calculation of secondary structure using three independent methods (CONTIN, SELCON3, and CDSSTR), allowing an internal check on the consistency of the analyses.
Analytical UltracentrifugationSedimentation equilibrium experiments were carried out in a Beckman XL-A analytical ultracentrifuge (Beckman, Palo Alto, CA) using an AnTi 60 rotor. Native and phosphorylated samples (1:100, PKA/Tir, or 1:300, PKA/C-Tir, mol/mol, respectively) were exhaustively dialyzed against (20 mM Tris, 50 mM NaCl, pH 7.5) and loaded into a six-channel centerpiece yielding 2 mm of data per channel. Radial absorbance scans at 230 nm were taken after 12 h and then after every 6 h at 17,000, 22,000, and 28,000 rpm using three different protein concentrations (5, 10, 20 µM). Successive pairs of scans were subtracted from one another, and equilibrium was judged to have been established if there were no systematic deviations across each of the cell channels at each speed. Buffer density and partial specific volume was calculated according to the method of Laue and Stafford (17). Data from each channel was analyzed using the software provided with the centrifuge. Sedimentation velocity experiments were carried out in two channel centerpieces at 40,000 rpm, and cells were scanned radially every 5 min. Sedimentation coefficient distributions were obtained using the maximum entropy method of Schuck and Rossmanith (18).
Protein UnfoldingEquilibrium unfolding of C-TirHSVHis was performed using 6 M guanidine hydrochloride (GdnHCl) stock solutions prepared in 20 mM Tris, 50 mM NaCl, pH 7.5. A series of protein samples (30 µM) were denatured in 05 M GdnHCl in 0.2 M steps and left to equilibrate for 3 h at 25 °C before fluorescence intensity measurements were taken. GdnHCl unfolding was also carried out by addition of aliquots of GdnHCl to a single protein sample, and the data re-scaled to take into account differences in volume of denaturant at each concentration. Unfolding of unmodified proteins in this manner resulted in similar unfolding curves to those obtained by incubating the protein at each denaturant concentration in the range 05 M.
Tryptophan FluorescenceGdnHCl dependent intrinsic tryptophan fluorescence was carried out using a Perkin Elmer Luminescence (LS50B) spectrofluorimeter equipped with a thermostatted cell holder. Spectra were recorded using the following parameters: excitation wavelength 290 nm, slit width 10, emission 300450 nm, and scan speed 60. The Raman spectral contribution was removed by subtraction of a buffer blank. All spectra were plotted using GraFit Data analysis and Graphics software, version 3.01 (Erithacus Software Ltd.).
MALDI-TOF Mass Spectrometry Analysis of C-TirA mass spectrum of the C-Tir protein was determined using a PE Biosystems Voyager-DE STR MALDI-TOF mass spectrometer with a Nitrogen laser operating at 337 nm. The matrix solution was freshly prepared Sinapinic Acid (Fluka), at a concentration of 1 mg/100 µl in a 50:50 mixture of acetonitrile (Rathburn) and 0.1% trifluoroacetic acid (Aldrich). Sample and matrix, 0.5 µl of each, were spotted onto the sample plate. The sample was calibrated against Aldolase (Sigma) run as an external standard. The spectrum was acquired under linear conditions with an accelerating voltage of 25,000 V and an extraction time of 750 ns.
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RESULTS |
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Deconvolution of the data into secondary structural components for both Tir
and C-Tir is shown in Table I.
Since phosphorylation has little effect on the CD spectra of either protein
the secondary structural components listed in
Table 1 apply equally to both
the phosphorylated and non-phosphorylated forms. Tir has secondary structure
typical of a soluble protein with a high level of -helical and
-sheet structure. The high helical content is consistent with the
expectation that two hydrophobic (probably helical) transmembrane domains
flank the extracellular intimin-binding domain. In contrast, the C-terminal
domain contains a large amount of unordered or undefined structure. The
accuracy of spectral deconvolution varies among the calculated secondary
structural elements; in general,
-helix can be calculated with greatest
accuracy, and the "unordered" component is not necessarily
equivalent to polypeptide in "random coil" structure
(17). These data suggest that
the C terminus of Tir has a significant amount of unordered structure with
phosphorylation in this region having no significant effect on secondary
structure.
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Attempts were made to obtain near UV data using the Tir protein in order to determine the effects of phosphorylation on the tertiary structure. Near UV data require large amounts of protein to obtain the concentrations (100 µM) necessary for measuring these weak signals and at these concentrations we found that Tir is prone to aggregation. However, the near UV data collected revealed no significant difference between the spectra of native and phosphorylated full-length Tir (not shown).
The C-terminal Domain of Tir Exists in a Monomer/Dimer EquilibriumGiven that PKA-phosphorylation induces shifts in the apparent molecular mass of Tir as determined by SDS-PAGE (11), both the native and phosphorylated Tir and C-Tir proteins were subjected to analytical ultracentrifugation to determine their molecular masses under non-denaturing conditions. Sedimentation velocity studies of native and phosphorylated Tir revealed the presence of multiple species of undeterminable mass, possibly indicative of aggregation (not shown). In contrast, the results with C-Tir show that phosphorylation using low PKA concentration (1:300, PKA/C-Tir, mol/mol) induces a shift in the sedimentation coefficient distribution to higher values (Fig. 2A). This is consistent with either a phosphorylation-induced conformational change and/or oligomerization. As each of these may be occurring, we carried out a sedimentation equilibrium experiment in order to assess the degree of oligomerization upon phosphorylation. The results are presented in Fig. 2B, which shows representative sedimentation profiles for unphosphorylated and phosphorylated forms of C-Tir. Each profile represents a global non-linear least-squares analysis of nine data sets collected at 3 protein concentrations and 3 speeds using the program NONLIN (19). The molecular mass determined from a single species fit were 27,562 (24,546, 30,553) and 46,601 (45,137, 48,058) for the unphosphorylated and phosphorylated forms, respectively. The figures in parentheses are confidence limits from the non-linear global fit quoted at the 67% level. Both of these are a non-integral value of the monomer molecular weight. A dissociation constant of 37 µM was calculated for the unphosphorylated C-Tir. The molecular mass of 27,562 kDa for unphosphorylated C-Tir is greater than that expected for a monomer (19,615 kDa), but less than that expected for a dimer (39, 227 kDa), indicative of a system in equilibrium (Table II). The best model to fit our data set was that of a homogenous reversibly associating monomer/dimer equilibrium. The data can also be fitted to other models such as that describing a monomer/trimer. However, chromatography analysis of C-Tir at concentrations (50 µM) above that of the calculated dissociation constant (37 µM) on a Superdex 75 HR 10/30 gel-filtration column revealed a single peak with an elution volume corresponding to that of a dimer. In addition, observation of a small amount of C-Tir dimer by MALDI-TOF mass spectroscopy as well as the presence of dimers observed on SDS-PAGE gels (Fig. 3), both indicate that the model used (monomer/dimer) is the most appropriate. As the molecular mass of phosphorylated C-Tir (46,601 kDa) as determined by analytical ultracentrifugation is higher than expected for a dimer (39,227) it appears that higher order forms are also present at the protein concentrations (520 µM) used.
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Stability and Association Properties of Tir and C-Tir on SDS-PAGEThe anomalous migration of Tir (apparent molecular mass of 78 kDa, predicted molecular mass of 57 kDa) on SDS gels was also reproduced by its C-terminal domain, (apparent molecular mass of 30 kDa, with a predicted molecular mass of 20 kDa) (Fig. 3). This indicates that at least some of the structural properties of Tir responsible for anomalous migration reside in the C-terminal domain. Moreover, these observations indicate that the proteins retain some structure on SDS gels with Fig. 3 indicating that this structure is resistant to boiling in SDS application buffer. In fact, the far UV CD spectra of Tir is surprisingly unaffected in SDS even after boiling in 0.1% SDS (not shown). Analytical ultracentrifugation data indicated that C-Tir exists in solution in a monomer/dimer equilibrium. We therefore assessed the possibility that SDS dissociates Tir and C-Tir protein into their monomeric forms under SDS-PAGE conditions. Fig. 3 reveals that both Tir and C-Tir migrate as monomers on SDS gels and as a series of bands that correspond to monomeric, dimeric, and in the case of Tir, higher oligomeric forms when SDS is omitted from the application buffer. The detection of dimers on SDS gels also indicates that non-covalent interactions are involved in the dimerization of Tir and C-Tir.
PKA-mediated Phosphorylation Affects the Self-association Properties of the C-terminal Domain of TirFigs. 3 and 4A reveal that Tir migrates as monomers, dimers, and higher oligomers on SDS-PAGE gels when SDS is omitted from the sample application buffer. PKA phosphorylation of Tir (1:1, PKA/Tir, mol/mol), as expected, induces shifts in the apparent molecular mass of the protein (7885 kDa), while the distribution of monomeric, dimeric, and higher oligomeric phosphorylated forms remained unaffected (Fig. 4A). Fig. 4B shows that phosphorylation of C-Tir, like Tir (14), induces equivalent shifts in apparent molecular mass with these forms designated C-Tir' and C-Tir'. Low concentrations of PKA (1:300, PKA/C-Tir, mol/mol) resulted in the majority of C-Tir being converted to the C-Tir' form with only low levels of C-Tir' evident. At higher PKA levels (1:15 and 1:3, PKA/C-Tir, mol/mol) all C-Tir is converted to C-Tir''. This result differs from that previously reported, and reproduced in this study (not shown), where Tir is only modified to the T' form at low PKA concentration (1:100, PKA/Tir, mol/mol) (11). The apparent enhancement in phosphorylation efficiency may be attributed to enhanced accessibility to PKA of serine residues in the isolated C-terminal domain.
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To further assess the analytical ultracentrifugation data, unmodified C-Tir was examined under native gel conditions and the detection of two distinct forms further supports an equilibrium (monomer/dimer) model (Fig. 4C). In vitro phosphorylation of C-Tir at low PKA concentration shifted this equilibrium predominately to a dimeric form (D1), as also suggested by analytical ultracentrifugation (Fig. 2), with two monomeric species (M1/M2) evident that presumably equate to the C-Tir' and C-Tir'' forms observed on SDS gels. The pI of unmodified C-Tir and the phosphorylated forms generated at low PKA concentration (i.e. M1/M2) were determined by two-dimensional gel electrophoresis revealing values of 5.54, 5.26, and 5.1 respectively (Table II). The pI value of 5.54 for the unmodified C-Tir is in close agreement with the theoretical pI of 5.45 predicted by the ExPASy Web software ProtParam. The pI of the two phosphorylated forms of C-Tir (M1/M2) confirms that both these forms are distinguished from the unmodified C-Tir by a significant difference in net charge.
At higher PKA concentrations (1:15 or 1:3, PKA/C-Tir, mol/mol) phosphorylation results in decreased level of the dimeric form of C-Tir as the protein is now predominately in a monomeric form (M3). This monomeric species now migrates more rapidly compared with the two phosphorylated forms observed following modification at the lower PKA concentration (M1/M2).
The detection of this M3 form is surprising as only two phosphorylated states are detected by SDS-PAGE analysis and further work including mass spectrum analysis is required to establish the number of residues that are phosphorylated in Tir. It is possible that PKA has low affinity for the serine residue involved in this modification perhaps as it is inaccessible due to location in a dimerization interface. To test this possibility the ionic strength of the phosphorylation buffer was altered from 50 to 150 mM NaCl (physiological ionic strength) to destabilize possible electrostatic interactions, and the effect on protein modification at low PKA concentration was assessed. This change in ionic strength resulted in the detection of the M3 form, in contrast to the detection of only M1/M2 forms following modification at low ionic strength.
Denaturation of C-Tir by Guanidine HydrochlorideThe CD data
indicate a high content of undefined structure within the C-terminal domain of
Tir. To assess the extent to which C-Tir displays properties of a globular
protein, including co-operative unfolding transitions, guanidine hydrochloride
unfolding was carried out. This experiment revealed a two-step unfolding
transition (Fig. 5). The first
transition occurring at very low concentrations of denaturant (01
M GdnHCl) was ascribed to dimer dissociation since
ultracentrifugation and native gel electrophoresis demonstrate that C-Tir is
largely dimeric at the protein concentration used in these experiments. The
second transition was ascribed to unfolding of the monomer and occurs through
a single unfolding step between 1 and 5 M GdnHCl. From the data the
transition midpoints, the free energy of unfolding
(GF-U) at 25 °C, and the change in
free energy of unfolding with GdnHCl concentration (m) were calculated as
previously described (20,
21). The free energy
associated with the first transition (dimer dissociation) was determined to be
0.5 kcal/mol with a transition midpoint of 0.1 M GdnHCl and
m value of 4.7 indicating weak dimeric interactions. The free energy of
unfolding of the second transition (unfolding of monomer) was determined to be
3.8 kcal/mol with a transition midpoint of 2.8 M GdnHCl and
m value of 2.6. The free energy of the native state relative to the unfolded
state, (
GF-U) and the small denaturant
dependence for unfolding (m) are each smaller than expected for compact,
dimeric proteins with a well packed hydrophobic core
(22,
23), and support the CD data
of a partially disordered or loosely packed domain.
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DISCUSSION |
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Phosphorylation-induced Changes on SDS and Native Gels The migration of C-Tir, and its phosphorylated forms, on SDS-PAGE and native PAGE appears to be inconsistent at first sight, since the phosphorylated forms migrate with apparently higher molecular mass in SDS, and with lower molecular mass on native gels. These apparently conflicting observations may result, either from phosphorylation-induced changes in the hydrodynamic properties of the proteins or may simply be due to charge effects. For example, a more compact or structured phosphorylated state will have increased migration on native gels, but decreased migration on SDS gels if the compact state has reduced binding sites for SDS (27). The pI values of the two phosphorylated forms of C-Tir confirmed that both these forms are distinguishable from the unmodified C-Tir by a significant difference in net charge. As no significant differences were observed in the circular dichroism spectra of the phosphorylated and non-phosphorylated Tir and C-Tir proteins, it is therefore more likely that phosphorylation-induced changes in the assembly-state of C-Tir may involve modulation of electrostatic interactions rather than large scale conformational changes.
PKA-mediated Phosphorylation Effects on the Self-association Properties of C-TirAnalytical ultracentrifugation data shows that unphosphorylated C-Tir exists in a dynamic equilibrium of monomers and dimers that is modulated in vitro by PKA-mediated phosphorylation. This is consistent with the observations from native gel electrophoresis. Upon phosphorylation of C-Tir with low concentrations of PKA (1:300, PKA/C-Tir, mol/mol) there is an increase of the dimeric form. It therefore appears that phosphorylation at this level of PKA activity promotes dimerization of C-Tir. The dissociation constant of 37 µM, calculated from the sedimentation equilibrium data set (Fig. 2B), suggest that at physiological concentrations of protein (1 pM - 1µM, Ref. 22) C-Tir is likely to exist in a monomeric form within host cells, with in vivo phosphorylation leading to dimerization. Such a mechanism has been reported both in vitro and in vivo for the Bacillus subtilis transactivator SpOA, which also displays phosphorylation-mediated shifts from monomers to an activated dimer conformation (28).
In previous studies, in vitro phosphorylation of Tir using high concentrations of protein kinase-A (1:1 PKA/Tir, mol/mol) reproduced the shifts in electrophoretic mobility (as assessed by SDS gels) that are displayed by Tir isolated from membrane fractions of EPEC-infected host cells (11). These shifts are also displayed by C-Tir (phosphorylated) using high concentrations of PKA (Fig. 4B). Interestingly, at these higher concentrations of PKA, C-Tir (as assessed by native gels) is now almost completely dissociated to a monomeric form compared with the dimeric state detected at low PKA concentrations (Fig. 4C). This monomeric form of C-Tir (M3) could also be generated using low concentrations of PKA and physiological ionic strength, perhaps due to salt-induced exposure of an inaccessible serine that is destabilized at higher salt concentrations. Although the relevance of this observation in the context of native Tir remains to be determined, PKA-mediated dissociation of domain contacts may function to expose binding sites for interaction with host proteins, for example the tyrosine kinase that mediates the final phosphorylation of Tir on tyrosine 474 (2). A role for phosphorylation-induced dimer destabilization is illustrated by studies with ezrin, a membrane cytoskeleton linker protein. Threonine phosphorylation of ezrin is shown to weaken the interactions between its C- and N-terminal domains through both electrostatic and steric effects (29). Similarly, phosphorylation of the sporulation response regulator Spo0A is viewed as altering an equilibrium between an inactive (monomeric) and active (dimeric) forms of the protein (28). Protein kinase A phosphorylation has also been found to induce the association of ADP-ribosylation factor 1 to Golgi membranes (30).
In conclusion, we have shown that the C-terminal domain of Tir exists in an equilibrium (monomer/dimer) that is modulated by in vitro phosphorylation. PKA-mediated phosphorylation induces changes in the association properties of the C-terminal domain that may facilitate interaction with host proteins in vivo, such as the tyrosine kinase that phosphorylates Tyr-474 (6, 7) and/or other proteins involved in Tir-intimin mediated actin rearrangements. However, the assembly state of Tir is not as clearly defined, and this is evident from our analytical ultracentrifugation and gel electrophoresis data. It may be the case that oligomerization of Tir is promoted by contacts in addition to those of its C-terminal domain. This is consistent with the observed dimerization of the intimin-binding domain bound to intimin (9), though this Tir-Tir interaction would presumably be a later step that occurs following intimin-binding domain insertion across the plasma membrane.
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FOOTNOTES |
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¶ Supported by a Royal Society University research fellowship.
|| To whom correspondence should be addressed: Dept. of Pathology & Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. Tel.: 44-117-9287530; Fax: 44-117-9287896; E-mail: B.Kenny{at}Bristol.ac.uk.
1 The abbreviations used are: EPEC, enteropathogenic E. coli; Tir,
Translocated intimin receptor; C-Tir, C-terminal domain (residues
385550) of Tir; PKA, cAMP-dependent kinase, GdnHCl, guanidine
hydrochloride; CD, circular dichroism; MALDI-TOF, matrix-assisted laser
desorption/ionization time-of-flight; CHAPS,
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid.
2 A. Hawrani, C. E. Dempsey, M. J. Banfield, D. J. Scott, and B. Kenny,
unpublished data.
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REFERENCES |
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