From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218
Received for publication, December 9, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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F factor TraM is essential for efficient
bacterial conjugation, but its molecular function is not clear. Because
the physical properties of TraM may provide clues to its role in
conjugation, we have characterized the TraM oligomerization
equilibrium. We show that the reversible unfolding transition is
non-two-state, indicating the presence of at least one intermediate.
Analytical ultracentrifugation experiments indicate that the first
phase of unfolding involves dissociation of the tetramer into folded monomers, which are subsequently unfolded to the denatured state in the
second phase. Furthermore, we show that a C-terminal domain isolated by
limited proteolysis is tetrameric in solution, like the full-length
protein, and that its loss of structure correlates with dissociation of
the TraM tetramer. Unfolding of the individual domains indicates that
the N- and C-terminal regions act cooperatively to stabilize the
full-length protein. Together, these experiments suggest structural
overlap of regions important for oligomerization and DNA binding. We
propose that modulating the oligomerization equilibrium of TraM may
regulate its essential activity in bacterial conjugation.
Bacterial conjugation is a plasmid-mediated mode of lateral gene
transfer. F factor was the first conjugative plasmid described (1). For
successful F factor conjugation, the mating signal, which initiates DNA
transfer upon formation of a stable mating pair, must be transmitted
(2). TraM is a plasmid-encoded protein believed to be involved in
mating signal transmission (3-5). TraM mutants form stable mating
pairs and nick the plasmid at oriT (origin of
transfer), but DNA transfer does not occur (4). Although
the identity of the mating signal is unknown, the signal is mediated by
the conjugative pilus and requires cell-cell contact (6). Furthermore,
signal transmission requires a step in addition to the interaction of
the pilus with the recipient cell (2).
F factor TraM can interact with other F factor proteins and plasmid
DNA, and its DNA-binding activity correlates with its in
vivo function. TraM binds to three sites in oriT
(7-10). The two highest affinity sites, sbmA and
sbmB, overlap with the two traM promoters
and negatively autoregulate the expression of TraM (11-13). Deleting
the oriT region that includes sbmA and
sbmB from a mobilizable plasmid decreases its transfer
efficiency by 100-fold (14). Further deletion of a region including
sbmC, the lowest affinity site, from this plasmid reduces
the efficiency of mobilization by another 100-fold (14). In similar
experiments with the oriT of F-like plasmid R100,
mutations in the site analogous to sbmC inhibited transfer
(15). These experiments indicate that TraM binding at sbmC
may be required for its essential role in conjugal DNA transfer. In
addition to binding DNA, TraM associates with the plasmid-encoded inner
membrane protein TraD in vitro (16). TraD is believed to be
involved in transferring the single-stranded plasmid through the
transfer pore to the recipient cell (5). The ability to interact with
oriT DNA and a membrane protein suggests that TraM may
function to tether the plasmid near the site of DNA transfer.
It is not clear how the putative tether function of TraM is important
in transmitting the mating signal. The TraM tether may be formed in
response to the mating signal, implying that the ability of TraM to
interact with oriT or TraD is modulated by the mating
signal. In vitro, TraM binds to DNA as a tetramer (17) and
to the three oriT sites cooperatively (9). Therefore, small changes in the cellular concentration of tetrameric TraM in response to
the mating signal could alter the occupancy of the lowest affinity binding site, sbmC. It is unlikely that the concentration of
TraM in the cell is increased transcriptionally because expression is
negatively autoregulated. Furthermore, conjugation efficiency is not
affected by inhibitors of RNA or protein synthesis (4), suggesting that
transcription and translation are not required for mating signal transmission.
We have performed a thermodynamic characterization of TraM
oligomerization and stability, a necessary first step toward
understanding how TraM might respond to the mating signal. We report
the first reversible equilibrium unfolding of TraM; and based on these
and other data, we propose that TraM unfolds via a folded monomeric intermediate. Moreover, we show that the DNA-binding and
oligomerization activities of TraM cannot be structurally separated.
The thermodynamic characteristics of TraM and the interaction between
structures that are important for DNA binding and oligomerization
suggest a mechanism for regulating the activity of TraM in
vivo.
Materials and Bacterial Strains--
Guanidine hydrochloride
(GndHCl)1 (Mallinckrodt
Chemical Works) solutions were prepared fresh and filtered (0.45 µm).
Chromatography supplies and the Gradifrac system were from Amersham
Biosciences. Oligonucleotides were synthesized by Integrated DNA
Technologies and used without further purification. DNA sequencing was
performed by the DNA Analysis Facility, mass spectrometry by the
Applied Biosystems-Mass Spectrometry Facility, and N-terminal
peptide sequencing by the Synthesis and Sequencing Facility, all of The Johns Hopkins University School of Medicine. Standard molecular biology
procedures were performed as described (18).
The bacterial strains used were BL21(DE3) (19), BL21(DE3)/pLysS
(Novagen), ER2738 and TB1 (New England Biolabs Inc.), JM109 (20), and
DY330 (21). DY330 F'ER was constructed by mating DY330 with
F' strain ER2738. The TraM open reading frame of DY330 F'ER
was replaced with a kanamycin cassette by homologous recombination as
described (21) to create DY330 F'ER M::kan. The
cassette was amplified by PCR from pET24a+ (Novagen).
Clones--
The TraM open reading frame was amplified from JM109
genomic DNA by PCR with primers that incorporated appropriate
restriction sites (22) and cloned into
NdeI/XhoI-digested pET21a+. For pET-TraM, the
reverse PCR primer included a stop codon; but for
pET-TraM-His6, the primer lacked a stop codon, which
allowed for expression with a C-terminal hexahistidine tag. The
N-terminal peptide (pET-NTP) and C-terminal domain (pET-CTD) clones
were constructed by removing unwanted sequence by PCR mutagenesis (22).
This procedure resulted in the addition of an N-terminal methionine to
the CTD open reading frame.2
Protein Overexpression and Purification--
Plasmids were
transformed into BL21(DE3) or BL21(DE3)/pLysS. To test for protein
overexpression, several colonies were individually transferred to 1 ml
of LB medium with 60 µg/ml ampicillin (LB/ampicillin) and grown to
A600 ~ 0.4. The culture was split, and protein
expression was induced in one-half for 3 h by adding
isopropyl-1-thio-
TraM and NTP were further purified by ion-exchange chromatography.
Buffer was generally kept on ice, especially for the purification of
NTP, to minimize degradation of the protein. Dialyzed samples were
centrifuged at 15,000 × g for 10 min and loaded onto a
5-ml HiTrap heparin column equilibrated in PNE buffer with 5 mM DTT (PNED buffer). A 100-ml linear gradient to 2 M NaCl was applied. Fractions enriched in the desired
protein were pooled, diluted 1:5 with PNED buffer, and applied to a
5-ml HiTrap Blue column equilibrated in PNED buffer. A 75-ml linear
gradient to 2 M NaCl was applied, and the purity of peak
fractions was assessed by SDS-PAGE. When necessary, one or both of
these two chromatography steps were repeated. The protein in the final
pooled fractions was concentrated by ammonium sulfate precipitation
when appropriate. Purified protein was dialyzed extensively against
PIPES buffer (25 mM PIPES (pH 7.0), 150 mM
NaCl, and 0.1 mM EDTA) or phosphate buffer (25 mM NaPO4 (pH 7.5), 150 mM NaCl, and
0.1 mM EDTA).
For purification of CTD, dialyzed cell extracts were cleared by
filtering (0.45 µm) and loaded onto a 5-ml HiTrap Q column equilibrated in PNED buffer. Protein was eluted with a 100-ml linear
gradient to 2 M NaCl. Enriched fractions were combined and
run over a 5-ml HiTrap heparin column followed in series with a 5-ml
HiTrap Blue column. The flow-through was collected and dialyzed
overnight against 4 liters of PNED buffer. The HiTrap Q column step was
repeated, and the concentrated protein (3-5 ml) was loaded onto a
Sephacryl S-200 HiPrep 16/60 size-exclusion column equilibrated in
PIPES buffer at room temperature and eluted at 0.2 ml/min.
TraM-His6 was purified by batch loading the soluble
fraction of sonicated cells onto Ni2+-NTA-agarose (QIAGEN
Inc.). The column was washed with 5 mM and then 20 mM imidazole in phosphate buffer with 1 mM DTT,
and protein was eluted with 600 mM imidazole in phosphate
buffer with 1 mM DTT. Imidazole was removed from the
protein solution by dialysis. Protein concentration was measured by
absorbance at 280 nm with extinction coefficients of 3960 M GndHCl-induced Unfolding--
Stock buffer solutions were
filtered (0.45 µm) before use. Concentrated protein solutions were
used within 2 weeks of purification and were centrifuged on the day of
the experiment for 1 h at 55,000 rpm in a Beckman Optima TL
ultracentrifuge using a TLA-55 rotor at 4 °C. Native and unfolded
(in 4 M GndHCl) protein samples were diluted to the
indicated concentrations immediately prior to the titration. When
indicated, sbmA DNA (5'-CGCTAGGGGCGCTGCTAGCGGTGCGT-3', annealed to its complement) (3, 9) was included at an equal molar ratio
to the TraM tetramer. All solutions were allowed to equilibrate for 5 min before the first measurement was recorded. Protein and DNA
concentration remained constant throughout the titration. All data were
collected at 37 °C, and titrant was kept at 35 ± 2 °C
during the experiment.
For titrations monitored by intrinsic tyrosine fluorescence,
protein was in PIPES buffer to prevent potential interactions between phosphate and tyrosine (24, 25). Titrations were monitored with
a SLM-AMICO 48000 spectrofluorometer in the L-configuration equipped
with a Neslab circulating water bath and magnetic stirrer. Polarizers
were set to the magic angle. The sample was excited at 275 nm (4-nm
excitation slit width), and emission was monitored at 302 nm (16-nm
emission slit width). Each fluorescence intensity measurement is the
average of at least 200 data points acquired over 2-5 s, and each data
point was averaged five times. The titration was performed manually as
described (26). The sample was allowed to equilibrate with stirring for
at least 2 min before each measurement.
Titrations monitored by CD were performed in PIPES or phosphate buffer
with similar results. Data from unfolding of full-length TraM were
acquired on an Aviv 62A DS spectropolarimeter with a computer-controlled Hamilton Microlab 500 titrator with two 500-µl syringes (27, 28). The samples were stirred for 90 s, and then
data were averaged for 30 s. This was sufficient to achieve equilibrium, as indicated by the stability of the CD signal. Manual titrations were performed on a Jasco J-710 spectropolarimeter equipped
with a PTC-348WI thermostat. The procedure was as described for the
fluorescence measurements, except that each measurement is the average
of 600 data points acquired over 30 s. The S.D. of each
measurement is indicated by error bars on the graphs.
Spectroscopic data were fit using the nonlinear least-squares analysis
curve-fitting function of Kaleidagraph Version 3.51. A signal-weighted
two-state model was employed (26, 29, 30). A similar three-state model
was derived to fit the observed spectroscopic signal
(Yobs) when appropriate.
Yobs is the sum of the fraction of each species
(native, fN; intermediate,
fI; and unfolded, fU) multiplied by its contribution to the signal
(YN, YI, and
YU) (Equation 1).
DNA Dissociation Titration--
The dissociation of
sbmA DNA (2.5 µM in PIPES buffer) upon
addition of GndHCl was monitored by absorbance at 260 nm with a Beckman
DU-70 spectrophotometer equipped with a temperature-controlled cell
holder. DNA was diluted into the buffer at 37 °C and allowed to
equilibrate for 5 min before each measurement.
Analytical Ultracentrifugation--
Protein solutions in PIPES
buffer with various concentrations of GndHCl and matching buffer
solutions were centrifuged at 55,000 rpm in a Beckman Optima TL
ultracentrifuge at 4 °C for at least 1 h. The top half of each
solution was removed for use in analytical ultracentrifugation
experiments. Sedimentation velocity analytical ultracentrifugation
(SV-AUC) experiments were performed in a Beckman XL-I analytical
ultracentrifuge using absorbance optics and an An-60Ti rotor.
All runs were performed at 35 °C and 60,000 rpm in cells with
charcoal-filled Epon 12-mm double-sectored centerpieces and sapphire
windows. The rotor and cells were prewarmed. Radial absorbance data
were collected at 275 nm at a nominal point spacing of 0.003 cm with no
averaging in continuous scan mode.
Weight average sedimentation coefficients
( DNA Binding Interference Assay--
TraM, preincubated with NTP
or CTD, was combined with sbmA DNA. Electrophoretic mobility
shift assays were performed essentially as described (22), except that
equilibration was at 37 °C.
Mating Assays--
DY330 F'ER and DY330
F'ER M::kan were transformed with pET21a+ (empty
vector), pET-TraM, pET-NTP, or pET-CTD by electroporation. Overnight
cultures from single colonies were diluted 1:100 into medium containing
all appropriate antibiotics and regrown to mid-log phase. Donor cells
(150 µl) were spun down and resuspended in 100 µl of warm LB
medium, and then 900 µl of mid-log phase TB1 recipient cells were
added. Mating cultures were incubated for 2 min at 37 °C without
agitation. Conjugation was interrupted by vigorous vortexing, and then
cells were incubated on ice for 10 min. Serial dilutions in cold
sterile phosphate-buffered saline (18) were plated to select for donors
and transconjugants. All mating experiments were repeated with
at least three independent donor clones.
Limited Proteolysis--
TraM or TraM-His6 was mixed
with trypsin at a molar ratio of 75:1 and incubated at room
temperature. Aliquots were removed at hour intervals, and the reactions
were stopped by addition of phenylmethylsulfonyl fluoride to 2 mM. The fragments generated were separated by
electrophoresis on an SDS-polyacrylamide gel (15% acrylamide) and
visualized by staining with Coomassie to monitor extent of digestion to
ensure that both proteins generated the same proteolysis pattern. The
C-terminal proteolytic fragments of TraM-His6 were purified
after 4 h. The reaction was stopped with phenylmethylsulfonyl
fluoride and mixed for 10 min at 4 °C with 25 µl of
Ni2+-NTA-agarose equilibrated in phosphate buffer with 5 mM Ni2+-NTA Coelution Assay--
TraM-His6
(5 µM) was combined with 15 µM TraM, NTP,
or CTD in 50-µl reactions in PIPES buffer with 10 mM DTT
and incubated for at least 30 min at 37 °C. Negative controls
included all components except TraM-His6. Each reaction was
mixed with 25 µl of Ni2+-NTA-agarose at room temperature
for 5 min; the agarose was washed once with 0.5 ml of PIPES buffer; and
then bound protein was eluted in 50 µl of PIPES buffer with 0.5 M imidazole. Protein that did not bind to
Ni2+-NTA-agarose (flow-through) was compared with retained
species by SDS-PAGE. The gels were scanned, and the band intensities
were quantitated with the FluorChem imaging system (Version 2.00)
controlled by AlphaEase FC software (Alpha Innotech Corp.). The
background was subtracted from each band, and the coelution efficiency
was calculated as the ratio of eluted test protein to
TraM-His6 in the same lane.
GndHCl-induced Unfolding of F Factor TraM--
The
reversible unfolding reaction of TraM was followed by CD and intrinsic
tyrosine fluorescence (Fig. 1). CD is a
measure of secondary structure, whereas tyrosine fluorescence can
report on close tertiary interactions, hydrogen bonding of the
fluorophore, and secondary structure. The GndHCl-induced unfolding
transition is biphasic, with an apparent intermediate state populated
near 1 M GndHCl. The congruence of the curves when
monitoring independent probes is consistent with each transition
observed in the unfolding curve being two-state (36). This suggests
that the full unfolding transition is three-state, involving only one
intermediate. These experiments were performed at 37 °C because the
unfolding transition at 20 °C exhibited pronounced hysteresis (data
not shown).
The equation describing a three-state model includes too many
parameters relative to the number of data points to obtain reliable fits. Therefore, the slope and intercept of the base line at high denaturant concentration were determined by fitting data from this
region directly to a linear equation, and these values were fixed
during subsequent fitting. It was not necessary to constrain the
shorter native base line in this manner. By fitting the constrained model to the CD data, we calculated that the overall stability of 10 µM TraM at 37 °C is 4.7 kcal mol
Although the unfolding data indicate two transitions in TraM unfolding,
we do not know how each transition reflects events of the unfolding
pathway. To determine which phase of TraM unfolding involves a change
in oligomerization, we performed the unfolding titrations of TraM at
protein concentrations of 1-45 µM (Fig. 2). 10 µM TraM had a
greater molar ellipticity at 222 nm than 1 µM, showing
that TraM is stabilized by increasing the protein concentration.
Although the offset between the two curves makes them difficult to
compare, the first transition began at a slightly lower concentration
of denaturant at the lower protein concentration, suggesting that the
first transition has shifted. Neither transition of the
fluorescence-monitored unfolding was dramatically shifted when the
concentration of TraM was raised from 10 to 45 µM. We also did not see a shift in either unfolding transition when
sbmA, the highest affinity binding site, was included in the
reaction. At this temperature and concentration, the midpoint of the
GndHCl-induced dissociation of sbmA is located at ~0.5
M (data not shown).
SV-AUC Analysis of TraM Oligomerization--
To directly evaluate
how the oligomeric nature of TraM changes during unfolding, we
performed SV-AUC at different concentrations of denaturant. The data
from these experiments can be used to determine the molecular weight of
the sedimenting species based on hydrodynamic models and can also be
used to monitor changes in oligomerization (37-40). We were not able
to perform complementary equilibrium analytical ultracentrifugation
because TraM aggregation occurred before the sample reached
equilibrium. The data for SV-AUC experiments were collected under
conditions similar to the unfolding of 45 µM TraM shown
in Fig. 2. This was the lowest protein concentration that could be
detected with the absorbance optics at 275 nm.
To determine whether the first transition involves a change in
oligomerization, we measured the sedimentation of TraM at 0.5, 0.75, and 1 M GndHCl (Fig. 3),
denaturant concentrations that span the first unfolding transition.
Visual examination of the curves suggests that, even at the lowest
concentration of GndHCl, the boundary shape is not symmetrical. This is
consistent with the solution containing a heterogeneous mixture of
species. As the concentration of GndHCl was increased, the distribution
first became more asymmetric, and then the peak shifted to the left. This translation of the g(s*) distribution toward
lower s* values is consistent with a change in
oligomerization concurrent with the first phase in the unfolding. The
g(s*) representation of the data shown is
model-independent and provides a qualitative comparison of TraM
sedimentation as a function of unfolding (34).
We performed a more rigorous, quantitative analysis by determining the
weight average sedimentation coefficient
( Isolating Structural Domains of TraM--
Using limited
proteolysis, we generated a stable C-terminal fragment beginning at
TraM residue 58 (data not shown). Expression vectors for this
C-terminal domain (pET-CTD) and the remaining N-terminal peptide
(pET-NTP) were constructed. The result from mass spectrometry of
purified CTD matched the value predicted from the DNA sequence (8100;
predicted molecular weight of 8103). However, mass spectrometry and
N-terminal peptide sequencing of purified NTP indicated that it does
not include the initial methionine and the final two residues coded for
in the genetic construct.
To determine whether the two phases observed in the full-length
denaturation experiments correlate with sequential unfolding of these
domains, we analyzed the unfolding of the individual domains (Fig.
4). Although the midpoint of the NTP
transition is similar to the second transition in the full-length
protein, both
The unfolding transition of CTD was completed by 0.5 M
GndHCl. At this concentration of denaturant, there was no observed loss
of TraM structure. The native base line in these unfolding curves is
not well defined, which makes fitting these data difficult. The
unfolding of 45 µM CTD was shifted relative to 10 µM, consistent with it being oligomeric. When the
two-state model was fit to the 45 µM data,
We used SV-AUC to determine whether CTD and NTP are oligomeric in
solution (Fig. 5). The concentration of
protein in these experiments (125 µM) was higher than for
the full-length protein (45 µM) to achieve sufficient
signal for data collection. These data indicate that CTD is a tetramer
in solution, consistent with the concentration dependence of the
unfolding curve. The SV-AUC data also suggest that NTP is dimeric in
solution. These data are at odds with the observation that the NTP
unfolding curve was not shifted when the initial protein concentration
was changed. Together with the unfolding data, these experiments show
that CTD structures are sufficient for tetramerization, although
N-terminal residues may be involved in stabilizing the tetramer.
Functional Analysis of Domains--
Our thermodynamic experiments
indicate that CTD and NTP act cooperatively to stabilize the TraM
protein. We proceeded next to see if both domains are required in
vivo for the function of TraM. Indeed, only the
full-length protein (and neither CTD nor NTP) was able to complement a
traM deletion mutant (Table
I). We also tested the effect of
including each construct in trans to a wild-type F' episome.
The transfer efficiency of DY330 F'ER was slightly, but
reproducibly, decreased in the presence of CTD, but not TraM or NTP.
This observed dominant-negative effect could indicate that CTD is
included in tetramers with the full-length protein in vivo
and that these tetramers are not active.
To determine whether the fragments interact with TraM in
vitro, we monitored the coelution of the untagged species with
TraM-His6 from Ni2+-NTA-agarose (Fig.
6). The concentration of protein in these
experiments was much lower than in the SV-AUC experiments. Both
fragments coeluted with TraM-His6, albeit at lower
efficiency than with TraM, although a genetic interaction was seen only
for CTD. We used a gel-retardation assay to address whether the
observed interactions affect the DNA-binding activity of TraM (Fig.
7). Although no in vivo effect
of NTP expression was observed, this fragment reduced the ability of
TraM to bind DNA. Conversely, the interaction of CTD with TraM did not
significantly affect its DNA-binding properties.
We have established conditions for the first reported reversible
unfolding of the TraM protein, allowing us to describe its stability
and oligomerization. Unfolding experiments were performed at 37 °C,
a temperature at which the unfolding transition is relatively free of
hysteresis, to allow for application of thermodynamic models. Moreover,
this is the physiological temperature at which TraM functions. Although
calorimetry experiments with R1 TraM (17) indicate that, at 37 °C,
the protein shows some structural alteration, this thermal unfolding
reaction is irreversible. In our equilibrium experiments, the
GndHCl-induced unfolding curve of TraM at 37 °C exhibits a well
defined base line at low concentrations of denaturant, which suggests
that the protein is in a native folded state under these conditions.
Our characterization of TraM in these experiments defines a
thermodynamic reference state that can be used in future studies to
correlate observed phenotypes with defects in stability or
oligomerization. The more precise characterization of TraM mutant
phenotypes could help further elucidate the molecular role of TraM in
mating signal transduction.
We have shown that monomeric and tetrameric forms of TraM are in
equilibrium in solution. The existence of a folded monomeric species
suggests that multiple forms of TraM may coexist inside a cell without
being degraded. The three-state unfolding of F factor TraM we report is
consistent with the observation that the irreversible thermal unfolding
of TraM from F-like plasmid R1 is non-two-state (17). We calculate
that, at 10 µM TraM, the energy difference between the
monomeric and tetrameric states is on the order of 2.5 kcal
mol The equilibrium between tetramer and monomer is two-state, indicating
that no other folding intermediates exist. These results are consistent
with studies of R1 TraM that show DNA binding only as a tetramer (17),
but argue against a model in which TraM initially binds to DNA as a
dimer (9). Interestingly, SV-AUC indicates that NTP is dimeric in
solution, although unfolding experiments suggest that it is not
oligomeric. The discrepancy between the unfolding and SV-AUC
experiments may indicate that dissociation of the NTP dimer does not
perturb the structure in a manner that can be detected by either CD or
intrinsic tyrosine fluorescence. An analogous fragment of R1 TraM,
TraMM26, was also shown to be dimeric by gel filtration (17), although
it was monomeric under conditions used to solve the NMR structure (42). The observation that TraM does not form dimers suggests that NTP structure may contribute to the stability of the TraM tetramer, but
is not sufficient to support oligomerization alone.
Our domain studies indicate that structures important for
oligomerization and DNA binding overlap. Although CTD is able to act as
a tetramerization domain, the TraM tetramer is more stable than the
isolated CTD tetramer, consistent with the assertion that N-terminal
structures influence the stability of the tetramer. Furthermore, it has
been shown that NTP contains residues that are important for DNA
binding (11, 43, 44), although neither this fragment nor CTD can
reproduce the DNA-binding activity of TraM. This suggests that the
DNA-binding domain of TraM includes structures that are involved in, or
stabilized upon, oligomerization. Consistent with this hypothesis, the
R1 TraM homolog is stabilized upon the addition of DNA (17).
We have shown that, in vitro, both NTP and CTD can interact
with full-length TraM. Although we have not ruled out the possibility of other types of interactions, it is possible that the constructs are
incorporated into tetramers with the full-length protein. Our
experiments demonstrate that, in vitro, CTD does not
interfere with TraM-DNA binding, although it does show a
dominant-negative interaction in mating experiments. We suggest that,
in vivo, CTD interferes with an essential function of TraM
that is separate from DNA binding. For example, it is possible that the
TraM-TraD interaction is disrupted in these cells. In contrast, NTP
interferes with the ability of TraM to bind DNA, although no genetic
interaction was observed. This apparent contradiction can be explained
if the interaction relieves the negative autoregulation of
traM. The increased expression of TraM would interfere with
any dominant-negative effect on conjugation.
We propose that the mating signal could modulate the oligomerization
equilibrium of TraM. In this model, the negative autoregulation of TraM
keeps the intracellular protein concentration at a level such that only
sbmA and sbmB, the high affinity binding sites, are occupied by TraM tetramers. After successful mating pairs have been
formed, the mating signal shifts the oligomerization equilibrium, and
the concentration of TraM tetramers is increased. This equilibrium
shift results in occupation of the sbmC binding site,
thereby allowing for DNA transfer to begin. Our thermodynamic analysis
of TraM oligomerization indicates that this model is consistent with
the physical properties of TraM. Future studies will determine whether
the activity of TraM is regulated by its oligomerization in
vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside to 1 mM, and the other half was kept on ice. Protein expression was assayed by SDS-PAGE, and the reserved portion of the culture that
demonstrated the best expression was diluted to 20 ml with LB/ampicillin, regrown to A600 ~ 0.5, and then
used to inoculate 2 liters of LB/ampicillin. When this culture reached
mid-log phase, isopropyl-1-thio-
-D-galactopyranoside was
added to 1 mM. After 3 h, cells were harvested by
centrifugation and stored at
80 °C. For protein purification,
500-ml cell pellets were resuspended in 25-50 ml of 50 mM
Tris-HCl (pH 7.5), 0.1 mM EDTA, 50 mM NaCl, 5 mM dithiothreitol (DTT), and 1 mM
phenylmethylsulfonyl fluoride and disrupted by sonication. The soluble
fraction was cleared by centrifugation; ammonium sulfate (0.25 g/ml)
was added; and precipitated protein was removed by centrifugation. The
remaining soluble protein was precipitated by adding ammonium sulfate
to saturation and collected by centrifugation. Protein was resuspended in 35 ml of PNE buffer (25 mM NaPO4 (pH 7.5),
25 mM NaCl, and 0.1 mM EDTA) with 1 mM DTT and dialyzed overnight against 4 liters of the same buffer.
1 cm
1 for TraM and
TraM-His6, 2560 M
1
cm
1 for NTP, and 1400 M
1
cm
1 for CTD (23).
The spectroscopic signal associated with each species
(YN, YI, and
YU) varied linearly with denaturant
concentration. K1 and K2
are equilibrium constants for the first and second steps of the
three-state equilibrium, respectively. According to the linear
extrapolation model (29), each K can be expressed as a
function of the standard state equilibrium constant,
K0, and the m-value.
(Eq. 1)
G0 was calculated from the fit
K0 values. An unfolding curve was determined to
exhibit hysteresis if the forward and reverse curves were reproducibly
offset greater than the error in the measurement.
s*20,w
), extrapolated to
t = 0, were calculated with the program
DCDT+ Version 1.14 (31, 32). Solvent density, viscosity, and
the buoyant molecular weight of the protein species were calculated with SEDNTERP Version 1.06 (33). Data for CTD and NTP were analyzed by
fitting to the modified Fujita-Machosham function using the dc/dt mode of SVEDBERG Version 6.39 (32, 34, 35),
which is better suited for small molecules. In all analyses, 14-20
scans were included in the fit. Goodness of fit was evaluated by
randomness in residuals, reduced
2, and visual
examination of the fits overlaid onto the experimental data. Regions of
the data that showed signs of meniscus effects or that indicated
accumulation at the bottom of the cell were excluded from the fits,
although, in the figures, the fit values are extended over the entire
s*20,w range.
-mercaptoethanol. The Ni2+-NTA-agarose
was washed once, and bound protein was eluted in phosphate buffer with
5 mM
-mercaptoethanol and 600 mM imidazole. Eluted protein was run on an SDS-polyacrylamide gel; stained with Coomassie; and then transferred to Hybond-P membrane (Amersham Biosciences) in 20 mM CAPS (pH 10), 1 mM DTT,
and 10% methanol at 300 V for 8 h at 4 °C. Bands were excised
from the membrane and subjected to N-terminal peptide sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TraM (10 µM) was
unfolded by titration with GndHCl at 37 °C. The transition was
monitored by CD (upper panel) or intrinsic tyrosine
fluorescence (middle panel). The unfolding reaction is
represented by open symbols, and refolding by closed
symbols. The line through the CD data shows
the constrained three-state model fit to the unfolding data. From this
fit, G1 = 2.7 ± 0.2 kcal
mol
1, m1 = 3.7 ± 0.4 kcal
mol
1 M
1 GndHCl,
G2 = 2.0 ± 0.3 kcal mol
1,
and m2 = 1.1 ± 0.1 kcal mol
1
M
1 GndHCl. The lower panel shows
normalized data (45) plotted on the same axes (
, CD;
,
fluorescence), demonstrating that both probes used to monitor the
unfolding report very similar curves, with an intermediate populated
near 1 M GndHCl. Error bars in the
upper and middle panels (and in other figures)
are the S.D. of the data points averaged for each measurement. In the
upper panel, the error bars are hidden by the
data markers. Linear correlation coefficient (R) values from
the fit are indicated on the graph. deg, degrees;
res, residue.
1. The
first transition (
G1 = 2.7 kcal
mol
1, m1 = 3.7 kcal
mol
1 M
1 GndHCl) results in a
greater decrease in free energy than the second transition
(
G2 = 2.0 kcal mol
1,
m2 = 1.1 kcal mol M
1
GndHCl). The fit of fluorescence data with the three-state model did
not converge. This is likely due to a lower signal-to-noise ratio in
these data. However, when the
G and m-values
derived from the CD fits were fixed, the fluorescence data were
reasonably well fit by the three-state model (data not shown).
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Fig. 2.
TraM at different initial concentrations was
unfolded as described in the legend for Fig. 1. The upper
panel shows unfolding of 1 µM ( ) and 10 µM (
) TraM monitored by CD. The more negative starting
value at higher protein concentration shows that TraM is stabilized at
increased concentrations. The middle panel shows the
fluorescence-monitored unfolding of 10 µM (
) and 45 µM (
) protein. The fluorescence data were normalized
so that both titrations had the same starting value. At all
concentrations tested, the shape of the curve is the same, and neither
transition midpoint varies dramatically. In the lower panel,
10 µM TraM was unfolded in the absence (
) or presence
(
) of 2.5 µM sbmA. deg, degrees;
res, residue.
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Fig. 3.
SV-AUC of TraM as a function of GndHCl
concentration. Upper panel,
g(s*) distribution from sedimentation of 45 µM TraM in 0.5 M ( ), 0.75 M
(+), or 1.0 M (
) GndHCl is shown. Each
g(s*) distribution was derived from 14-16 radial
absorbance scans at approximately the same
2t
and was area-normalized (37). As the concentration of GndHCl was
increased, the peak in the g(s*) distribution
became more asymmetric and shifted to lower s* values. The
beginning and end points of the shift are marked with vertical
lines. Lower panel, the weight average sedimentation
coefficient is plotted as a function of GndHCl concentration (
). The
two-state model was fit to these data to derive
Gtet(app) = 2.9 ± 0.8 kcal
mol
1 and mtet(app) = 3.1 ± 2.3 kcal mol
1 M
1 GndHCl.
AU, absorbance units.
s*20,w
) as a function of
denaturant concentration, shown in Fig. 3. The
s*20,w
represents the equilibrium
distribution of all species, regardless of the rate of interconversion,
as long as the system is at equilibrium at the beginning of the run
(41). In our experiments, the protein samples were incubated in the cell at 35 °C for 20-45 min before the experiment was initiated. When native TraM was diluted into 2 M GndHCl at this
protein concentration, the fluorescence-monitored unfolding was
complete within 3 min (data not shown). The
s*20,w
data indicate that the change in oligomerization that occurred upon unfolding of TraM was
completed by 1.5 M GndHCl under these conditions. We fit
the two-state model to the
s*20,w
data to derive the apparent tetramerization energy
(
Gtet(app)). From this fit, we determined
Gtet(app) to be 2.9 kcal mol
1
and mtet(app) to be 3.1 kcal mol
1
M
1 GndHCl. These values agree well with
G1 (2.7 kcal mol
1) and
m1 (3.7 kcal mol
1
M
1 GndHCl) from the chemical unfolding,
providing further evidence that the dissociation of the native tetramer
is two-state. Together, these data indicate that the first phase in the
unfolding of TraM involves dissociation of the native tetramer to a
monomeric form.
GNTP (3.0 kcal
mol
1) and mNTP (2.0 kcal
mol
1 M
1 GndHCl) are greater
than the corresponding
G2 (2.0 kcal
mol
1) and m2 (1.1 kcal mol
M
1 GndHCl). The unfolding of 45 µM NTP overlays the data collected with 10 µM protein, suggesting that this protein does not undergo a change in oligomerization upon unfolding. Furthermore, unlike TraM,
the molar ellipticity of NTP did not change when the protein concentration was increased (data not shown).
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Fig. 4.
Unfolding of NTP and CTD. A,
the CTD unfolding transition was monitored by fluorescence (left
panel, 10 µM (diamonds) and 45 µM (circles)) and CD (right panel,
10 µM (squares)). The unfolding curve is
shifted to the right at higher protein concentrations, suggesting that
this domain is oligomeric. At 45 µM,
GCTD(F) = 2.2 ± 0.1 kcal
mol
1 and mCTD(F) = 3.6 ± 0.8 kcal mol
1 M
1 GndHCl. The
m-value derived from the fit to the data at 45 µM was fixed in the fits of the data at 10 µM to compensate for the short native base line in
these unfolding curves. For 10 µM CTD, the fit of
the CD data (right panel) indicates that
GCTD(CD) = 0.9 ± 0.4 kcal
mol
1, and the fluorescence data (left panel)
yield
GCTD(F) = 0.6 ± 0.4 kcal
mol
1. B, NTP unfolding was also monitored by
fluorescence (left panel, 10 µM
(inverted triangles) and 45 µM
(circles)) and CD (right panel, 10 µM (triangles)). The unfolding curve of NTP is
not sensitive to the initial protein concentration. At 10 µM NTP, the fit to the fluorescence data (left
panel) indicates that
GNTP(F) = 3.0 ± 0.2 kcal mol
1 and mNTP(F) = 2.0 ± 0.1 kcal mol
1 M
1
GndHCl. The fit to the CD data (right panel) is very similar
(
GNTP(CD) = 3.0 ± 0.6 kcal
mol
1 and mNTP(CD) = 2.1 ± 0.3 kcal mol
1 M
1 GndHCl). The
unfolding reaction is indicated by open symbols, and
refolding by closed symbols. Lines through the
data points are the two-state model fit to the unfolding data.
mdeg, millidegrees; res, residues.
GCTD(45) = 2.0 kcal mol
1 and
mCTD(45) = 3.6 kcal mol
1
M
1 GndHCl. The m-value derived
from these fits is very similar to m1 (3.7 kcal
mol
1 M
1 GndHCl) of the
full-length protein. Because the m-value reflects the amount
of surface area exposed upon unfolding, which should be the same at
both protein concentrations, we fixed this parameter in fits of the
two-state model to the data collected at 10 µM. In these
fits,
GCTD (0.6-0.9 kcal mol
1)
is lower than
G1 (2.7 kcal
mol
1). These experiments suggest that loss of CTD
structure coincides with dissociation of the tetramer.
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Fig. 5.
SV-AUC data of isolated domains (125 µM) were fit using the dc/dt mode
of SVEDBERG. Each panel shows the one-species model fit
(solid lines) overlaid on one data set (the difference
between two scans); however, the fit was generated starting with 12-14
scans after the boundary had moved almost halfway down the cell. CTD
(upper panel) is tetrameric in this fit, and NTP
(lower panel) is dimeric.
Mating assay data
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Fig. 6.
Ni2+-NTA coelution assay shows
that both NTP and CTD can interact with the full-length protein.
The indicated protein (15 µM) was combined with
TraM-His6 (5 µM) and equilibrated at
37 °C. The mixtures were then added to Ni2+-NTA-agarose,
which retained TraM-His6. The arrow and
arrowheads indicate the extent of migration for
TraM-His6 (TraMH6), TraM (M), CTD
(C), and NTP (N). The label above each lane
indicates which protein was equilibrated with TraM-His6.
The three lanes labeled ELUTE show proteins retained on
Ni2+-NTA-agarose, and this coelution is quantitated in the
graph. Both CTD and NTP were retained with TraM-His6,
suggesting that they can interact with the full-length protein. The
three lanes labeled FT (flow-through) show proteins that
were not bound to Ni2+-NTA-agarose.
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Fig. 7.
The ability of TraM (50 nM) to
bind DNA was measured after it was equilibrated with either CTD
(third through fifth lanes) or NTP
(seventh through ninth lanes). The
TraM:fragment molar ratio is indicated above each lane. TraM alone (50 nM; TraM lane), but neither NTP nor CTD (500 nM; NTP and CTD lanes), could bind
DNA. The appearance of free DNA (first lane,
arrow) in the lanes with TraM indicates that an interaction
with the fragment inhibited DNA binding by TraM. Equilibration of TraM
with NTP severely affected DNA binding, especially at a ratio of 1:10
(seventh lane, arrow).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 at 37 °C. This is only four times RT,
the thermal energy at this temperature. The concentration of TraM in
the cell is 2-3 orders of magnitude lower than in our experiments
(12), which would decrease the energy difference between the monomer
and tetramer. However, there are also factors in vivo that
could stabilize the tetramer, such as specific interaction with DNA or
other proteins or the cytoplasmic environment. The interplay of these
factors will ultimately determine which oligomeric state is favored in the cell, although our experiments demonstrate it is feasible that both
states are populated in vivo. TraM was not apparently stabilized by the presence of its DNA ligand in vitro (Fig.
2). However, in the absence of DNA, the first unfolding transition did
not begin until 0.5 M GndHCl, which is near the midpoint of the sbmA DNA dissociation transition. Furthermore, it is
likely that GndHCl can interfere with DNA binding by TraM. The effects of GndHCl on either DNA dissociation or TraM-DNA interactions could
prevent observation of TraM stabilization by a TraM·DNA complex.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Ludwig Brand for access to the fluorometer; Dr. Doug Barrick for discussions of thermodynamics and for use of the spectropolarimeter; and Drs. Beverly Wendland, Ernesto Freire, and Evangelos Moudrianakis for use of equipment. We are grateful to Dr. Michael E. Rodgers for guidance in analytical ultracentrifugation data collection and discussion of data and to Olivia Doyle and members of the Schildbach laboratory for comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB-9733655, American Cancer Society IRG 58-005-39, and National Institutes of Health Grant GM61017.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: Dept. of Biology, The
Johns Hopkins University, Mudd Hall 235, 3400 N. Charles St.,
Baltimore, MD 21218. Tel.: 410-516-0176; Fax: 410-516-5213; E-mail:
joel@jhu.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212502200
2 Primer sequences are available upon request.
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ABBREVIATIONS |
---|
The abbreviations used are: GndHCl, guanidine hydrochloride; CTD, C-terminal domain; NTP, N-terminal peptide; DTT, dithiothreitol; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); NTA, nitrilotriacetic acid; SV-AUC, sedimentation velocity analytical ultracentrifugation; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid.
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REFERENCES |
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