From the Departamento de Bioquímica, Facultad
de Medicina, Universidad Nacional Autónoma de México,
México City 04510, Mexico and § Max-Planck-Institut
für Biochemie, Martinsried 82152, Germany
Received for publication, August 23, 2000, and in revised form, December 23, 2000
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ABSTRACT |
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The effects of pH and temperature on the
stability of interdomain interactions of colicin B have been studied by
differential-scanning calorimetry, circular dichroism, and fluorescence
spectroscopy. The calorimetric properties were compared with those of
the isolated pore-forming fragment. The unfolding profile of the
full-length toxin is consistent with two endothermic transitions.
Whereas peak A (Tm = 55 °C) most likely
corresponds to the receptor/translocation domain, peak B
(Tm = 59 °C) is associated with the
pore-forming domain. By lowering the pH from 7 to 3.5, the transition
temperature of peaks A and B are reduced by 25 and 18 °C,
respectively, due to proton exchange upon denaturation. The isolated
pore-forming fragment unfolds at much higher temperatures
(Tm = 65 °C) and is stable throughout a wide
pH range, indicating that intramolecular interactions between the
different colicin B domains result in a less stable protein
conformation. In aqueous solution circular dichroism spectra have been
used to estimate the content of helical secondary structure of colicin
B ( Membrane proteins are characterized by water-exposed
extramembranous as well as intramembranous regions, which are embedded in the interior of the lipid bilayer. As a consequence, the energetics of stabilization of membrane-inserted proteins is expected to include
contributions from these two regions in different proportions. Whereas
the extramembranous region is more likely to be organized by forces
similar to those described for water-soluble proteins (1, 2), the
membrane-inserted regions are very stable because of considerable
strengthening of hydrogen-bonding interactions within the lipidic
milieu (3).
The transition of proteins from a soluble to a membrane-associated
state offers a new challenge for describing the energetics involved in
the process of membrane insertion, which are critical for the proper
folding of membrane proteins (3, 4). A key aspect in lipid-protein
interactions is the partitioning of the protein from water into the
membrane. The conformational changes, however, that are associated with
protein insertion into, transport across, and maintenance within
membranes remain poorly understood.
Pore-forming colicins are water-soluble proteins that insert into
membranes. Merely by offering a different environment and without the
need of chemical modifications, these proteins can be investigated as
well in solution as in their membrane-inserted state. Therefore, they
provide excellent model systems to study the structural changes during
membrane insertion. Pore-forming colicins (e.g. colicin A,
B, N, E1, and Ia) invade Escherichia coli cells by
parasitizing receptors of the outer membrane and use the Tol or Ton
protein complexes for translocation. In a final step these proteins
cause voltage-dependent pore formation. The three functions
correlate with three distinct domains of the polypeptide chain, which
are arranged along the primary sequence in the following order:
translocation, receptor recognition, and channel formation.
Here we study colicin B
(ColB),1 which is a 54.6-kDa
protein secreted by Escherichia coli as a monomeric unit.
Primary overall sequence comparison and functional studies indicate
that ColB is most closely related to colicins A, N, and Ia (5-7). It
has been suggested that, after binding to the target cell but before insertion occurs, the colicin pore-forming domains undergo
conformational changes to adopt an insertion-competent state. This
transition is induced in vitro by acidic pH. Membrane-active
colicins thereby exhibit close similarity to other proteins such as
diphtheria, tetanus, and botulinum toxins (8). Previous calorimetric
studies have contributed some understanding of the structural
transitions of the colicin A and colicin E1 pore-forming domains (9,
10). However, the conformational changes of full-length colicin B or its thermolytic fragments, which are necessary for membrane binding and
insertion, have so far not been characterized.
Macromolecules are stabilized by the cooperative action of numerous
weak forces. Because such highly cooperative structures undergo phase
transition-like conformational changes upon exposure to heat, relevant
thermodynamic, and structural information is obtained from
differential-scanning calorimetry (DSC) and circular dichroism (CD)
studies. Unlike the many calorimetric studies conducted with
water-soluble proteins, the stability of only a few membrane proteins
has been measured calorimetrically, and even less is known about the
thermal stability of proteins that insert into membranes (9-14).
Investigations of full-length colicin B as well as its C-terminal
fragment both in solution and the membrane-associated state allows for
a direct comparison of the thermal characteristics in these different
environments. In addition, by investigating the
pH-dependent properties of full-length colicin and its
pore-forming fragment, important information on the domain structure
and inter-domain interactions is obtained (1). The observed modulations
of thermal stability by interactions between domains and with the
membrane have important implications also for the insertion and
translocation competence of colicin B.
Preparation of ColB and ColB PFF--
ColB was expressed in
E. coli. The full-length protein as well as its thermolytic
fragment were purified following procedures described elsewhere (15,
16). This latter domain of the homologous colicin A protein has been
shown to exhibit pore-forming activities (e.g. Ref. 17) and
the thermolytic fragment is, therefore, abbreviated PFF.
SDS-polyacrylamide gel electrophoresis of ColB and its pore-forming fragment shows one single band in Coomassie Blue-stained gels.
Preparation of Lipid Vesicles--
All phospholipids were from
Avanti Polar Lipids (Birmingham, AL) and used without further
purification. Small unilamellar vesicles of POPC:cardiolipin:POPE
(5:1:2, w/w) were prepared from a homogeneous solution of the three
lipids in chloroform/methanol (1:1). The solvent was completely removed
with a stream of nitrogen and by exposure to high vacuum overnight.
Thereafter, buffer was added and the suspension was subjected to
several freeze-thaw cycles. A clear suspension of small unilamellar
vesicles was obtained after repeated extrusion first through 100-nm (5 times) and thereafter 50 nm polycarbonate filters (Avestin, Milsch,
Laudenbach, Germany). The lipid concentration for DSC, CD, and
fluorescence experiments was 50 mg/ml.
Differential-scanning Calorimetry--
DSC was used to measure the
transition temperatures (Tm) of full-length ColB
and its pore-forming fragment. Tm is defined as
the temperature at which the excess heat capacity is maximal. Lyophilized protein was dissolved in buffer (0.1 M KCl,
0.02 M glycine at pH 3.5-4.5; or 0.1 M KCl,
0.02 M tris-malate at pH 6.0-7.0), followed by dialysis
overnight against the same buffer. Excess heat (Cp)
versus temperature scans were obtained from 37 µM protein solutions using a high sensitivity
differential scanning calorimeter MicroCal VP-DSC. The sample and
reference solutions were carefully degassed under vacuum for 5 min
before loading the cells (0.56 ml). After equilibration of the system
at 10 °C, the temperature was increased to 100 °C and decreased
to 10 °C at scan rates of 1 °C/min. Thereafter another
10-100 °C scan was obtained. This rescan, which in all cases showed
no evidence of reversibility, was subtracted from the initial scan, and
the resulting baseline was corrected.
Circular Dichroism (CD) Spectropolarimetry--
CD spectra
(190-250 nm) were recorded on a Jasco J-715 spectropolarimeter using a
quartz cuvette of 0.1-mm path length. The protein concentration was 3 µM. For temperature scans at 222 nm, a heating rate of
1 °C/min was used (20-85 °C). CD spectra were recorded every
5-10 °C in the temperature range 20-80 °C as indicated in the
figures. The helicity of the peptides was determined quantitatively only for spectra recorded at 25 °C, because limited protein
aggregation was observed above the transition temperatures. All
measurements were corrected for buffer contributions. The lipid
concentration was kept constant.
Intrinsic Emission Fluorescence Measurements--
All emission
fluorescence experiments were performed using a PerkinElmer LS-50
luminescence spectrometer with a water-thermostat cell holder.
Excitation wavelengths of 290 nm were used, and the emission spectra
were recorded within the range 300-500 nm. The temperature dependence
of the fluorescence intensity was recorded at a constant heating rate
of 1 °C/min from 25-85 °C at 320 nm. The protein concentration
of ColB or its pore-forming fragment was 3 µM.
To test for the effect of pH on the thermal stability of ColB and
its pore-forming fragment in solution as well as in the presence of
lipids, the proteins were investigated in a
temperature-dependent manner by DSC, fluorescence, and CD spectroscopy.
Differential-scanning Calorimetry--
The temperature
dependencies of the excess molar heat capacity of the full-length ColB
between 10 and 100 °C are shown in Fig.
1 as a function of pH. The scans were
obtained at heating rates of 1 °C/min. Denaturation was
irreversible, because the transitions are absent during cooling or
reheating of the samples. In this study the "heat of transition,"
The heat absorbed during unfolding is obtained by integrating the area
under the fitted curves of each transition. Table
I lists the unfolding parameters
determined using linear and quadratic baselines in a multiple-curve
fitting routine of the DSC data. The calculated
To study the origin of the two transitions of ColB, a 203-amino acid
C-terminal fragment of ColB that contains the pore-forming domain was
also investigated. Fig. 4 shows the
temperature dependence of the excess molar heat capacity of the
pore-forming fragment at different pH values at a heating rate of
1 °C/min. Irreversible denaturation was observed after heating to
100 °C. The denaturation profile of the pore-forming fragment in
solution shows one main transition corresponding to 90% of the total
The denaturation profile of ColB in the presence of small unilamellar
lipid vesicles is shown in Fig.
6A. At pH 7.0 ColB exhibits the same two transitions observed during its unfolding in aqueous solution in agreement with weak membrane-association of the protein at
neutral pH. The Tm values of peaks A and B are
55.4 ± 0.4 °C and 57.9 ± 0.4 °C, respectively. At pH
3.5, however, peak A exhibits a Tm of 53.3 ± 0.9 °C and peak B is absent (Fig. 6B). In the presence of lipids the total
Fig. 6C shows the DSC profile of the pore-forming fragment
in the presence of lipids. The transition occurs over a wide
temperature range and CD Spectropolarimetry--
Far UV-CD spectra of ColB and the
pore-forming fragment in solution and in the presence of lipids at pH
7.0 display a maximum at 191 nm as well as minima at 208 and 222 nm
(Figs. 7C, 7D,
8B, and 8C). The CD line shapes are, therefore,
characteristic of a large proportion of
The relative intensity changes of the ellipticity at 222 nm, however,
were recorded as a function of temperature and compared with the DSC
thermal transitions (Fig. 7, A and B). Fig.
7A presents the temperature dependence of the ColB
ellipticity at 222 nm. At temperature scan rates of 1 °C/min
(25-85 °C) a single transition is observed for ColB at pH 7.0 with
Tm values of 56.4 °C and 54.6 °C in the
absence or presence of lipids, respectively (Fig. 7A). In
both experiments the ellipticity at 222 nm of ColB sharply decreases.
The corresponding full-wavelength scans (195-255 nm) are shown in Fig.
7 (C and D).
Fig. 7B shows the temperature dependence of the 222-nm
intensity of the far UV-CD spectra of ColB at pH 3.5 in both aqueous solution and in the presence of lipids. Transitions are observed at
Tm values of 39.5 °C and 55.2 °C,
respectively. However, the maximal level of unfolding is reached at a
slower rate when compared with ColB at neutral pH (Fig. 7, A
and B). The inset shows the derivatives of the
experimental curves. Fig. 7 (E and F) corresponds to the full-wavelength scans for ColB in solution and in the presence of lipids, respectively, at different temperatures and at pH 3.5.
The temperature dependence of the CD spectra of the pore-forming
fragment in solution at pH 7.0 exhibits single transitions at
64.4 °C and 62.3 °C in the absence or in the presence of lipids, respectively (Fig. 8A). This former value is in good
agreement with the transition temperature of 65.7 °C observed in DSC experiments.
Although in the absence of lipids much of the helix contributions are
lost at temperatures above Tm (Fig.
8B), in the presence of lipids a high helix content of the
pore-forming fragment is conserved even at elevated temperatures (Fig.
8C). The inset in Fig. 8A shows the
derivatives of the melting curves.
Intrinsic Emission Fluorescence--
The temperature dependence of
the intrinsic emission fluorescence of ColB and its pore-forming
fragment in solution was also tested at scanning rates of 1 °C/min
from 25 to 85 °C. ColB contains eight tryptophan residues, five of
which are located in the R/T domain. The temperature dependence of the
tryptophan fluorescence of ColB at pH 7.0 reveals a nearly linear
decrease in fluorescence intensity as a function of increasing
temperature (not shown). However, the temperature-induced quenching
also exhibits a sharp increase in intensity at around 50-55 °C,
thereby matching Tm of ColB peak A. The curves
obtained during cooling to 25 °C or during a second heating period
did not show the stepwise increase in intensity. This is in agreement
with the irreversible unfolding observed by DSC. In the presence of
lipids, the same transition is observed. The tryptophans of the
pore-forming fragments experience a temperature-induced quenching in
fluorophore intensity in solution or in the presence of lipids.
However, a stepwise change in fluorescence intensity is absent during
the melting process (not shown).
Structural and Functional Characteristics of Colicin B and Related
Colicins--
The high resolution x-ray structures of several
channel-forming domains of colicins (A, N, E1, Ia) exhibit a 10-helix
arrangement with 2 hydrophobic helices being surrounded by two layers
of hydrophilic and amphipathic helices (19-22). These conformations,
therefore, represent water-soluble forms of the proteins. The primary
sequences of the C-terminal parts of colicins A and N exhibit
However, when the receptor and translocation mechanisms of colicins are
compared with each other, the molecules group in a different manner.
Although group A colicins (including colicin A and ColN) use the TolA
system for translocation, group B colicins (including ColB and ColIa)
parasitize the Ton B machinery (6). Despite this functional similarity
between ColB and ColIa, the primary sequence homology is modest and the
helix content of ColB (Fig. 7B) is considerably lower than
that of colicin Ia, in particular when considering the R/T domains
alone (22).
Structural details, such as the location of the Ton recognition
sequence in ColIa (22), or the distribution of hydrophilic and
hydrophobic residues within the pore-forming domains of colicins indicate that upon receptor-binding, translocation and
membrane-insertion conformational changes occur. For example, a
multitude of investigations show that the helical conformation of ColA
PFF is preserved during pH-dependent membrane insertion
when at the same time refolding of the tertiary structure occurs
(23).
Thermal Transitions of Colicin B--
The thermal melting
characteristics of colicin B and its PFF provide valuable information
about the conformational stability and the interactions between the
domains also when high resolution structures have not been obtained.
The heats of transition of colicin B and its thermolytic fragments in
solution are
The DSC traces of colicin B exhibit two well-separated transitions
(Fig. 1) suggesting denaturation of two discrete cooperative units. The
R/T domain is known to account for 61% of the total length of colicin
B, which corresponds closely to the 68% value obtained for the heat of
transition A when averaged between pH 7 and 4. Comparison with the DSC
traces obtained from the isolated PFF provides further support for this
assignment. Although the transition temperatures of the isolated PFF
are considerably higher when compared with peak B of full-length ColB,
this difference is largely annihilated after transition A has been
uncoupled from transition B by incubation at intermediate temperatures
(Fig. 3). In addition, the size and pH dependence of the heat of
transition (
The CD spectra of colicin B and its pore-forming fragments indicate a
high content of helical secondary structures of the C-terminal domain
at room temperature (Figs. 7 and 8). This is in excellent agreement
with previous structural findings for the colicin A pore-forming domain
(19, 25) as well as the high degree of primary sequence homology
between the pore-forming fragments of ColA and ColB (16). The
calorimetric, fluorescence, and circular dichroism studies presented in
this paper demonstrate that the two reported transitions of ColB are
due to denaturation of two discrete domains of the protein. Far UV-CD
spectra indicate that during this process the ColB-pore-forming
fragment in solution loses most of its secondary structure (Fig.
8B). This is in contrast to the colicin A pore-forming
domain, which at 90 °C and at pH 5 retains a considerably higher
proportion of secondary structure in CD spectra (9).
Three out of eight tryptophans of colicin B are located within the
pore-forming fragment of colicin B at positions analogous to those in
colicin A. Structural comparison, therefore, strongly suggests that at
ambient temperature the tryptophan side chains at positions 395, 439, and 449 are buried within the hydrophobic interior of the protein (19,
25). A continuous decrease in fluorescence intensity is observed upon
heating, which partly persist when the sample is equilibrated back to
room temperature (not shown). This indicates a pronounced change in
environment. The remaining five Trp residues in ColB are located in the
R/T domain. At the transition temperature a stepwise increase in the fluorescence intensity is observed thereby paralleling the DSC thermal
transition (not shown).
pH Effects on the Thermal Stability of Colicin B--
At pH values
above the pI of full-length ColB (pI = 4.65), the protein
conserves its thermal stability within a wide pH range. However, the
temperatures of both ColB transitions are lowered when the pH of the
samples is decreased even before the pH-induced molten globule state of
the ColA PFF is reached (26). The pH dependence of the R/T domain (peak
A) is more pronounced when compared with peak B, therefore, upon
acidification an increased resolution of both transitions is observed.
At the same time the heat of transition of peak B increases and the one
associated with peak A decreases (Fig. 2). The decrease in transition
temperatures at constant
The decreased thermal stability of ColB at lowered pH correlates with
the previously observed requirement of acidic conditions for optimal
channel and molten globule formation of ColB (30), increased membrane
association of ColA (31, 32), augmented membrane-insertion rate of
ColIa (33, 34), and pH-sensitive conformational changes of ColE1 in the
presence of membranes (35). The unfolding profile of diphtheria toxin
has been published previously (36). This protein shares similarities
with membrane-active colicin B such as acidic pH-triggered membrane
insertion or the secondary and tertiary structure of the
membrane-inserting domain (37). Diphtheria toxin also exhibits two
transitions, which have been assigned to the A and B fragment (at pH 8, Tm = 54.5 and 58.4 °C, respectively) (36). In
a similar manner the transition temperatures and transition enthalpies
exhibit a strong pH dependence, and the isolated A fragment exhibits a
6.5-10 °C reduction in Tm when compared with
the full-length protein.
Thermal Transitions of the Isolated Pore-forming Domain of Colicin
B--
Investigation of the isolated ColB PFF indicates that only the
heat of transition is pH-dependent, whereas the transition temperature remains unaffected between pH 3.5 and 7. In a similar manner, between pH 5 and 7 the heat of transition of ColA PFF exhibits
a small tendency to decrease with increasing pH, at transition temperatures similar to those of ColB PFF (9). At pH < 5, however, both parameters of ColA PFF strongly decrease with pH. The
ColB PFF-melting characteristics, therefore, do not provide direct indications of conformational instabilities that might be important during membrane insertion. It should be kept in mind, however, that the
slow time scale of the DSC experiment is not sufficiently sensitive to
detect the increased membrane insertion kinetics at acidic pH of ColA
PFF or ColB PFF (26, 38). Nevertheless, reduced numbers of negative
charges at the surface of the protein might be important for low pH
interactions with the membrane surface also of this pore-forming
domain, in particular when these contain acidic phospholipids (39, 40).
Localized electrostatic interactions might help to increase membrane
association and to orient the protein with respect to the lipid
bilayer. Such interactions thereby open directed avenues for molten
globule formation and membrane insertion (26, 38, 41).
Interactions between R/T and C Domains--
At low pH the
transition of the isolated ColB PFF occurs at considerably higher
temperatures than transition B of full-length ColB (Tables I and II).
Preincubation at temperatures between the transition temperatures of
peaks A and B, however, shifts Tm of peak B from
40 to 62 °C, thereby closely approaching the transition temperature
of ColB PFF (Figs. 1D and 3). Similarly, interaction of the
PFF with lipid bilayers at low pH results in an increase of
Tm of peak A by >20 °C (Figs. 1D
and 6B). These data indicate that interaction between
domains results in a reduction of the stability of both cooperative
units. The decrease in conformational entropy when two protein domains
are tightly coupled to each other is expected to contribute to the
destabilization of large protein domains, but more specific
interactions between the proteins, such as van der Waals, hydrogen
bonding, or electrostatic interactions, can also be important. In a
related manner, functional differences in ColA PFF and colicin A have
been attributed to interactions between the pore-forming and the
receptor binding domains (32, 42).
Although the isolated C-terminal fragment by itself is competent to
insert into membranes (16), and pore-forming domains of other colicins
exhibit pore-forming activities in vitro (17, 43-45), all
three domains are required for recognition, translocation, and pore
formation at intact cells in vivo. It appears that mutual interactions between different regions of colicin B help to maintain the translocation and the pore-forming domains in less stable but
functionally competent conformations. Intra- and intermolecular interactions thereby reduce the energies involved in conformational changes during protein translocation and insertion (cf.
e.g. Ref. 22).
Thermal Stability of Colicins in the Presence of Lipids--
At pH
3.5 the denaturation profile of ColB shows a decrease in total heat of
transition, disappearance of transition B, and stabilization of
transition A due to the presence of lipids. Similarly, the heat of
transition of membrane-associated ColB PFF is decreased by two orders
of magnitude. In addition, CD spectra indicate that a large fraction of
its secondary structure is preserved at higher temperatures. These
observations are similar to those reported for the pore-forming domains
of ColA (9), ColE1 (10), as well as the pore-forming proteins
equinatoxin II (46), staphylococcal
Proton-decoupled 15N solid-state NMR spectra of oriented
membrane samples indicate that the helices of the ColB and ColE1
C-terminal domains are predominantly orientated parallel to the bilayer
surface (16). In addition, neutron scattering experiments show a
location of large parts of the ColA PFF close to and beyond the
membrane surface (47). We, therefore, suggest that the small but
detectable decrease in
The residual change in heat of transition observed in denaturation
profiles of lipid-associated C-terminal domains of ColB (this study),
ColA (9), and ColE1 (10), therefore, strongly suggest that a fraction
of the membrane-inserted protein is located in extramembranous or
interfacial regions. The reproducible fine structure encompassing at
least four peaks in the DSC traces of ColB PFF is to our knowledge
observed for the first time for membrane-associated proteins and could
derive from, for example, independent helical structures at the
membrane interface (Fig. 6, C and D).
In summary, this study provides thermal denaturation profiles of
proteins in solution and in their membrane-associated states. The same
protein reveals pronounced differences in stability and thermodynamic
properties when investigated in solution or in the membrane. The
membrane-inserted parts of the proteins seem to a large extent
resistant to thermal denaturation and exhibits an interesting fine
structure in DSC experiments. In contrast, those regions exposed to the
aqueous environment lose much of their native conformation. The domain
structure of colicin B is also reflected in two well-separated thermal
transitions in aqueous buffer. Nevertheless, at low pH the interaction
between these domains results in their mutual destabilization and
transition temperatures well below those of the domains when uncoupled
from each other. Intramolecular interactions may, therefore, play an important role during conformational changes that are associated with
translocation and insertion into the inner E. coli membrane.
40%) or its pore-forming fragment (
80%). Upon heating, the
ellipticities at 222 nm strongly decrease at the transition
temperature. In the presence of lipid vesicles the
differential-scanning calorimetry profiles of the pore-forming fragment
exhibit a low heat of transition multicomponent structure. The heat of
transition of membrane-associated colicin B (Tm = 54 °C at pH 3.5) is reduced and its secondary structure is
conserved even at intermediate temperatures indicating incomplete
unfolding due to strong protein-lipid interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL METHODS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES
H, therefore, relates to the heat absorbed
during the process of denaturation and cannot be fully interpreted as
the enthalpy of a reversible
process.2 Two superimposed
endothermic transitions give the best theoretical fit of the scans
obtained at pH 7.0: peak A with Tm = 54.9 ± 0.5 °C,3 and peak B
with Tm = 58.7 ± 0.8 °C
(n = 3) (
Tm(B-A) =3.9 °C). At pH 3.5 both transitions occur at lower temperatures and with better
resolution. The transition temperature of peak A is 29.8 °C and that
of peak B 40.9 °C (
T(B-A) = 11.1 °C). The highest Tm was observed at pH
values of >4.5. When the pH is decreased in a stepwise manner from 7.0 to 3.5, peaks A and B decrease by 25.0 °C and 17.8 °C,
respectively. Therefore, although both components are affected by pH,
peak B is less sensitive to changes in proton activity and more
resistant to thermal denaturation than peak A. The transition
temperatures obtained at pH 2.5 and pH 9.0 are very similar to those of
pH 3.5 and pH 7.5, respectively (not shown).
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Fig. 1.
Effect of pH on the DSC profile (excess Cp
versus temperature) of the full-length ColB in aqueous
solution. The temperature dependence of the excess molar heat
capacity of the protein is depicted at different pH values:
A, 7.0; B, 6.0; C, 4.5; and
D, 3.5. The protein was dissolved in 100 mM KCl
and 20 mM glycine (C and D) or 100 mM KCl and 20 mM tris-malate (A and
B). Solid lines correspond to the experimental
data; dotted lines correspond to the best-fit analysis. The
scan rate was 1 °C/min.
H, for peaks A and B at different pH are
included in Table I. The pH dependence of
H
for peak A and B is depicted in Fig. 2.
Although
HA increases,
HB decreases at the same rate. The
observation of two well-distinguishable transitions could be due to
different protein conformations or due to separate conformational unfolding steps of discrete domains within the same protein. To further
discriminate between these possibilities, ColB was heated to 35 °C,
slightly above the Tm of peak A at pH 3.5, and
was kept at this temperature for 10 min. A DSC experiment with
partially unfolded ColB was then performed at a heating rate of
1 °C/min between 25 and 85 °C (Fig.
3). A single transition with
H = 98 kcal/mol and
Tm = 62.1 ± 0.1 °C (S.D.,
n = 3) was observed, which is 21 °C increased from
TmB of the continuous scan. This result indicates that the transition of peak B is subject to interactions with
components contributing to peak A.
Calorimetric data of ColB in aqueous solution at 1 °C/min
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Fig. 2.
Calorimetric heat of transition
( H) as a function of pH. The
H values of transition A
(
HA;
) and transition B
(
HB;
) were derived from the
integral of the fitted curves of the DSC profiles of ColB in aqueous
solution.
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Fig. 3.
DSC profile (excess Cp versus
temperature) of ColB in aqueous solution at pH 3.5. ColB, at
a protein concentration of 37 µM, was dissolved in 100 mM KCl and 20 mM glycine, pH 3.5, and heated
for 10 min at 35 °C. Measurements from 25 to 85 °C at 1 °C/min
showed a single transition at 62 °C. The solid line
corresponds to the experimental data; the dotted line
corresponds to the component obtained from the best-fit analysis.
H. The transition temperature for the main
transition is 65.7 ± 1.5 °C at pH 7.0. A minor transition with
Tm = 55.5 ± 1.5 °C is consistently
present and corresponds to the remaining 10 ± 4% of the total
heat of transition. The transition temperature of the pore-forming
fragment is pH-independent between pH 3.5 and 9.0 (Fig. 4 and Table
II). The pH-dependent changes
in
H of the PFF and peak B of ColB exhibit comparable characteristics (Tables I and II). By changing the pH from
3.5 to 7.0 the relative decrements in
H are 63 and 59% for ColB peak B and the pore-forming fragment, respectively. A minor increase in heat of transition when the pH was lowered from 7 to
5 was observed for the related ColA PFF (Fig. 2 in Ref. 9). The effect
of pH on ColB reveals more than one protonation state, suggesting the
existence of several conformational states (Fig. 5A). In contrast, between pH
3.5 and 7.0 both transitions of the PFF occur in a pH-independent
manner (Fig. 5B).
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Fig. 4.
Effect of pH on the DSC profile of the
colicin pore-forming fragment. The temperature dependence of the
excess molar heat capacity of the pore-forming fragment is depicted at
different pH values: A, 7.0; B, 6.0;
C, 4.5; and D, 3.5. The pore-forming fragment was
dissolved in 100 mM KCl and 20 mM glycine
(C and D) or 100 mM KCl and 20 mM tris-malate (A and B). The
solid lines correspond to the experimental data; the
dotted (individual components) and hatched lines
(sum) correspond to the best theoretical fit.
Calorimetric data of the pore-forming fragment in aqueous solution at
1 °C/min
View larger version (13K):
[in a new window]
Fig. 5.
pH dependence of
Tm of colicin B and its pore-forming
fragment. A, dependence of the
Tm of ColB on pH. The Tm
used in this analysis corresponds to the temperature at the peak of the
transition. The solid symbol ( ) illustrates the pH
dependence of transition A; the open symbol (
)
illustrates the pH dependence of transition B. B, dependence
of the Tm of ColB pore-forming fragment on pH.
The solid symbol (
) corresponds to the pH dependence of
transition A and (
) corresponds to the pH dependence of transition
B.
H of ColB at pH 7.0 is 172 kcal/mol. At pH 3.5
H equals 140 kcal/mol in
the presence of lipids, a value similar to the
H observed for peak A at pH 3.5 in aqueous
buffer alone (Fig. 1D and Table I). However, in the presence
of lipids this latter transition has shifted to higher temperatures by
at least 20 °C (Fig. 6B).
View larger version (15K):
[in a new window]
Fig. 6.
Temperature dependence of the excess molar
heat capacity of ColB and the pore-forming fragment in the presence of
small unilamellar lipid vesicles. ColB at (A) pH 7.0 and (B) 3.5; pore-forming fragment at pH 7.0 (C)
and 3.5 (D). The solid line corresponds to the
experimental data; the dotted line corresponds to the
best-fit analysis. Note that panels A and B, and
C and D, are shown at two different scales. The
vesicles were prepared by extrusion as described under "Experimental
Methods."
H is significantly
decreased (Table III). After rescan subtraction and baseline correction line-fitting analysis reveals a
pattern of multiple transitions at pH 7.0 or pH 3.5 (Figs. 6, C and D). The calculated values of
Tm and
Htotal are listed in Table
III.
Calorimetric data of the pore-forming fragment in the presence of
lipids at 1 °C/min
-helical secondary
structures. When the mean residue ellipticity at 222 nm is taken as an
indicator, helix contents of 39 and 82% are obtained for ColB and its
PFF, respectively, in aqueous solution before starting the heating scan
(Figs. 7C and 8B).
In the presence of liposomes optical distortions by absorption flattening and light scattering would result in an underestimation of
the helical content (18). These effects are minimized when small
unilamellar vesicles prepared by extrusion through 50-nm filters are
used (18). Nevertheless, no attempt was made to calculate the helix
content quantitatively from the CD spectral line shape in the presence
of lipids or after thermal denaturation.
View larger version (34K):
[in a new window]
Fig. 7.
Ellipticity of ColB at 222 nm as a function
of temperature. A, in aqueous solution (trace
a) and in the presence of lipids (trace b) at pH 7.0. B, in aqueous solution (trace a) and in the
presence of lipids (trace b) at pH 3.5. The inset
plots represent the derivative of the helical content of ColB
versus temperature plots. C and D,
show far UV-CD spectra of ColB at different temperatures in aqueous
solution and in lipids, respectively, at pH 7.0. E and
F, show far UV-CD spectra of ColB at different temperatures
in aqueous solution and in lipids, respectively, at pH 3.5.
View larger version (17K):
[in a new window]
Fig. 8.
A, ellipticity of ColB PFF at 222 nm as
a function of temperature in aqueous solution (trace a) and
in the presence of lipids (trace b) at pH 7.0. The
inset shows the derivative of 222
versus temperature plots. CD spectra of the pore-forming fragment
at pH 7 and at different temperatures in aqueous solution
(B) and in the presence of lipids (C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES
54%
identity and 71 or 62% homology with ColB PFF, respectively. In
addition, both the high resolution x-ray structure of ColA PFF (19) and the ColB PFF CD spectra (Fig. 8B) exhibit
75%
-helix
contributions. It is, therefore, reasonable to assume that the colicin
A and B pore-forming domains adopt closely related structures. In
contrast, the length and amino acid composition of the pore-forming
regions of colicins A, B, and N exhibit considerable differences when compared with those of colicins Ia, Ib, and E1 (e.g. Ref.
16). Therefore, these C-terminal fragments are assigned to different families.
3.6 and
1.3 cal/g, respectively, and, therefore,
depending on the experimental conditions, are smaller or within the
range previously observed for globular proteins in solution at
comparable transition temperatures (11, 24).
H) of peak B of ColB parallel
those of the main transition observed for the isolated pore-forming
fragment (Tables I and II). The large reduction in heat of transition
of both peak B and the ColB pore-forming fragment during membrane
association of the corresponding proteins further demonstrate that
transition B is closely associated with the pore-forming domain of
full-length ColB (Fig. 6, B-D).
Htotal
indicates that entropic changes during the thermal transition increase
when the pH is lowered (11). Models for changes in entropy during
thermal transitions include alterations of the side-chain and backbone
conformational freedom as well as changes in the hydration shell of
macromolecules (11, 24). Augmentation in
H, as
observed for peak B, has been taken as an indication of increased
exposure of apolar residues during the thermal transition (11).
Correspondingly, the decrease in
HA suggests an increasing amount
of solvent-exposed hydrophilic areas and/or augmentation of protonation
reactions at low pH when compared with neutral conditions (11). The
observed proton exchange during the unfolding of ColB below its pI,
indeed, indicates that residues buried in the native structure are
protonated during the ColB thermal transition (27). Similarly,
pH-dependent changes in the thermodynamic parameters have
been observed with other soluble proteins (1, 28, 29).
-toxin (12),
diphtheria toxin (36), or other transmembrane proteins that appear to
be resistant to complete thermal denaturation (11). The hydrogen bonds
of helices within hydrophobic environments are considerably stronger
than in water (4), therefore, the helical structures of residues buried
inside the lipid bilayer are often remarkably stable. In contrast, the
least stable regions of membrane proteins are generally those that are
extramembranous. As a consequence, these regions seem to unfold in a
similar manner than soluble proteins (11).
-helix content and the heat of transition
observed by calorimetric measurements of the ColB pore-forming fragment in the presence of lipids (Figs. 6 and 7) is associated with unfolding of loop structures, hydrophilic ends of membrane-associated helices, and/or complete unfolding of those helices that are weakly associated with the membrane interface (Ref. 11 and references cited therein).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael Bärmann and Prof. Erich Sackmann of the Department of Physics, E22, Technische Universität München, Germany, for discussions and for allowing us to use the DSC instrument of their laboratory; Luis Moroder for providing access to CD and fluorescence spectrophotometers; Volkmar Braun for the colicin B strain; Mechthild Linnemann for technical help during protein purification; Leopold Ilag, Ulrike Harzer, Bas Vogt, Pieter Jasperse, and Sathish Hasigae for their help and discussions; Lissy Wahey for the helpful advise during UV-CD measurements; and Dr. Miriam Gitler of NOVA Research Company, United States, for the review of this manuscript. The comments by Jeremy Lakey were very valuable and helped improve the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Alexander von Humboldt Foundation (DGAPA-IN218397), Universidad Nacional Autónoma de México, Mexico (to A. O.), the Max-Planck-Institut für Biochemie, Germany, and a grant from the Deutsche Forschungsgemeinschaft (Be1247 to B. B.). The DSC instrument of the Department of Physics, E22, Technische Universität München, Germany, was supported by Sonderforschungsbereich 266.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: Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, Martinsried 82152, Germany. Tel.: 49-89-8578-2466; Fax: 49-89-8578-2876; E-mail: bechinge@biochem.mpg.de.
Permanent address: Departamento de Bioquimica, Facultad de
Medicina, Universidad Nacional Autónoma de México,
México D.F. AP 70-159, C.P. 1045, México. Tel.:
52-5-623-2511; Fax: 52-5-616-24-19; E-mail:
aortega@servidor.unam.mx.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M007675200
2
In equilibrium processes the area under the
transition endotherms (Cp versus temperature) represents the
change of enthalpy of systems at thermodynamic equilibrium throughout
the temperature-induced unfolding process (48). Therefore, in the
literature reference is often made to "transition enthalpies" also
in cases of irreversible protein denaturation during the heating scan
(e.g. Refs. 9, 14). One of the reviewers pointed out that
this approach might only be valid under some circumstances. Although
the excellent line fit obtained during the deconvolution of our traces
seems to suggest that application of a reversible model bears some
justification (Figs. 1D, 3, 4) we prefer to make reference
to "heat of transition" (H) throughout the text.
3 Compare Tables 1-3 for standard deviations of transition temperatures.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ColB, colicin B; ColA, colicin A; ColIa, colicin Ia; CD, circular dichroism; DSC, differential-scanning calorimetry; PFF, pore-forming fragment (thermolytic C-terminal fragment); POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; R/T, receptor/ translocation; Tm, transition temperature.
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