From the Groningen Biomolecular Sciences and Biotechnology
Institute and the Department of Biochemistry, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
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INTRODUCTION |
The transport of some carbohydrates from the environment into
bacterial cells is mediated by the proteins from the
phosphoenolpyruvate-dependent phosphotransferase system
(PTS).1 This system
couples the translocation of substrates across the cytoplasmic membrane
to concomitant phosphorylation, using phosphoenolpyruvate (PEP) as the
energy source. Thus, the overall reaction catalyzed by the PTS system
is best described as follows.
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The equilibrium of this reaction is shifted far to the right.
The transport proteins of the PTS, termed enzyme
IIs,2 are specific for the
carbohydrate they transport but nonetheless show a remarkably similar
architecture throughout. In almost all systems, a membrane-spanning
domain and two cytoplasmic phosphotransfer domains are found, in some
cases covalently bound and in other cases as separate proteins or as
combinations of these two possibilities. The phosphoryl group is
transferred to the first of the phosphotransfer domains or proteins,
the A domain, via two general PTS proteins, enzyme I and HPr, and from
there to the second phosphotransfer domain, the B domain. The incoming
substrate is bound by the C domain, translocated, and phosphorylated
directly by the B domain. Reviews on the PTS systems can be found in
Refs. 1-5.
The subject of this study is the mannitol-specific PTS protein from
E. coli, enzyme IImtl. In this protein, the
three domains are all part of the same polypeptide chain, with the C
domain at the N terminus and the A domain at the C terminus. The
phosphorylation sites are His554 on the A domain and
Cys384 on the B domain. All three domains as well as the
binary combinations, IICBmtl and IIBAmtl, have
been subcloned and overexpressed separately and were shown to be
enzymatically active in the presence of the other constituents of the
PTS (6-10).
In contrast to the large number of studies on the stability of
water-soluble proteins, comparatively little is known about the factors
determining the stability of membrane proteins in their natural
environment. This is mostly caused by the fact that membrane proteins
are more difficult to handle than their water-soluble counterparts due
to their inherent hydrophobicity, often resulting in aggregation and/or
precipitation. The effect is even stronger in the unfolded state, since
unfolding results in exposure to the solvent of hydrophobic parts that
are normally buried in the interior of the protein. In this study, we
circumvented this problem by reconstituting EIImtl, as well
as IICBmtl and IICmtl, in a lipid bilayer,
enabling us to study the thermal stability of the enzyme by
differential scanning calorimetry and CD spectroscopy.
Data from unfolding studies can also be used to obtain information on
the extent of the interactions between the domains in a protein. The
energy required to take the enzyme from its native state to the
unfolded state equals the sum of the unfolding energies of each of the
domains and the interaction energies between the domains. If, in a
separate experiment, the unfolding energy of each of the domains can be
determined, the interaction energy can in principle be calculated from
the difference in energy required in each case (11). EIImtl
is well suited for this type of study, since the individual domains of
EIImtl are available in subcloned form and are
enzymatically active and, therefore, are expected to retain their
structural integrity.
It is obvious from what is known about the catalytic mechanism of
EIImtl that domain interactions play an important role in
the functioning of the enzyme. Therefore, these have been, and still
are, the subject of extensive studies (12-14). It was shown that (i)
the B and C domains influence each other's conformation as a function of the phosphorylation state of the B domain, (ii) binding of mannitol
leads to a large conformational change in the protein, part of which
occurs in the B domain, and (iii) in the absence of the C domain there
are no extensive stabilizing interactions between the B and A domains
in the unphosphorylated or phosphorylated state. In this study, we
focus on the B and C domains in order to get better insight into their
mutual interactions.
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EXPERIMENTAL PROCEDURES |
Materials--
Dimyristoylphosphatidylcholine (DMPC) was
purchased from Avanti Polar Lipids.
N-Decyl-
-D-maltoside, tosylphenylalanyl
chloromethyl ketone-treated trypsin from bovine pancreas and soybean
trypsin inhibitor were purchased from Sigma. Nickel-nitrilotriacetic
acid-agarose was obtained from Qiagen. Perseitol was purchased from
Pfanstiehl Laboratories Inc. (Waukegan, IL). Decyl-PEG was synthesized
by B. Kwant at the Department of Chemistry, University of
Groningen.
Protein Purification--
EIImtl and
IICmtl were purified as described previously (8-10).
Construction of the IICBmtl Overexpression
System--
The plasmid, pMamtlA, containing the wild type
mtlA gene and its promoter was used in a site-directed
mutagenesis procedure, according to the method described by Kunkel
(15). An amber stop codon in combination with a HindIII
restriction site was created at position 1462 in the wild type
mtlA gene with the primer
5'-GAACAGGTTAAGCTTGGACTAGTCAAAGCT-3'. The choice of this position was based on the stable expression of the B
domain of EIImtl (8). The creation of an extra
HindIII site in the pMamtlA(am) plasmid was confirmed by a
restriction analysis. The new HindIII site was used to
delete the part of the mtlA gene that encodes for the A
domain. The resulting PmaIICB consists of the Pmtl promoter followed by an open reading frame coding for the first 487 amino acids
of the N-terminal part of EIImtl. Overexpression was
achieved by the insertion of the
-Pr promoter with the
cI857 repressor gene into the pMaIICB plasmid (9, 16). For this
purpose, an EcoRI-SalI fragment containing the Pr promoter and repressor gene was excised from pJRD187 and
ligated into the corresponding restriction sites in the plasmid,
pMaIICB. The resulting overexpression vector, pMaIICBPr,
containing IICBmtl behind a tandem promoter was sequenced
as described (17) and was identical to the previously published
sequence of this portion of the gene (18).
Growth, Expression, and Purification of
IICBmtl--
Growth, expression, and purification
procedures for IICBmtl were identical to those described
for the wild type EIImtl (8).
PmaMtlIICHis Plasmid Construction--
In order to obtain a
construct with a C-terminal His tag, the 51-mer polymerase chain
reaction primer
5'-TGCTCTAGATTAGTGATGGTGATGGTGATGGCTGGTTTTCAGCAAAATAGC-3' was designed, which was based on the PmaIIC plasmid constructed earlier (9). In this way, the six histidines were positioned directly
behind the last predicted membrane-spanning helix at position 337 in
the amino acid sequence (19). The XbaI site in the primer
was used for cloning. A 3'-primer was designed, directed against the
beginning of the MtlA gene (3'-ATGTCATCCGATATTAAGATCAAA-5'). The
MltA-encoding part of PmaMtlA was used as a template for the polymerase
chain reaction. Together with the Pwo polymerase, this yielded a 1-kilobase pair fragment, which was cloned into the pSK
vector with EcoRV. This fragment was then
digested with NcoI (position 390) and XbaI and
cloned into the PmaMtlaPr plasmid, resulting in PmaIIChisPr. The entire
gene was sequenced and showed the expected sequence.
Growth and Expression of His-IICmtl--
The
procedures to grow and express His-IICmtl were identical to
those described for the wild type EIImtl (8). Preparation
of membrane vesicles and extraction of the protein from the membrane
were performed as described (8, 20); the supernatant containing the
extracted membrane protein was used for the purification.
Purification of His-IICmtl by
Nickel-Nitrilotriacetic Acid-Agarose Affinity Chromatography--
1.5 ml of
nickel-nitrilotriacetic acid-agarose suspension was used per ml of
membrane vesicles and was equilibrated with 20 mM Tris-HCl,
pH 8.4, prior to use. The nickel-nitrilotriacetic acid-agarose was
added to the extracted membrane fraction together with 20 mM imidazole and incubated on a rotator. After 1 h,
the mixture was poured into a column and washed with the extraction buffer containing 20 mM imidazole until the
A280 became constant. The column was then washed
with at least 2 column volumes of 20 mM Tris-HCl, 150 mM NaCl, 2 mM
-mercaptoethanol, 1 mM NaN3, 20 mM imidazole, and 6 mM decylmaltoside. The latter was added to change the
detergent. Batchwise elution was performed with the same solution after
raising the imidazole concentration to 200 mM. The
His-IICmtl concentration was determined as described
(21).
Activity Determinations--
The PEP-dependent
mannitol phosphorylation rate and the mannitol/mannitol 1-phosphate
exchange rates were determined as described (20, 22). The temperature
was controlled by a water bath, and samples were equilibrated at the
desired temperature for 5 min prior to starting the reaction.
Reconstitution of EIImtl, IICBmtl,
IICmtl, and His-IICmtl in DMPC Vesicles--
A
small amount of DMPC in chloroform, sufficient to yield a concentration
of 5 mg/ml in the final solution, was dried under argon.
EIImtl, IICBmtl, IICmtl, or
His-IICmtl, solubilized in decylmaltoside and buffer (10 mM NaPi, pH 7.6, 10 mM
mercaptoethanol, 1 mM EDTA, 1 mM
NaN3, and, if desired, 100 µM mannitol) were
added to the dried lipid to yield a solution with the desired
concentrations of enzyme and lipid. A small volume of a concentrated
solution of decylmaltoside in water was added to this solution until it
became clear, usually at a final detergent concentration of
approximately 7 mM. This solution was left at room
temperature for 30 min and then stirred for 30 min with 40 mg/ml
Biobeads SM2 that had been pretreated as described (23). After removal
of the Biobeads, the solution was stirred with 80 mg/ml fresh Biobeads
for 1 h. This last step was repeated (usually three times) until
no further change could be detected by DSC in the gel to liquid
crystalline transition of DMPC. The solution was diluted in
reconstitution buffer in order to obtain a volume that could easily be
handled and then spun down at 150,000 × g, after which
the pellet was resuspended in 1.8 ml of the buffer. Finally, in order
to homogenize the preparation, the solution was extruded through a 200- or 400-nm polycarbonate filter. If desired, mannitol or perseitol was
added shortly prior to the extrusion step.
Reconstituted preparations with very low mannitol concentrations were
also obtained in an alternative way. The preparations were
reconstituted as described above but in the presence of 100 µM mannitol, and they were diluted in buffer that did not
contain any mannitol prior to centrifugation. This was repeated three or four times to yield a final mannitol concentration of approximately 10 nM; the Kd of EIImtl for
mannitol is approximately 100 nM.
Protein concentrations of all reconstituted preparations were
determined by quantitative amino acid analysis at Eurosequence (Groningen, Netherlands) as described (24).
In order to ascertain whether the reconstitution procedure affected the
protein, small samples were taken in the course of the process and
assayed for mannitol phosphorylation activity in the presence of the
detergent decyl-PEG. The activity of the protein slowly decreased to
80% of the initial activity during incubation and removal of the
detergent by Biobeads and then dropped to 45% after extrusion over a
polycarbonate filter. This sharp drop in activity is caused by the fact
that part of the protein is absorbed, probably because it is present in
large aggregates or membrane sheets that fail to pass the filter. It
is, however, necessary to remove these aggregates and/or sheets,
because they interfere with the DSC and CD measurements. Therefore, if
it is assumed that the passage through the filter does not
significantly change the specific activity of the protein, one can
conclude that no more than 20% of the protein is irreversibly
inactivated during the reconstitution process.
The enzymatic activity of aliquots of EIImtl taken during
reconstitution after solubilization in detergent does not give any information on the activity of the protein in the membrane itself. In
order to check this, the mannitol phosphorylation and exchange activity
of reconstituted EIImtl and IICBmtl were
determined. The specific phosphorylation activity ranged from 230 to
300 (nmol of mtl-1-P) min
1 (nmol of
EIImtl)
1 in the membranes but increased to
approximately 2300 (nmol of mtl-1-P) min
1 (nmol of
EIImtl)
1 after solubilization of the vesicles
in decyl-PEG. Boer et al. (9) observed a specific
phosphorylation activity of approximately 400 (nmol of mtl-1-P)
min
1 (nmol of EIImtl)
1 using
EIImtl solubilized in decyl-PEG but under slightly
different buffer conditions. The specific transphosphorylation activity
of EIImtl was in the range 1.3-2.7 (nmol of mtl-1-P)
min
1 (nmol of EIImtl)
1 at an
enzyme concentration of 5 nM and at 30 °C, which
compares well with a value of 0.54 (nmol of mtl-1-P) min
1
(nmol of EIImtl)
1, determined in the presence
of decyl-PEG but also under slightly different buffer conditions (9).
The specific transphosphorylation activity of IICBmtl after
reconstitution was 0.17-0.35 (nmol of mtl-1-P) min
1
(nmol of IICBmtl)
1 at 30 nM
enzyme concentration.
The vesicle solutions were further analyzed by electron microscopy,
using negative stain to visualize the lipid structures. Mostly
multilamellar structures were found that did not disappear after
extrusion through polycarbonate filters. Flow dialysis measurements to
determine the extent of binding indicated that the vesicles are
permeable to small molecules, since no difference in the binding could
be detected between reconstituted preparations before and after
solubilization in the detergent decyl-PEG (data not shown). It is
unlikely that large molecules like enzyme I and HPr are able to
permeate the first layer and reach EIImtl molecules
inserted in one of the inner layers of a multilamellar vesicle. This
would explain the large differences between the effects of detergents
on the mannitol exchange and phosphorylation activities, since in the
former, only mannitol and mannitol-1-P are needed, whereas in the
latter, the transfer of a phosphoryl group from P-HPr to the A domain
of EIImtl is the first step. We therefore conclude that the
protein has been reconstituted in the lipid bilayer largely in its
functional form and that the preparations are suitable for further
analysis.
Limited Proteolysis of Reconstituted EIImtl
Preparations--
Preparations of reconstituted IICmtl
could also be obtained by limited proteolysis of reconstituted
EIImtl by trypsin. To 1 ml of freshly prepared
EIImtl vesicles, reconstituted using Biobeads in the
presence of 100 µM mannitol, 2 ml of reconstitution
buffer and 1 ml of a freshly prepared 10 µg/ml solution of trypsin
from bovine pancreas were added. This mixture was left at room
temperature for 45 min, and then the reaction was quenched by adding 1 ml of a 10 mg/ml solution of soybean trypsin inhibitor (1000-fold
excess). Trypsin and soybean trypsin inhibitor were removed by
dilution, and subsequent concentration of the vesicle solution by
ultracentrifugation as described for mannitol. Samples were analyzed
for EIImtl, trypsin and soybean trypsin inhibitor by
SDS-polyacrylamide gel electrophoresis. None of these components could
be detected on Coomassie-stained gels.
Circular Dichroism Spectroscopy--
All CD measurements were
performed on an Aviv 62A DS circular dichroism spectrometer equipped
with a thermoelectric cell holder for adequate temperature control.
Far-UV CD spectra were recorded from 250 to 200 nm, and thermal
unfolding between 25 and 95 °C was monitored at 222 nm. In all
cases, a 1-mm path length cell and a 1-nm bandwidth was used. The
Tm of the protein under study was extracted from the thermal
unfolding curves by smoothing the curves and differentiation.
Differential Scanning Calorimetry--
All DSC measurements were
performed on an MCS differential scanning calorimeter from Microcal
(Northampton, MA) with buffer in the reference cell under a 1.5-bar
nitrogen pressure. Samples were degassed by stirring gently under
vacuum prior to measurements. The gel to liquid crystalline transition
of the lipid was scanned between 5 and 40 °C using a scan rate of
30 °C/h and a 2-s filter time. Protein unfolding events were
recorded between 40 and 95 °C with a scan rate of 60 °C/h, unless
indicated otherwise, and 15-s filter time. In order to check for
reversibility of the observed transitions, rescans of the samples were
performed after slowly cooling to 30 °C. The scans were analyzed
after subtraction of an instrument base line recorded with water in
both cells using the software packages ORIGIN (Microcal) or
Mathematica (Wolfram Research).
Modeling of DSC Data--
A general way to describe an
irreversible protein unfolding transition is the model proposed by
Lumry and Eyring (25). In this model, an unfolding reaction at
thermodynamic equilibrium is followed by an irreversible reaction of
the unfolded state, 5521
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(Eq. 1)
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where N, U, and D are the
native, unfolded and denatured states, respectively;
Keq is the equilibrium constant of the unfolding reaction, and k is the first order rate constant of the
irreversible step and can be described by the Arrhenius equation,
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(Eq. 2)
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where Ea is the activation energy and
Tk is the temperature at which the rate equals 1 min
1. A detailed theoretical analysis of this model
applied to DSC data has been published (26), and in this study it was
shown that, under the assumption that the equilibrium is established at
all temperatures and that the irreversible step proceeds with a
negligible heat effect, the excess heat capacity function could be
described by the equation,
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(Eq. 3)
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where v is the scanning rate and T0 is a
temperature at which both k and the concentration
D are negligible.
Depending on the kinetics of the reversible and irreversible steps, two
limiting cases can be defined; the rate of the irreversible step is
either 0 or much faster than the rate of the refolding reaction. In the
former case, the irreversible step after the unfolding of the protein
does not occur, and the unfolding transition is completely reversible.
Equation 1 reduces as follows,
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(Eq. 4)
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and Equation 3 as follows,
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(Eq. 5)
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which can be used in the deconvolution of DSC data (27). This
model predicts that the heat absorption peak in a scan and a rescan of
the same sample are equal and that, if the sample is in thermodynamic
equilibrium, the result is independent of the scanning rate used in the
experiments. Both of these properties can be used to test the model in
practice.
In the case of very fast kinetics of the irreversible versus
the equilibrium reaction, Equation 1 effectively reduces as
follows,
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(Eq. 6)
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since the equilibrium is never established and the intermediate
state, U, will not be populated at any temperature. It has been shown that the excess heat capacity function in this case can be
described by the equation,
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(Eq. 7)
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again assuming that the irreversible step proceeds with a
negligible heat effect (28). To test whether the model is appropriate, the activation energy can be calculated in four different ways: (a) from a curve fit using Equation 7; (b) from
the slope of an Arrhenius plot, where the rates are calculated
according to the equation,
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(Eq. 8)
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(c) the ratio of the maximum and area of the peak
using the equation,
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(Eq. 9)
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or (d) the dependence of the temperature of maximum
heat absorption on the scanning rate,
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(Eq. 10)
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For the model to hold true, the activation energies calculated
by all four methods should be equal (see Ref. 29 for a detailed discussion).
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RESULTS |
Reconstitution of EIImtl--
Membrane proteins have a
pronounced effect on the heat absorption peak of the gel to liquid
crystalline transition of liposomes (30); in general, the Tm
shifts downward, the peaks are broadened, and the total enthalpy
associated with the phase transition decreases. In Fig.
1, the phase transitions of DMPC vesicles, as monitored by DSC, are shown in the absence of protein (A) and in the presence of 9 µM
EIImtl, 21 µM IICBmtl, or 7.5 µM His-IICmtl (B). In each case,
the broadening of the transition is clear, indicating that indeed all
three proteins are inserted into the proteoliposomes. Further evidence
for insertion into the membrane was obtained from the fact that the
broadening effect is dependent on the concentration of the protein (not
shown). The peaks observed in the presence of protein can best be
described by a broad peak at approximately 23 °C with a sharper peak
superimposed. Analysis of these data yielded a linear dependence
(R2 = 0.99) of Tm of the sharp
transition on the protein:lipid molar ratio, assuming that the final
concentrations of the protein and lipid do not differ significantly
from the starting situation, whereas the Tm of the broad
transition was virtually independent of the protein concentration. The
dependence of the lipid phase transition enthalpies on the molar ratio
of protein and lipid can be analyzed according to Ref. 31,
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(Eq. 11)
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where
H0 is the enthalpy change in the
absence of protein, Na is the number of lipid
molecules withdrawn from the phase transition per EIImtl
monomer, and P/L is the protein:lipid molar
ratio. This analysis yields an estimate of 40 for Na
in the case of IICmtl.

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Fig. 1.
A, the DMPC gel to liquid crystalline
phase transition as observed with DSC. B, the gel to liquid
crystalline transition of DMPC after reconstitution of 9 µM EIImtl (1), 21 µM
IICBmtl (2), and 7.5 µM
His-IICmtl (3).
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Thermal Unfolding of EIImtl, Reconstituted in the
Presence of 100 µM Mannitol--
The thermal unfolding
of EIImtl and its separate domains was first studied after
reconstitution in the presence of 100 µM of the substrate
mannitol. For the wild type enzyme, two transitions could be observed,
one centered at about 62 °C, the other at about 82 °C (Fig.
2A). In the absence of the A
domain, almost all of the first transition disappeared, whereas
reconstitution of the C domain led to only one observable transition at
76 °C. To ensure that the observed heat absorption peaks were due to
structural rearrangements within the protein, the changes of the CD
signal at 222 nm, indicative of
-helix content, were also monitored as a function of temperature. The results are shown in Fig.
2B, as well as the first derivative of these data (Fig.
2C). The Tm values of the high
temperature transition observed with CD are somewhat lower than
observed with DSC, but this is caused by the slower scan rate in the CD
experiments (see below). From the resemblance between the DSC and CD
data, it can be concluded that the heat absorption peaks indeed arise
from unfolding events in the proteins.

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Fig. 2.
Heat-induced unfolding of EIImtl,
IICBmtl, and HisIICmtl, reconstituted in
DMPC. A, raw DSC data. Traces have been displaced along
the y axis for clarity. B, change in CD signal at
222 nm, normalized to the signal at 25 °C. C, first
derivative of the data in B. Experimental conditions were as
follows: 50 mM NaPi, pH 7.5, 10 mM
-mercaptoethanol, 1 mM NaN3.
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Multiple transitions observed by DSC are usually analyzed using the
deconvolution method (27). This method is only applicable to cases
where the protein solution is in thermodynamic equilibrium during the
complete scan. To satisfy this condition, two criteria must be met; the
transition must be reversible (i.e. data from a scan and
rescan of the same sample should be identical), and the results must be
independent of the scanning rate used in the experiment. In our case,
no protein unfolding signal could be detected in the rescan after
heating the sample to 95 °C (Fig. 3A). However, if the DSC scan
was stopped at 67 °C (i.e. directly after completion of
the first transition), cooled, and rescanned, the first transition was
fully reversible. Furthermore, the Tm of this peak was
independent of the scanning rate used to record the data (Fig.
3B), and therefore it can be concluded that this peak can be
analyzed using a model derived from equilibrium thermodynamics. In
contrast, the high temperature peak is both irreversible and dependent
on the scanning rate, indicating that the kinetics of the unfolding
reaction of this part of the protein also contribute to the shape and
the observed Tm of this transition, and therefore this will
have to be taken into account in modeling the transition.

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Fig. 3.
A, raw DSC data for EIImtl,
reconstituted in DMPC. The preparation was scanned to 67 °C
(solid line), cooled to 30 °C, and rescanned
to 90 °C (dashed line) and again cooled and
rescanned (dotted line). No mannitol was present
in this preparation. B, scan rate dependence of the thermal
unfolding of EIImtl, reconstituted in the presence of 100 µM mannitol. The scan rates were 59 °C/h
(solid line) and 120 °C/h (dashed
line). Experimental conditions were 50 mM
NaPi, pH 7.5, 10 mM -mercaptoethanol, 1 mM EDTA, 1 mM NaN3.
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Modeling of DSC Data Obtained in the Presence of Mannitol--
The
data of Fig. 3B were interpreted using Equations 5 and 7 for
the low and high temperature transitions, respectively. In total, three
independently unfolding units were observed, two in the low temperature
region of the spectrum and one in the high temperature region, as
indicated by the quality of the fits (Fig. 4A). The estimates for the
activation energy of the high temperature transition were 363 and 386 kJ mol
1 at 59 and 120 °C h
1,
respectively. To further test the model, an Arrhenius plot was constructed, using Equation 8 for both data sets, resulting in a value
of 368 kJ mol
1 for the activation energy (Fig.
4B). The estimates for the activation energy from the height
and area of the high temperature peak (Equation 9) and the dependence
of Tm on the scanning rate (Equation 10) were 391 and 400 kJ
mol
1, respectively, but the latter can, in view of the
limited data set, only be regarded as a rough estimate. From the
agreement of the values of the activation energy calculated by the four different methods, we conclude that the two-state irreversible model
(Equation 6) describes the data for the high temperature transition in
the presence of mannitol reasonably well.

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Fig. 4.
A, analysis of the thermal unfolding of
EIImtl in the presence of mannitol, using the two-state
reversible model (Equations 4 and 5) for the low temperature transition
and the two-state irreversible model (Equations 6 and 7) for the high
temperature transition, as described under "Experimental
Procedures." The data are indicated by the open
circles, the solid line represents the
fit of the data, and the dotted lines show the
contribution of each of the individual transitions. The raw data were
plotted in Fig. 3B. B, Arrhenius analysis of the high
temperature unfolding peak of the data in A. The rates were
calculated from Equation 8. The slope of the solid
line corresponds to an activation energy of 368 kJ
mol 1. Black squares correspond to
the data obtained at 120 °C h 1; open
circles correspond to the data obtained at 59 °C
h 1.
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Thermal Unfolding of EIImtl in the Absence of
Mannitol--
In order to study the effect of the substrate on the
thermal stability of EIImtl, the unfolding of the enzyme
was also studied in the absence of mannitol. To this end, a preparation
of EIImtl, reconstituted in the presence of 100 µM mannitol, was washed several times by dilution in a
large volume of buffer without mannitol followed by
ultracentrifugation. The DSC scan of this preparation is shown in Fig.
5. Removal of the substrate results in a large shift of the high
temperature peak from ~80 to 73 °C and a decrease in the total
enthalpy of unfolding from approximately 1500 to 1200 kJ
mol
1. The unfolding process was irreversible, since no
reappearance of the heat absorption peaks in rescans of samples after
cooling could be observed. Varying the scan rate between 30 and
120 °C/h showed a clear dependence of the temperature of maximum
heat absorption for the high temperature transition but did not
influence the shape or position of the unfolding transition at 63 °C
(data not shown); this is similar to the behavior in the presence of
the substrate.
Ligand-free EIImtl samples were also obtained by directly
reconstituting the enzyme in the absence of the substrate. The results obtained in this way were very similar to those obtained from samples
prepared by the washing procedure outlined above (Table I). It can be concluded that the results
are independent of the method used to prepare the samples.
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Table I
Differential scanning calorimetry of EIImtl and separate
domains
The data were collected at 60 °C/h unless indicated otherwise and
analyzed as described under "Experimental Procedures" and
"Results." The S.D. values of the parameters derived from the fits
of the data were around 1 °C and 10% for Tm,1 and
H1, respectively, 0.2 °C for
Tm,2 and Tm,3, and 5% for
H2 and H3 and
Ea in the two-state irreversible model. Due to the
small contribution of Tk and Ea to the
overall shape of the peak in the Lumry-Eyring model compared with the
two-state reversible model, these parameters are not well determined
from the fits; variations can be as large as 3 °C for Tk and
50% for Ea, and reasonable fits are still obtained.
The values in the table for these parameters should therefore be
regarded as indicative numbers only. The main source of errors is
variations arising from differences in the method of preparation, in
the concentration determination, and the choice of the base line that
is used to calculate the excess heat capacity function from the raw
data. A better estimation of the errors is therefore derived from
averaging results obtained for different preparations; these S.D.
values are 1.7 °C for Tm,1, 70 kJ mol 1
(27%) for H1, 0.5 °C for
Tm,2, and 60 kJ mol 1 (13%) for
H2. For Tm,3 and
H3, the S.D. values are 1.9 °C and 170 kJ
mol 1 (23%) in the presence of mannitol, 1.2 °C and 70 kJ
mol 1 (12%) in the absence of mannitol, and 0.8 °C and 120 kJ mol 1 (18%) in the presence of perseitol. The S.D. for
Ea in the presence of mannitol is 110 kJ
mol 1 (25%).
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Modeling of DSC Data Obtained in the Absence of
Mannitol--
Deconvolution of the observed excess heat capacity
function using the model described above for the data obtained in the
presence of mannitol did not result in good fits of the C domain peak
(Fig. 5C). Fitting the data with a model
that describes the unfolding of the C domain as a transition that is in
thermodynamic equilibrium throughout the unfolding process, however,
showed that the shape of the observed peak deviates only slightly from
this at the high temperature side, i.e. at temperatures
where the rates of the irreversible reaction are the fastest (Fig.
5A). It appears that the two-state reversible model
(Equation 4) is an underestimation of the rate of the irreversible
reaction, whereas the two-state irreversible model overestimates the
reaction rate; therefore, a model that is intermediate between the two
extremes seems appropriate. Such a model is the Lumry-Eyring model
(Equation 1; Ref. 25). The result of this fitting procedure is shown in
Fig. 5B; the values of the parameters obtained with each
model are listed in Table I. The variations of Tm and
H for the three transitions with the three different
models are relatively small, with the largest variation found for the B
domain transition. The latter is not surprising, since the enthalpy of
this transition is the smallest, and therefore the contribution of this
transition to the overall profile is the least pronounced. The analysis
outlined above was also applied to data obtained in the presence of the substrate analog perseitol (see below).

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Fig. 5.
Deconvolution analysis of the unfolding of
EIImtl in the absence of mannitol. The high
temperature peak is described by the two-state reversible model
(A), the Lumry-Eyring model (B), or the two-state
irreversible model (C).
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Assignment of Unfolding Transitions to Structural Domains--
In
order to assign the individual transitions observed in the unfolding of
EIImtl to structural entities in the protein, the unfolding
of the separate domains was also investigated and compared with the
unfolding pattern of the intact protein. To exclude effects arising
from differences between reconstituted preparations, reconstituted IICmtl was prepared from reconstituted EIImtl
by proteolytic cleavage. Exposure of EIImtl to trypsin
initially results in the formation of two products of approximately
equal size, one of which is water-soluble and has been identified as
the binary combination of the B and A domains, whereas the other is
embedded in the proteoliposome and corresponds to the C domain. Upon
longer exposure to the protease, the B and A domains are fully
degraded, but the C domain stays intact, apparently because it is
protected from further hydrolysis by the lipid environment. The
degradation products and protease can easily be removed by washing the
preparation several times with a large volume of buffer and
recollecting the proteoliposomes by ultracentrifugation. In this way,
reconstituted C domain was obtained that could be directly compared
with the intact EIImtl. Stephan and Jacobson (32) showed
that tryp-IICmtl, in their case obtained from inside-out
membrane vesicles, is still able to bind and translocate mannitol and
that upon complementation with IIBAmtl full
phosphorylation and transport activity of the system is restored.
Fig. 6 shows the deconvolution analysis,
using Equations 5 and 6, of EIImtl reconstituted in the
presence of mannitol (Fig. 6A) as well as the unfolding of
tryp-IICmtl (Fig. 6B) and subcloned
IIBAmtl in the presence of DMPC liposomes (Fig.
6C). Unfolding of tryp-IICmtl results in a
single peak at 74.9 °C, the shape of which is close to the shape
predicted by Equation 7. For the water-soluble IIBAmtl, two
closely separated transitions around 64 °C are observed, arising
from the independent unfolding of the B and A domains at 59 and
64 °C, respectively (14). Together with the data from Fig. 2, it can
be concluded that, when reconstituted in the presence of mannitol, the
domains of EIImtl unfold independently; the hydrophilic B
and A domains unfold at approximately 63 °C, and the
membrane-embedded C domain unfolds between 71 and 82 °C, depending
on conditions.

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Fig. 6.
DSC data after base-line correction and
analysis of EIImtl (A), tryp-IICmtl
(B), and IIBAmtl (C). The
EIImtl and tryp-IICmtl data were obtained in
the presence of 100 µM mannitol. Experimental conditions
were 50 mM NaPi, pH 7.5, 10 mM
-mercaptoethanol, 1 mM EDTA, 1 mM
NaN3.
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Enzyme-Substrate and Domain Interactions--
To gain better
insight into the interaction of the enzyme with its substrate, mannitol
was added to mannitol-free samples of the native enzyme and to
tryp-IICmtl derived from the same preparation (preparation
3 in Table I). This experiment was repeated using a structural analog
of mannitol, perseitol (Fig. 7), a linear
C-7 sugar that is bound by EIImtl with a comparable
affinity to mannitol (Kd is approximately 100 nM for mannitol and 200 nM for perseitol; Ref.
33) but is not transported or phosphorylated (34). The addition of
mannitol to the wild type enzyme led to an increase of the Tm of the C domain from 73 to 78 °C with a concomitant increase in the
enthalpy of unfolding of approximately 125 kJ mol
1,
depending on the model used to interpret the data; the unfolding transitions of the B and A domains remain largely unaffected. The
addition of perseitol has a much smaller effect on the unfolding behavior; the Tm of the C domain increases ~1 °C, and
H increases approximately 40 kJ mol
1 (Fig.
8). These changes are reflected in the
results obtained for the reconstituted tryp-IICmtl,
obtained from the same preparation. The removal of the A and B domains
by trypsin results in a decrease of 1 °C for Tm and 120 kJ
mol
1 for the enthalpy of unfolding of the C domain. Upon
the addition of mannitol, both Tm and
H increase
but remain smaller than observed for the C domain in intact
EIImtl under the same conditions. The addition of perseitol
has little effect on the Tm of tryp-IICmtl, but
H appears to be smaller than in the absence of
ligand.

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Fig. 8.
DSC data after base-line correction of
EIImtl (solid line) and
tryp-IICmtl (dotted line), obtained
from the same preparation, in the absence of mannitol or perseitol
(A), in the presence of 100 µM mannitol
(B), and in the presence of 100 µM perseitol
(C). Experimental conditions were 50 mM
NaPi, pH 7.5, 10 mM -mercaptoethanol, 1 mM EDTA, 1 mM NaN3.
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 |
DISCUSSION |
Reconstitution of EIImtl--
In the reconstitution
process, three factors play a role: the protein, the lipids, and the
detergent. We have chosen DMPC for two important reasons:
(a) the protein was shown to be stable when inserted in
membranes composed of this
lipid3 and (b) the
gel to liquid crystalline phase transition of the lipid bilayer can be
monitored to obtain information on the reconstitution process without
affecting the protein. We chose the detergent n-decyl-
-D-maltoside despite its 2 mM critical micel concentration because, unlike for example
decyl-PEG, which is used during the purification, it is a homogeneous
preparation, and, more importantly, EIImtl is stable and
exhibits a good activity in this detergent.
The insertion of EIImtl, IICBmtl, and
IICmtl into the lipid bilayer of DMPC vesicles leads to a
decrease in Tm of the gel to liquid crystalline phase
transition, the development of a second component to the heat
absorption peak, and a general broadening of the transition. In the
case of IICmtl, approximately 40 lipid molecules per
inserted membrane protein molecule are withdrawn from the cooperative
phase transition. The same analysis applied to DMPC/bacteriorhodopsin
systems yields values of approximately 20-40 (30). Since the membrane
parts of EIImtl and bacteriorhodopsin are of similar size,
a comparable number of lipid molecules can be expected to bind to these
proteins when inserted into the membrane. The fact that this is indeed
observed and the fact that both proteins are enzymatically active
support our conclusion that EIImtl is inserted in a way
that is comparable with its physiological state.
The Assignment of Unfolding Transitions to Structural
Domains--
A critical step in the analysis of the data is the
assignment of the unfolding events to structural entities in the
protein. Although each of the three separate domains undergoes an
unfolding transition between 40 and 95 °C, we observed only two
peaks in this temperature interval. The removal of the A domain from
the intact protein leads to a large decrease of the transition at 62 °C (Fig. 2), and on the basis of this result it can be concluded that this transition is, at least partly, due to the unfolding of the A
domain. Furthermore, the Tm of this transition is independent
of the amount of mannitol or perseitol present, as is to be expected
for a part of the protein that is far removed from the mannitol binding
site. A very small transition remains around 60 °C that disappears
if the B domain is removed, indicating that this transition originates
in the B domain. Further evidence for the assignment of the first
transition to both B and A domain unfolding comes from the striking
resemblance of the deconvolution of the unfolding profiles of
IIBAmtl and the wild type enzyme (Fig. 6). The high
temperature transition for His-IICmtl at 76 °C is
assigned to the unfolding of the C domain. Removal of the B domain in
the presence of mannitol leads to a decrease in melting temperature and
a smaller width at half-height for this transition, which is most
convincingly seen in the comparison of samples before and after
treatment with trypsin (Fig. 8). This is discussed in detail below.
Modeling of the DSC Data--
The unfolding of the B and A domains
can be described by standard equations, derived from equilibrium
thermodynamics, since the transitions are reversible and independent of
the scanning rate. The unfolding of the C domain, however, is
kinetically controlled (both in the presence and absence of the
ligand), and therefore a model that specifically takes this into
account is the appropriate choice in a deconvolution type analysis. In
the presence of the substrate mannitol, the data can be described well
by the simple two-state irreversible model of Equation 2, as shown
under "Results." This model does not take into account effects of
dissociation of ligands or the monomerization of the EIImtl
dimer upon unfolding. The fact that the data can be described by this
model suggests that these events either do not take place or only
happen after the rate determining step in the unfolding reaction has
taken place. From our data, we cannot discriminate between these two
possibilities, and therefore we have chosen the simplest model that
accurately describes the data.
In the absence of the substrate, the simple two-state irreversible
model no longer holds, and a more complicated model has to be invoked
to describe the data. We have used the Lumry-Eyring model in this case,
since it can be regarded as intermediate between the two-state
equilibrium and irreversible models. We do not have evidence other than
the goodness of fit that this is appropriate, because the analysis of
scanning rate-dependent data in the absence of mannitol was
hampered by conformational heterogeneity of the C domain, obscuring the
shape and Tm of the main peak. However, the values for the
thermodynamic parameters obtained from an analysis assuming a two-state
reversible model, a two-state irreversible model, or the Lumry-Eyring
model did not vary much; the largest variations are found for the
transition characterized by the smallest enthalpy of unfolding (B
domain). This makes us confident that the basic assumption of our
analysis, the existence of three independent unfolding transitions, is
correct and that the parameters determined are reasonably accurate.
The analysis of the DSC data in this work yields values for Tm
and
H for the water-soluble A and B domain in the wild
type enzyme. The unfolding of IIAmtl, IIBmtl,
and IIBAmtl has been studied previously in detail but under
slightly different buffer conditions (14). The results obtained here
are, within the error of the experiment, in agreement with those data,
indicating that the presence of the DMPC vesicles does not influence
the stability of the A and B domains in the absence of the C domain. Also the addition of mannitol to IIBmtl in the absence of
the C domain does not alter the stability of this protein (data not
shown), and therefore changes in the stability or conformation of the
enzyme upon the addition of mannitol are all mediated through the C
domain.
The Thermal Stability of EIImtl--
The enthalpy of
unfolding of the C domain in EIImtl in the absence of
ligands, averaged over all estimates in Table I is 600 ± 70 kJ
mol
1 or 18 ± 3 J g
1. This value is
low compared with the average value of 33 J g
1 for
water-soluble proteins (35) but higher than the values obtained for
other membrane proteins such as cytochrome C oxidase from beef heart,
yeast, and Paracoccus denitrificans (11.3, 10, and 12 J
g
1, respectively; Refs. 36-39) or bacteriorhodopsin
(13-16 J g
1, depending on pH; Ref. 40). The low values
of the enthalpy of unfolding compared with water-soluble proteins have
been attributed to the higher stability of the membrane-embedded parts.
The stability of these parts is so high that they do not completely
denature but only partially unfold, leaving large parts of the
structure intact, thus contributing only moderately to the total
enthalpy of unfolding. In the case of EIImtl, this is
reflected in the change of the CD signal at 222 nm, the wavelength
specific for
-helices. Almost 60% of the signal at 30 °C is
still present after heating to 95 °C (Fig. 2), indicating that a
large part of the putative six transmembrane helices (19) is still
intact after heat denaturation. However, these structures only account
for approximately 50% of the total mass of the C domain; the remainder
may protrude into the aqueous phase. It is likely that this is the part
of the protein that is responsible for the observed unfolding
transition, explaining the somewhat higher enthalpy of unfolding
compared with other membrane proteins.
Domain Interactions in EIImtl--
The enthalpy of
unfolding of the transition assigned to the B domain in
EIImtl is also small, 18 ± 5 J g
1
averaged over the entire data set of Table I. This small value was also
reported in an earlier paper on the interactions between the A and B
domains (14), where it was attributed either to the high flexibility of
the B domain necessary for the domain to be able fulfill its phosphoryl
group transfer function or to missing B/C domain interactions. Of these
two possibilities, the latter now seems the more likely. Fig. 8 shows
the raw DSC data for the unfolding of EIImtl and
tryp-IICmtl, obtained from the same preparation of
reconstituted enzyme. This type of comparison ensures that only those
differences arising from the removal of the A and B domains will be
observed. In each case, the enthalpy of unfolding of the C domain in
the absence of the B domain is lower than when the B domain is
covalently attached, most notably so when the substrate mannitol is
bound. In the latter case, the Tm of the C domain in the wild type enzyme is ~2.7 °C higher, and
H is ~200 kJ
mol
1 larger than in the case of tryp-IICmtl
(Fig. 8 and Table I). This suggests that an interaction between the C
and B domains of EIImtl exists, which is only broken upon
unfolding of the C domain. However, at the Tm of the C domain,
the unfolding of the B domain has already taken place, and therefore
the results can only be explained in two ways: in the presence of
mannitol a part of the B domain undergoes an unfolding transition that is strongly coupled to the unfolding of the C domain and therefore cannot be observed in its absence, or the C domain interacts with the
unfolded polypeptide chain from the B domain. In the latter case,
however, it is difficult to understand why the effect is so much larger
in the presence of mannitol than in its absence.
On the basis of the large difference of the heat capacity increment of
binding to wild type EIImtl and tryp-IICmtl,
determined from titration calorimetry (
4.0 and
0.5 kJ
K
1 mol
1, respectively), we have recently
proposed a model for EIImtl in which the binding of
mannitol leads to the formation of a complementary surface between the
B and C domains (33). The newly formed interaction could result either
from folding of previously unstructured parts of the polypeptide chain
or from docking of two existing surfaces in the protein. The large
change of the enthalpy of unfolding of the C domain in the wild type
enzyme upon the addition of mannitol compared with
tryp-IICmtl from the same preparation seems to suggest the
former possibility as the most likely, with the thermal unfolding of
the newly formed structure coupled to the partial unfolding of the C
domain. This interpretation would also explain the very low enthalpy of
unfolding observed for the B domain if it is assumed that the
unstructured part is localized in this part of the protein.
Unfortunately, the quality of the data and the small enthalpy of
unfolding of the B domain do not allow us to accurately assess the
effects of the binding reaction on this transition.
The results obtained in the presence of perseitol differ considerably
from those obtained after the addition of mannitol. In general, the
effects are much smaller; an increase of 0.5-1 °C for Tm
and 6-12% for
H for the wild type enzyme are observed.
In fact, these differences are so small that we cannot be sure whether
perseitol is still bound at the melting temperature of the C domain,
although binding of perseitol at room temperature was confirmed by flow
dialysis in a competition experiment with radioactively labeled
mannitol (data not shown). So despite the fact that the thermodynamics
of binding of the two compounds, as measured in inside-out membrane
vesicles by titration calorimetry, are almost identical (33), the
binding of mannitol leads to a much larger shift in Tm and
H than the binding of perseitol. It therefore appears
that the binding proceeds in two steps, with the second, slow, step
leading to a stabilization of the complex and the change in Tm
and
H and only occurring in the case of mannitol. Indeed
such a two-step mechanism has been proposed from an analysis of the
binding kinetics of mannitol and perseitol (41), the second step being
a conformational change to an occluded state in which mannitol is not
accessible from either side of the membrane. It was also proposed that
the change to the occluded state cannot take place when perseitol is
bound, presumably because of steric repulsion with the C-7 moiety of this ligand (34).
Interactions between the B and C domains of EIImtl have
been demonstrated in a number of studies. Lolkema et al.
(12) reported that the mannitol translocation rate catalyzed by the C
domain increases by 3 orders of magnitude upon phosphorylation of the B
domain, while Boer et al. (13) showed a dependence of the kinetics of mannitol binding on the nature of the side chain at position 384, normally the cysteine at the active site of the B domain.
These data are in agreement with our findings here and support the
proposed model. A definitive answer to questions regarding the
mechanism of substrate binding and transport, however, awaits the
elucidation of the structure of this enzyme in its substrate bound and
free state.
We thank Prof. M. Blandamer (University of
Leicester, United Kingdom) for many useful discussions and for making
available a calorimeter in the early stages of this study.