From the Department of Biochemistry and Groningen Biomolecular
Science and Biotechnology Institute (GBB), University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
This paper reports that the aggregation state of
a membrane protein can be changed reversibly without the use of
chaotropic agents or denaturants by altering the attractive
interactions between micelles of polyethylene glycol-based detergents.
This has been documented using mannitol permease of
Escherichia coli (EIImtl), a protein
whose activity is dependent on the dimerization of its
membrane-embedded domains. We show that the driving force for the
hydrophobic interactions responsible for the dimerization can be
decreased by bringing the protein into a less polar environment. This
can be done simply and reversibly by increasing the micelle cluster
size of the solubilizing detergent since the micropolarity in the
micelle decreases upon clustering and is directly related to the
cluster size.
The micelle cluster size was varied at a fixed temperature by adding
sodium phosphate or a second detergent with a distinct clustering
behavior, and the changes were quantified by quasi-elastic light
scattering and by determining the cloud point or demixing temperature
(Td) of the detergent. Maximal EIImtl
activity was found when no micelle clustering occurred, but the activity gradually decreased down to 5% of the maximal activity with
increasing cluster size. The inactivation was found to be completely
reversible. The kinetics of heterodimer formation were also
significantly affected by changes in the micelle cluster size as
expected. Increasing the cluster size resulted in faster formation of
functional heterodimers by increasing the rate of homodimer
dissociation. This phenomenon should be generally applicable to
controlling the oligomeric state of membrane-bound proteins or even
water-soluble proteins if their subunit association is dominated by
hydrophobic forces.
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INTRODUCTION |
It is well documented that the micropolarity of
PEG-based1 detergent micelles
decreases when they form clusters and that this decrease is directly
related to the micelle cluster size (1-3). In principle, this decrease
in micropolarity should be able to be used to transfer a membrane
protein, solubilized by such a detergent, to a less polar environment.
Only PEG-based detergents show this clustering behavior. It can be
induced by heating where, at a certain temperature, the cloud point or
demixing temperature (Td), the cluster size reaches
a value which initiates separation of the micellar phase into two new
micellar phases, one consisting of the micellar clusters and one
containing a low concentration of micelles (2). Phase separation or
demixing of PEG-based detergents into two new micellar phases at
Td upon heating has been studied in detail with
various techniques such as light, neutron and x-ray scattering,
viscosity measurements, and NMR spin lattice relaxation (2, 4-8). Such
studies suggest that the intermicellar forces become stronger upon
heating due to a decrease of the hydration of the PEG chains. This
leads to stronger van der Waals interactions between the micelles and
the formation of micelle clusters. It is believed that the size of the
micelle does not change upon heating (7), but the size of the clusters
increases asymtotically and follows a power-law given by
(Td
T)/Td (4, 6).
The attractive forces between micelles can also be increased at a fixed
temperature, T, by the addition of certain inorganic salts,
especially phosphate or fluoride salts, or by mixing with a detergent
with a lower Td (2). Whether one increases
T or lowers Td, the result in the same,
the interval Td
T decreases, and as
the two approach one another, the micelle cluster size increases until,
at T = Td, phase separation occurs. This demixing property has been used in biochemical studies to separate
hydrophobic proteins from more hydrophilic ones (9). Hydrophobic
proteins concentrate in the detergent-rich phase upon heating the
detergent solution above Td. After centrifugation, this phase can be easily separated from the aqueous phase, containing the hydrophilic proteins. Triton X-114 with Td = 22 °C is often used in such procedures.
Here we demonstrate that these same intermicellar attractive forces can
be used in a more subtle way to disrupt the hydrophobic forces
responsible for subunit interactions and thereby reversibly control the
association state of EIImtl in PEG-based detergents.
EIImtl is inactive as a monomer and phophorylates mannitol
when in the dimeric state. Hydrophobic forces are involved in the
dimerization process (10). Both the activity of EIImtl and
the rate of formation of EIImtl heterodimers can be
controlled by choosing a specific micellar cluster size. The results
are explained by relating the decrease in micropolarity of the micelles
upon clustering with the decrease in the driving force for hydrophobic
bonding, the interactions responsible for EIImtl
dimerization.
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EXPERIMENTAL PROCEDURES |
Materials--
dPEG, C10E6,
C10E5, and C10E4 were
supplied by B. Kwant (Kwant High Vacuum Oil Recycling and Synthesis,
Bedum, The Netherlands). dMal was from Sigma.
Na3PO4 solutions were adjusted to pH = 7.6 with concentrated H3PO4.
Q-Sepharose Fast Flow and S-Sepharose Fast Flow were from Pharmacia
(Sweden); hexyl-agarose was from Sigma.
D-[1-3H]Mannitol (976.8 GBq/mmol) was from
NEN Life Science Products. EI and HPr were purified as described
previously (11-13). All other reagents were analytical grade. The
purification of EIImtl was as described previously for
EIImtl(C384S) (14).
Mannitol Phosphorylation Assays--
The
PEP-dependent mannitol phosphorylation activity of
EIImtl was measured as described (15). The assay buffer
contained 25 mM Tris·HCl, pH 7.6, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 0.25% (v/v) detergent, 20 µM HPr, and 0.33 µM EI. The
reaction was started with 60 µM
[3H]mannitol (1 mM for the experiments
presented in Fig. 2). Concentrated stock solutions (1-2
µM) of EIImtl were used, which were diluted
1,000-fold when the enzyme was assayed. Therefore, the influence of the
dPEG (Td = 58 °C) in this stock solution during
the assay conditions can be neglected. All reactions were performed at
30 °C in buffers with Td> 30 °C. Changes in
Td (and thus Rh) are known to
occur abruptly (7) as was checked under the conditions used in this
study.
Concentration Determinations on EIImtl
Samples--
The EIImtl concentrations were determined by
flow dialysis which quantitates the number of mannitol binding sites
(16), assuming one high affinity binding site (KD
~100 nM) per EIImtl dimer in accordance with
the observations of Pas et al. (14).
Light Scattering Experiments--
Light scattering experiments
were performed at 30 °C by using a DynaPro-801TC instrument (Protein
Solutions Inc., Charlottesville, VA), equipped with a thermostated
cell. Detergent (0.25%, v/v) was dissolved in 25 mM
Tris·HCl, pH 7.6, 5 mM DTT, 5 mM
MgCl2, and 5 mM PEP. In one case, 0.5% dPEG
was used since no stable signal at 0.25% was observed. Solutions were
filtered through 0.1 µm Anotop10 filters (Whatman). Data were
analyzed using the software supplied by the manufacturer. Data could be
resolved by the theoretical single exponential autocorrelation function (monomodal analysis), indicating that the solutions were monodisperse. Each sample was measured at least seven times. Standard deviations in
Rh were 0.1 nm or less.
Cloud points (± 0.5 °C) were determined by heating a detergent
solution in a test tube with a thermometer.
 |
RESULTS |
Effect of Micelle Cluster Size on the Activity of
EIImtl--
Micelles clustering is reflected in an
increase in the apparent hydrodynamic radius (Rh) of
the micelle, and this occurs as the temperature of the solution
approaches the cloud point (Td). The phosphorylation
activity of EIImtl has been determined at 30 °C in
PEG-based detergent mixtures with various cloud points, and the
apparent hydrodynamic radii of the micelle clusters of these solutions
under the assay conditions has been determined by quasi-elastic light
scattering experiments. The Td of PEG-based
detergents was lowered by increasing the concentration of sodium
phosphate. Addition of up to 250 mM Na3PO4 (pH = 7.6) lowered the
Td of dPEG from 58 to 33 °C with a corresponding
increase in Rh from 3.4 to 8.2 nm (Fig.
1A,
). A plot of the enzyme
activity against Rh shows that increased values of
Rh result in a lowering of the enzyme activity down
to 10% of the activity found in dPEG with Rh = 3.4 nm (Fig. 1A,
). The activity in the latter buffer
corresponds with the highest specific activity found for EIImtl solubilized in a detergent (2000-3000
nM/min/nM enzyme when assayed with 60 µM mannitol). Increasing the dPEG concentration from 0.25 to 1% at 200 mM Na3PO4 did not
change the EIImtl activity (not shown). Fig. 1B
shows that the sensitivity of the EIImtl activity to a
change in Td was not only observed in the
polydisperse detergent, dPEG, but also in the monodisperse PEG-based
detergent, C10E5. The activity of
EIImtl in buffer containing pure
C10E5 (Td = 44 °C) was
the same as that found in dPEG with Td = 58 °C
(17). Lowering the Td via the introduction of
Na3PO4 again resulted in increased values of
Rh and a lowering of the EIImtl
activity. Finally, the Td of these solutions could
also be changed by mixing with another PEG-based detergent with a
different Td rather than with
Na3PO4. Fig. 1C shows that mixing C10E5 (Td = 44 °C) with
C10E4 (Td = 16 °C) also resulted in an increase in Rh and a decrease in
EIImtl activity.

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Fig. 1.
Relationship between the cloud point
(Td) and apparent hydrodynamic radii
(Rh) of solution containing dPEG or
C10E5 and the PEP-dependent
phosphorylation activity of EIImtl in these solutions.
Rh measurements were performed at 30 °C on a
solution containing 25 mM Tris·HCl, pH 7.6, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 0.25% (v/v) detergent, and eventually Na3PO4 or C10E4 to
lower the Td. The Td measurements were performed on the same solutions. The PEP-dependent
phosphorylation activity of EIImtl was determined by
incubating the enzyme for 5-10 min at 30 °C in 25 mM
Tris·HCl, pH 7.6, 5 mM DTT, 5 mM
MgCl2, 5 mM PEP, 0.25% (v/v) detergent, 20 µM HPr, 0.33 µM EI, and eventually
Na3PO4 or C10E4 to
lower the Td. The reaction was started after the
incubation period by adding [3H] mannitol with a final
concentration of 60 µM (final volume 100 µl). At four
different times, 20 µl-aliquots were taken, and the amount of
mannitol-1-phosphate was quantified. A, relationship between
Rh and Td ( ) and the activity
of EIImtl (1.0 nM) versus Rh
in these buffers with 0.25% dPEG and various amounts of
Na3PO4 to lower the Td
( ). The Td decreased linearly from 58 °C at 0 mM Na3PO4 to 33 °C at 250 mM. B, relationship between
Rh and Td ( ) and the activity
of EIImtl (0.08 nM) versus
Rh in buffer with 0.25% C10E5 and
various concentrations of Na3PO4 to lower the
Td ( ). The Td decreased
linearly from 44 °C at 0 mM to 31 °C at 250 mM Na3PO4. C,
relationship between Rh and Td
( ) and the activity of EIImtl (0.08 nM)
versus Rh in buffer with 0.25% pure
C10E5 or a mixture of
C10E5/C10E4 ( ). The
Td was lowered by increasing the percentage of
C10E4. The Td decreased linearly from 44 °C for pure C10E5 to
31 °C for 60% C10E5, 40% C10E4 (v/v). The specific enzyme activities
under these conditions in the absence of Na3PO4
or C10E4 were 2270, 2965, and 3090 nM mannitol-1-phosphate/min/nM enzyme in Fig.
1, A, B, and C, respectively.
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The decreasing EIImtl activity with increasing
Rh should be reflecting the change in
oligomerization state of EIImtl from active dimers into
less active or inactive monomers. This is supported by the behavior of
the specific activity of EIImtl as a function of the enzyme
concentration in buffer containing detergent with high (Fig.
2,)
) and low (
)
Td. These experiments were performed under
Vmax conditions (1 mM mannitol instead of 60 µM), which allowed the enzyme to be assayed
between 0.05 and 50 nM at a single mannitol concentration.
In dPEG with a Td = 58 °C (Fig. 2,
), an
activity of ± 4000 nM/min/nM enzyme was
found that was constant between 0.05-5 nM
EIImtl. In dPEG buffer with 200 mM
Na3PO4 (Td = 35 °C) (Fig. 2,
), the specific activity increased from less than 670 nM/min/nM enzyme at 0.05 nM enzyme
to 2000 nM/min/nM enzyme at 50 nM
EIImtl, confirming that lowering the Td
results in dissociation of active dimers into less active or inactive
monomers. Increasing the EIImtl concentration shifts, by
mass action, the equilibrium back to the active, dimeric form.

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Fig. 2.
Relationship between the EIImtl
concentration and the PEP-dependent specific
phosphorylation activity of EIImtl in assay buffer with
various Td. The specific activity of
EIImtl was determined at 30 °C by incubating the enzyme
for 5-10 min in 25 mM Tris·HCl, pH 7.6, 5 mM
DTT, 5 mM MgCl2, 5 mM PEP, 0.25% dPEG (v/v), 20 µM HPr, 0.33 µM EI, and 200 mM ( ) or 0 mM Na3PO4 ( ). Td is 38 and 58 °C, respectively. The
reaction was started after the incubation period with 1 mM
mannitol.
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The reversibility of the change in association state upon a change in
Td has been demonstrated in
C10E5 buffer. EIImtl (0.8 nM) was incubated for 10 min at 30 °C in buffer
containing 120 mM Na3PO4
(Td = 34 °C) to cause the protein to dissociate. One portion of this solution was then diluted 10-fold with assay buffer
containing the same Na3PO4 concentration while
another portion was diluted with assay buffer lacking
Na3PO4. After 10 min at 30 °C, the
phosphorylation reaction was started by adding 60 µM
mannitol. As expected, a low activity (240 nM/min/nM enzyme) was found for the enzyme
incubated with buffer containing 120 mM
Na3PO4 and diluted into the same buffer. A high
activity (2055 nM/min/nM enzyme) was observed
for the enzyme incubated with buffer containing 120 mM
Na3PO4 but then diluted with the low salt
buffer. The same high activity was measured for the control enzyme that had not been exposed to high Na3PO4 in the
incubation or dilution phase. Therefore, the effect of a variation in
Td on the activity of EIImtl is
completely reversible.
Fig. 3 presents the effect of a rapid
change in Td on the EIImtl activity. The
enzyme was preincubated in a dPEG buffer with high Td (58 °C) along with PEP, E1, and HPr to
generate phosphorylated EIImtl (P-EIImtl) and
then diluted into buffers with mannitol and dPEG with the same or lower
Td. The control (Fig. 3,
) diluted into dPEG
buffer with a Td of 58 °C showed a rapid linear increase of mtl-P with time. But when the samples were diluted into
dPEG buffers with a lower Td, the time dependence showed two phases. (i) In the first 4-5 min, the specific activity gradually decreases. (ii) After 5 min, the specific activity was stable, the lower the Td then the lower the
activity.

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Fig. 3.
Change in PEP-dependent
phosphorylation activity of EIImtl upon a rapid change in
apparent hydrodynamic radii (Rh) of the assay
buffer. 1.4 nM EIImtl was incubated at
30 °C in 25 mM Tris·HCl, pH 7.6, 5 mM DTT,
5 mM MgCl2, 5 mM PEP, 0.25% (v/v)
dPEG (Td = 58 °C), 20 µM HPr, and
0.33 µM EI for 5 min. The reaction was started after a
1.4-fold dilution in a solution containing mannitol and
Na3PO4 resulting in final
Na3PO4 concentrations of 0 ( ), 215 ( ),
235 (×), and 250 mM ( ). The Rh
values were 3.4, 6.4, 7.3, and 8.2 nm, respectively, and the
corresponding Td were 58, 37, 35, and 33 °C.
Final mannitol concentration was 60 µM.
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Since the new Td values were reached immediately
after mixing (see "Experimental Procedures"), these phases most likely reflect the time-dependent changes in the
association state of the enzyme. During the first phase, the
concentration of monomer in solutions with lower Td
increases until a new monomer-dimer equilibrium establishes itself.
After 5 min, the new equilibrium is established, and the rates are
linear with time. The results presented so far show a reversible
dissociation of active EIImtl dimers into less active
monomers upon increasing Rh or correspondingly
decreasing Td. In the next section, the effect of
variation of Td on the formation of heterodimers will be presented.
Effect of Variation in Micelle Cluster ;size in Complementation
Assays--
The influence of micelle cluster size on the kinetics of
heterodimer formation of EIImtl has been studied by using
two EIImtl mutants, each of which lack
PEP-dependent activity but become active upon formation of
a heterodimer (complementation assay, see Fig.
4, inset). One such couple is
EIImtl (G196D), with drastically reduced binding affinity
for mannitol, and IICmtl,
2 which is inactive by virtue
of the missing A and B domains (18). Upon formation of a heterodimer,
the phosphoryl group can proceed from PEP via HPr and E1 and the A and
B domains of EIImtl to mannitol bound in the C domain (see
"Discussion"). Fig. 4 follows the rate of appearance of
phosphorylation activity when these two enzymes are incubated in
detergent of varying Td. EIImtl (G196D)
and IICmtl were incubated for 10 min in phosphorylation
buffer with dPEG and various concentrations of
Na3PO4 shifting the Td between 58 and 35 °C (Rh = 3.4-7.3 nm). The
reaction was started with mannitol, and the accumulation of
mannitol-1-phosphate over time was monitored. In detergent solutions
with a high Td (58 °C), a slow initial activity
was observed that gradually increased in time and then became constant
(Fig. 4,
). When the Td was decreased, initial
activities increased, resulting in faster attainment of a constant
PEP-dependent phosphorylation activity (
). In a
detergent solution with a Td of 35 °C, a high and
constant activity was observed immediately (Fig. 4, ×). These results
suggest a more rapid formation of functional heterodimers from inactive
homodimers in detergent solutions with a lower
Td.

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Fig. 4.
Dependence of the rate of appearance of the
phosphorylation activity of EIImtl
(G196D)/IICmtl heterodimers on the apparent hydrodynamic
radii (Rh) of the buffer.
EIImtl(G196D) (20 nM) and IICmtl
(100 nM) were incubated at 30 °C for 10 min in buffer
containing 25 mM Tris·HCl, pH 7.6, 5 mM DTT,
5 mM MgCl2, 5 mM PEP, 0.25% (v/v)
dPEG, 20 µM HPr, 0.33 µM EI, and 0 mM ( ), 90 mM ( ), or 235 mM
Na3PO4 (×) with Td = 58, 50, and 35 °C corresponding to Rh = 3.4, 4.1, and
7.3 nm, respectively. The reaction was started with 60 µM
mannitol. Inset, schematic representation of the
complementation of IICmtl (1) and
EIImtl(G196D) (2) yielding EIImtl
(G196D)/IICmtl heterodimers (3). The nomenclature of the
EIImtl domains (A, B, C) has been used.
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The following experiments were performed to examine the formation of
heterodimers in more detail (Fig. 5).
EIImtl (G196D) and IICmtl were incubated for 10 min in buffer containing 25 mM
Na3PO4, 5 mM DTT, and 0.25% dPEG
(Td = 58 °C). This solution was diluted 20-fold
in the same buffer that now contained 5 mM MgCl2 and 5 mM PEP, 0.33 µM EI,
and 20 µM HPr for an additional 5 min to generate
P-EIImtl. Upon addition of mannitol, a low initial activity
was found, which gradually increased with time (Fig. 5A,
). But if the initial solution was diluted in buffer with high
Na3PO4 to produce a low Td
(37 °C), maximal phosphorylation activity was observed immediately
(Fig. 5A, ×). Similarly, if the incubation was performed under low Td conditions and the assay at either high or low Td conditions, maximal initial activity was
found immediately (Fig. 5A,
and
, respectively).
Therefore, as long as both enzymes have been incubated with each other
under low Td conditions, formation of heterodimers
is complete within the 5-min incubation period and does not reverse
when the Td is subsequently increased. Although more
inactive monomers are expected in detergent solutions with a low
Td (Figs. 1 and 3), the high protein concentration
used in these experiments will result in high heterodimer
concentrations. These experiments show that formation of heterodimers
is very fast when the Td of the buffer is low;
moreover, the specific activity of the heterodimer is not affected at
high enzyme concentrations when the Td is varied
between 37 and 63 °C (Rh = 6.4-3.4). Incubation
of EIImtl with mannitol has been reported to stimulate the
dissociation of EIImtl dimers (19) and could also be
expected to stimulate heterodimer formation. To test this,
EIImtl (G196D) and IICmtl were incubated for 10 min at 30 °C in assay buffer with dPEG (Td = 58 °C) that included PEP, EI, and HPr. Upon starting the reaction
with mannitol, a low initial activity was found (Fig. 5B,
). However, when mannitol replaced PEP in the incubation step and
the reaction was started with PEP, maximal activity was found almost
immediately, indicating that formation of heterodimer was nearly
completed at the moment that PEP was added (Fig. 5B,
).
When both PEP and mannitol were omitted during the incubation of the
two enzymes, and the reaction was started with these two components, a
similar pattern was found as when the reaction was started with
mannitol (Fig. 5B, ×). The similarity in activity profiles
of
and × shows that prior phosphorylation of
EIImtl does not affect the process of heterodimer formation
as probed under the conditions used.

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Fig. 5.
Dependence of the rate of appearance of the
phosphorylation activity of EIImtl
(G196D)/IICmtl heterodimers on the apparent hydrodynamic
radii (Rh) of the micelles and the presence of
mannitol and/or PEP. A, EIImtl (G196D) (200 nM) and IICmtl (1.45 µM) were
incubated at 30 °C for 10 min in buffer containing 0.25% dPEG, 5 mM DTT, and 25 mM
Na3PO4 (solution 1) or 215 mM Na3PO4 (solution 2). , solution 1 was
diluted 12-fold into 25 mM Na3PO4,
pH 7.6, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 0.25% dPEG, 20 µM HPr, 0.33 µM EI, and after 5 min at 30 °C, the reaction was
started with 60 µM mannitol (final Rh
and Td are 3.4 nm and 58 °C, respectively). ×,
solution 1 was diluted 12-fold into 215 mM
Na3PO4, pH 7.6, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 0.25% dPEG, 20 µM HPr, 0.33 µM EI, and after 5 min, the
reaction was started with mannitol (final Rh and
Td are 6.4 nm and 37 °C, respectively). ,
solution 2 was diluted 12-fold into 25 mM
Na3PO4, pH 7.6, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 0.25% dPEG, 20 µM HPr, 0.33 µM EI,, and after 5 min at
30 °C, the reaction was started with 60 µM mannitol
(final Rh and Td are 3.4 nm and
58 °C, respectively). , solution 2 was diluted 12-fold into 215 mM Na3PO4, pH 7.6, 5 mM
DTT, 5 mM MgCl2, 5 mM PEP, 0.25%
dPEG, 20 µM HPr, 0.33 µM EI, and after 5 min, the reaction was started with mannitol (final
Rh and Td are 6.4 nm and
37 °C, respectively). B, 20 nM
EIImtl((G196D) and 100 nM IICmtl
were incubated at 30 °C for 10 min in 25 mM Tris·HCl,
pH 7.6, 5 mM MgCl2, 5 mM DTT, 5 mM PEP, 0.25% dPEG (Td = 58 °C), 20 µM HPr, and 0.33 µM EI ( ). As shown in
, 5 mM PEP was replaced by 60 µM mannitol,
whereas in ×, no PEP or mannitol was present in the incubation period.
The reaction was started with 60 µM mannitol ( ); 5 mM PEP ( ), or 60 µM mannitol and 5 mM PEP (×).
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Finally, we have investigated the formation of heterodimers in
n-decyl
-D-maltopyranoside (dMal), a
detergent which does not form micelle clusters and experience phase
separation. The specific activity of EIImtl in 3 mM dMal is the same as when the enzyme is solubilized in 0.25% dPEG (Td = 58 °C) or
C10E5 (Td = 44 °C), detergents in which maximal EIImtl activity is observed.
EIImtl (G196D) (20 nM) and IICmtl
(145 nM) were incubated for 5 min at 30 °C in assay
buffer containing 0.25% dPEG and 140 mM
Na3PO4 (Td = 44 °C)
(
), 3 mM dMal (
), 6 mM dMal (×), or 6 mM dMal and 140 mM
Na3PO4 (
), and the phosphorylation assay was
started with mannitol (Fig. 6). While a
high activity was observed in dPEG, replacement of this detergent by
dMal results in an 8-16 times lower activity, indicating that heterodimer formation was strongly suppressed in dMal presumably via suppression of homodimer dissociation.

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Fig. 6.
Effect of detergent on the
PEP-dependent phosphorylation activity of
EIImtl((G196D)/IICmtl heterodimers. 20 nM EIImtl (G196D) and 145 nM
IICmtl were incubated for 5 min at 30 °C in 25 mM Tris·HCl, pH 7.6, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 20 µM HPr, 0.33 µM EI containing 0.25% dPEG
( ) and 140 mM Na3PO4
(Td = 44 °C), 3 mM dMal ( ), 6 mM dMal (×), and 6 mM dMal ( ) and 140 mM Na3PO4, respectively. The
reaction was started with 60 µM mannitol.
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DISCUSSION |
Numerous papers have been dedicated to study the kinetics of
mannitol translocation and phosphorylation of Escherichia
coli, EIImtl (20). Various biophysical techniques like
radiation inactivation (21), size-exclusion chromatography (22, 23),
chemical cross-linking (24), Fourier transfer infrared spectroscopy
(25), and single tryptophan fluorescence spectroscopy (17) have been
used to study the domain structure and oligomerization state of
EIImtl. While some work was carried out on proteoliposomes
of EIImtl, most of these studies have been performed while
the enzyme was solubilized by a PEG-based detergent, especially Lubrol
PX and dPEG. EIImtl has been observed both as a monomer and
a dimer (19, 21, 26). The crucial contacts resulting in the dimer
appear to be between the hydrophobic C domains (23, 27). Stephen and
Jacobson (19) found that, upon mild extraction of the enzyme from
vesicles, the percentage of dimer increased with increasing ionic
strength but decreased upon introduction of the PEG-based detergent,
Lubrol PX, or mannitol or upon phosphorylation of the enzyme. Kinetic experiments have shown that the dimer is primarily responsible for the
PEP-dependent phosphorylation and mtl/mtl-P exchange (10, 28). The formation of heterodimers is another approach used to show
that EIImtl dimers are functional (18, 27, 29, 30). Mutants
of EIImtl, each inactive by virtue of a mutation in the A,
B, or C domain, could be reactivated by mixing with another mutant form
carrying the mutation on another domain (see Fig. 4, inset).
Apparently, the phosphoryl group can cross the dimer interface when
proceeding from the A domain, via the B domain, to mannitol bound at
the C domain.
The following evidence indicates that an increased tendency of the
micelles to form clusters (high Rh conditions) induces monomerization of EIImtl dimers. 1) A lowering of
the EIImtl activity was observed with increasing
Rh. 2). The specific activity of EIImtl,
under conditions of high Rh, increased with
increasing enzyme concentration. A higher percentage of (active) dimers
is expected upon an increase in enzyme concentration, due to mass action. Under conditions of minimal Rh (maximal
Td), maximum activity was observed at all
EIImtl concentrations monitored, indicative of a completely
dimeric enzyme. The increase of specific activity which Boer et
al. (27). observed upon addition of high concentrations of
IICmtl to active EIImtl can also be explained
in these terms; a population of EIImtl monomers were
present under their specific detergent conditions, which were titrated
by high concentrations of IICmtl to form active
heterodimers. 3) The rates of heterodimer formation, from two
homodimers, were significantly increased under conditions of high
Rh due to the increased rate of dissociation of the
homodimers. In line with this, the kinetics of heterodimer formation
were also increased by the introduction of mannitol, a substrate known
to dissociate EIImtl (19).
The relationship between the EIImtl activity and the
Td of dPEG or C10E5 was
found to be similar, whether the lowering of Td was
caused by C10E4 or
Na3PO4 (Fig. 1). This, and the observation that
the EIImtl activity at low Td can be
completely converted to the high activity found in buffer with a high
Td, supports the view that the enzyme is sensitive
to changes in the micellar properties rather than the chemical
composition of the detergent. EIImtl exhibits maximal
activity when solubilized in dPEG, C10E5, or C10E6 (not shown). The micellar size of dPEG
(Td = 58 °C), C10E5
(Td = 44 °C), and C10E6
(Td = 62 °C) at 30 °C, where almost no
clustering of micelles is expected, differs significantly
(Rh is 3.4 nm, 4.9 nm, and 2.9 nm, respectively). Apparently, the size of the micelle is not important for the monomer to
dimer equilibrium. The concentration of micelles is 4 orders of
magnitude higher than the enzyme concentration used in these experiments. Clustering of micelles, including the ones containing EIImtl, will result in partial solvation of the
EIImtl-detergent micelles by other detergent micelles
instead of by bulk water. This shift in solvation is expected to
increase with increasing micelle cluster size. Since hydrophobic forces
are involved in the formation of the EIImtl dimer (10, 19),
the shift to a more hydrophobic environment most likely explains the
dissociation of dimers.
Recently, Boer et al. (18). demonstrated that the
simultaneous expression of EIImtl (G196D) and
IICmtl in E. coli. resulted in cells that were
able to take up mannitol. No mannitol uptake was observed if the
mutants were expressed separately. This experiment showed that
heterodimer formation between EIImtl (G196D) and
IICmtl occurs in vivo. Heterodimer formation
also proceeds in dPEG and C10E5 and is
facilitated by an increase of Rh (decrease in
Td). Almost no heterodimer formation was found in dMal although the wild-type enzyme is highly active when solubilized in
this detergent. Therefore, PEG-based detergents are probably the best
class of detergents for mechanistic investigations on EIImtl since the activity is comparable with the activity
for EIImtl in vesicles (31), and heterodimer formation is
readily achieved in these detergents.
In conclusion, a "new" detergent parameter relevant for membrane
protein chemistry, i.e. the tendency of micelles to cluster, has been shown to control the oligomerization state of
EIImtl. Proper control of this variable can result in
almost complete monomerization or dimerization of the protein. It
provides a unique tool to study mechanistic aspects of membrane protein
oligomerization.
We thank B. Kwant for supplying the various
detergents used in this study, T. Pijning for assistance in the light
scattering experiments, and M. G. Sonetti for the purification of
EIImtl.