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INTRODUCTION |
The cytotoxic function of the bactericidal protein colicin E1 is
found in the ability of the protein to form voltage-gated, ion-conductive channels within the cytoplasmic membrane of susceptible cells. However, despite recent strides in the elucidation of the soluble structures of whole colicin (1) and various channel domain
fragments (2-4), the current state of knowledge of the structure and
function of membrane-associated colicins is modest at best. Colicin E1,
secreted by Escherichia coli that possess the naturally
occurring colE1 plasmid, consists of three functional domains: the translocation, receptor-binding, and channel-forming domains. Initially, the receptor-binding domain (5) interacts with the
vitamin B12 receptor of target cells (6). After
recognition, the translocation domain interacts with the
tolA gene product, which permits the translocation of
colicin E1 across the outer membrane and into the periplasm (7). In the
periplasm the channel domain undergoes a conformational change to an
insertion-competent state and then inserts spontaneously into the
cytoplasmic membrane, forming an ion channel. The channel translocates
monovalent ions, resulting in the dissipation of the cationic gradients
(H+, K+, Na+) of the cell, causing
depolarization of the cytoplasmic membrane (8, 9). In an effort to
compensate for the membrane depolarization effected by the colicin E1
channel, Na+/K+ ATPase activity is increased in
the target cell, resulting in the consumption of ATP reserves, without
concomitant replenishment (10). The final outcome is host cell death.
Treatment of colicin E1 with thermolysin (11) generates a compact
fragment composed of the COOH-terminal channel-forming domain, or
"channel peptide," which forms functional channels in osmotically
shocked cells (10), membrane large unilamellar vesicles
(LUVs),1 (12) and within
planar bilayers (13). Before insertion into model membrane systems, low
pH-induced activation (14) is required.
Previous studies have generated evidence in support of both a monomeric
and an oligomeric colicin E1 channel; however, the number of
channel-forming domains required to form a functional colicin E1
channel has not been clearly established. In favor of a monomeric
channel, Jacob et al. (15) demonstrated that cytotoxicity is
first order with respect to colicin E1 concentration and proposed that
a single channel is capable of killing a cell. In addition, the
in vivo (16) and in vitro (17) efflux of K+ and intravesicular markers through colicin E1 channels
also obey first-order kinetics. The implications of these kinetic
studies were confirmed by Levinthal and co-workers (18), who
demonstrated a 1:1 stoichiometry for active peptides forming channels
in both small unilamellar vesicles and LUVs based on the discharge of a
membrane potential in these target membrane systems. Also, steady-state conductance through colicin E1 channels is linear with respect to
colicin E1 concentration (12). Finally, ultracentrifugation sedimentation equilibrium studies (19) and hydrodynamic Stokes radii
comparison (20) indicated that colicin E1 is monomeric in solution over
a broad pH range.
Despite this evidence in support of a monomeric channel, the ability of
the colicin E1 channel to accommodate large organic molecules,
including NAD+ (21), is not easily reconcilable with a
monomeric structure. Carboxyl-terminal colicin E1 peptides, with as few
as 88 amino acid residues, can form modestly functional channels
in vitro (22); however, it is unlikely that a channel formed
by such a short fragment would have a diameter sufficient to permit the passage of large organic molecules. More recently, the dimensions of
the colicin Ia channel were mapped using a method based on the filling of the channel by nonelectrolytes from either side of the
membrane. The channel was found to have cis and
trans entrance diameters of 18 and 10 Å, respectively, with
a constriction size of 7 Å (23). Notably, a channel with such a large
lumen is not consistent with a two- or four-helix bundle assembled from
a monomeric protein.
The colicin E1 channel peptide is a globular protein that consists of
10 
-helices organized into three layers (3). Layer A is composed of
helices 1, 2, and 10; layer B contains helices 8 and 9; and layer C
contains helices 3, 4, 6, and 7. Helices 5a and
5b transverse part of the protein structure by running across the layers and are not included in any of the designated layers.
The orientation of the channel domain helices in the membrane-bound state of the channel peptide has been the subject of a number of
investigations. Hydrophobic helices 8 and 9 and the loop connecting them have been proposed to create a hydrophobic hairpin anchor for the
induction of the membrane channel (24, 25). A model for colicin A has
been proposed wherein most helices open like an "umbrella" on the
surface of the membrane (26). An alternate model, called the
"penknife model," has been suggested which proposes that helices 8 and 9 only partially penetrate into the membrane bilayer (27, 28).
In an earlier study, Palmer and Merrill (29) used nitroxide-labeled
phospholipids as fluorescence quench probes to determine the membrane
depths of Trp residues in single Trp mutant proteins of the colicin E1
channel peptide by a parallax analysis method (30). Recently, this
experimental approach was extended in an investigation where a new
shallower, nitroxide quencher (attached to the phospholipid headgroup)
(31) was used as an additional quench species to probe Trp depths
within the membrane bilayer. In the latter report, it was suggested
that helices 8 and 9 are embedded in the membrane in a tilted fashion
and do not protrude through the bilayer to the trans side.
It was also found that the bilayer thickness could affect the structure
of the colicin E1 channel domain in the membrane.
In the present investigation, one goal was to use fluorescence
resonance energy transfer (FRET) in an attempt to reconcile the dispute
over whether the colicin E1 channel is monomeric or oligomeric. A
second objective was to use the water-soluble fluorescence quencher,
acrylamide, to probe the solvent accessibility of Trp residues within
the membrane-bound state of the colicin E1 channel domain. The second
objective was rendered achievable by the use of single Trp mutant
channel proteins of colicin E1 as characterized previously (31, 32).
The fluorescence quench data were also correlated with various other
Trp fluorescence parameters for the Trp mutant proteins, including
fluorescence lifetimes, fluorescence quantum yields, and fluorescence
emission maxima (
emmax) values.
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EXPERIMENTAL PROCEDURES |
Preparation and Purification of WT, Single Trp, and
Trp
Colicin E1 Channel Peptides--
The Trp-deficient
(Trp) and single Trp mutant proteins of colicin E1 were
prepared using site-directed mutagenesis to replace the three native
Trp residues (W-424,2 W-460,
and W-495) with Phe residues, or leave/substitute a Trp residue at a
single site, as described previously (14, 20). Colicin E1 was purified
from a lex A strain of E. coli
(IT3661) which contained the pSKE1 plasmid
with the appropriate mutation (except for the WT protein) and the
channel peptide isolated by digestion with thermolysin (32).
Preparation of LUVs--
LUVs composed of 60:40 (mol:mol)
POPC:POPG (Avanti Polar Lipids, Birmingham, AL) were prepared by
extrusion, using a hand-held extruder (LiposofastTM,
Avestin Inc., Ottawa, ON) according to a method described previously (29). Phospholipid concentration was determined using a microBartlett assay (33) following degradation of the phospholipids by ashing (34).
Preparation of AEDANS-labeled Trp Peptide--
The
AEDANS-labeled Trp channel peptide was prepared,
purified, and characterized according to methods recently developed in our laboratory (35, 36).
Spectroscopic Measurements--
Steady-state fluorescence
measurements were performed using a PTI-Alphascan-2 spectrofluorometer
(Photon Technologies Inc., South Brunswick, NJ) equipped with a
thermostated cell holder. Fluorescence spectra were obtained using
295-nm excitation light, which selectively excites the Trp residue(s)
of single Trp or WT channel peptides. The excitation and emission slit
widths were 2 nm and 4 nm, respectively, and the temperature was
maintained at 20 °C. For all fluorescence measurements, a wedge
depolarizer (Oriel Corp., Stratford, CT; 95% transmittance) was placed
on the exit side of the excitation monochromator, and fluorescence emission was detected at right angles. Steady-state Trp fluorescence emission was scanned from 303 to 450 nm upon 295-nm excitation. Corrections were made for the appropriate blanks and for the
wavelength-dependent bias of the optical and detection
systems. The fluorescence emission maxima values were determined by
calculating the first derivative of the smoothed, corrected emission
spectra for all samples and recording the wavelength where the
derivative was zero.
FRET Measurements of Membrane-bound Donor and Acceptor Channel
Peptide--
Titrations were performed according to one of two
methods. In one method, AEDANS-labeled Trp channel
peptide in LUVs was titrated with WT channel peptide while monitoring
the AEDANS fluorescence emission. In the other method, WT channel
peptide in LUVs was titrated with AEDANS-labeled Trp
channel peptide, and the fluorescence emission of the Trp residues of
WT channel peptide was monitored.
Samples were prepared in quartz cuvettes, by first preparing 0.24 µM AEDANS-labeled Trp channel peptide (or
0.24 µM WT channel peptide) in 10 mM
dimethylglutarate, 100 mM NaCl, pH 4.0, followed by the
addition of 400 µg of 60:40 (mol:mol) POPC:POPG LUVs. After the
addition of LUV, the samples were incubated for 5 min with magnetic
stirring to allow protein-LUV binding to reach equilibrium. Stirring
was discontinued during spectroscopic measurements to minimize
background signal. A control sample was prepared to test for energy
transfer in the absence of LUVs. Blanks to correct for LUV-induced
scatter contained LUVs and unlabeled Trp channel peptide.
During the titration experiments, aliquots (5 µl each) of WT channel
peptide (or AEDANS channel peptide) were added to the stirred samples,
allowing 15 min of equilibration time between additions, prior to
spectroscopic measurements.
Fluorescence Lifetime Measurements--
Time-resolved
fluorescence measurements were performed using a PTI LaserStrobe model
C-72 lifetime fluorometer (Photon Technology International). The
excitation source was a pulsed nitrogen laser pumping a dye laser with
a frequency doubler. The laser operated at 10 Hz, and the detection
channel consisted of an emission monochromator with a stroboscopic
detector. The data analysis was performed with a 1-to-4 exponential
fitting program involving iterative reconvolution and minimization of
chi-square. The excitation wavelength was 295 nm, and the emission was
set at 340 nm with 1-nm band passes. The <
a> was
calculated from the relationship <a> = 
ii/i, and because
i = 1 (normalized preexponential values) then
<a> = ii. The
<a> parameter was used in all cases except for the calculation of kq from the Stern-Volmer constant
(KSV). The <f> parameter was
calculated from the equation: <f> = i2/ii.
The <f> value was used only for the calculation of the kq values from the KSV
data shown in Table I (37) where kq = KSV/<f>.
Model for Peptide Association--
The degree of association of
colicin E1 channel peptides within the membrane bilayer of LUVs was
assessed by measuring FRET from a donor population (the three Trp
residues in WT channel peptide) to an acceptor population
(AEDANS-labeled Trp channel peptide). The derivation of
the model for association of channel peptides (Equation 1), adapted
from Veatch and Stryer (38), has been presented previously (36).
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(Eq. 1)
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QF/Q0, the
relative quantum yield of the donor species, is a ratio of the quantum
yield of the donor species in the presence (QF)
and absence (Q0) of the acceptor species.
E represents the efficiency of the FRET process,
d is the distance separating the centers of the donor and
acceptor chromophores, and n is the number of colicin E1
channel peptides that compose each functional colicin E1 channel.
Relative Quantum Yields (RQF)--
The relative Trp
fluorescence quantum yields were measured for membrane-associated
channel peptides by initially determining the quantum yield of the
proteins in solution using
N-acetyl-L-tryptophanamide as the quantum
standard (QF = 0.14) in order to quantum
calibrate the proteins as reported previously by our laboratory (14). This was conducted by exciting the samples at 295 nm (absorbance at the
excitation wavelength for the various samples ranged between 0.03 and
0.05) while scanning the fluorescence emission from 305 to 450 nm in
0.5-nm increments (2-nm slits for both excitation and emission). To
determine the RQF values for the membrane-bound peptides, an aliquot of the calibrated protein solution (quantum yield
calibrated) was mixed with vesicles as described above. After a 15-min
equilibration time, the Trp fluorescence from the proteoliposome
solutions was scanned, and the quantum yield was calculated based on
the previous determination for the membrane-free protein solution. The
RQF was determined by a simple ratio,
QF,mem/QF,sol where
QF,mem is the QF value
for the protein-lipid solution, and QF,sol is
the QF for protein in solution.
Acrylamide Quenching in LUVs--
Samples were prepared by the
addition of 60:40 DOPC:DOPG LUVs (40 µg) to the colicin E1 channel
peptide (60 µg) in 20 mM dimethylglutarate, 130 mM NaCl, pH 4.0, in a 1-cm path length quartz cuvette (21 µM LUV; 1.27 µM colicin E1 channel
peptide). The samples were incubated at 25 °C for 15 min to ensure
complete binding of colicin E1 channel peptide to the membrane LUVs. A
blank that did not contain protein was prepared to correct for the
contribution of LUVs to the fluorescence emission. Aliquots of an
acrylamide stock solution (4.0 M acrylamide in water) were
added to the cuvette. Mixing was performed with a digital pipette set
to 300 µl. Acquisition of fluorescence data was initiated immediately
after the mixing because it was found that the signal was constant as a
function of time after mixing. Fluorescence measurements were conducted with 295-nm excitation light, and the emission was monitored at 340. A
309-nm cutoff filter (Oriel Corporation, Stratford, CT) was placed in
front of the emission monochromator entrance slit to minimize the
contribution of LUV-induced light scattering to the fluorescence
emission signal. The fluorescence signal was collected for 30 s,
and the data were analyzed by integrating the area under the 30-s time
trace. The contribution of the LUVs blank was automatically subtracted
by the instrument/software. Quenching data were plotted as the ratio of
fluorescence in the absence of quencher (F0) to
the intensity in the presence of quencher (Fi)
against quencher concentration as described previously (39). The
calculated slope was equated to dynamic parameters according to the
modified Stern-Volmer equation:
F0/Fi = 1 + KSV[Q], where KSV is equal to
kqf (39, 40).
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RESULTS |
Colicin E1 Single Trp Mutant Channel Peptides--
The primary
sequence for the colicin E1 thermolytic channel peptide (molecular
weight 19,700; NH2 terminus, Ile-345) is shown in Fig.
1A. The three naturally
occurring Trp residues are located at positions 424, 460, and 495. 14 single Trp mutant peptides were chosen for investigation by Trp
fluorescence spectroscopy in order to obtain site-specific topological
information on various segments (helices) within the channel peptide
upon the transition from the water-soluble to the membrane-bound state.
The specific locale for the single Trp residues within these mutant
peptides include: F355W, Y367W, F404W, F413W, Trp-424, F431W, F443W,
Trp-460, Y478W, F484W, L492W, Trp-495, I499W, and Y507W. Phe-355 is
located in the middle of helix 1 where this residue faces inward and is nestled between helices 5b and 8. Tyr-367 is located at the
NH2 terminus of helix 2 and is largely buried at the
interface among helices 8, 9, and 10 in the x-ray structure. Phe-404 is
located in the loop region between helices 3 and 4 and is nearly fully exposed; Phe-413 is located in the middle of helix 4 and appears to be
partially exposed to solvent and relatively close to Trp-495. Trp-424
is largely buried in the middle of helix 5a, a small polar helix. Phe-431 is located in the middle of the small helix
5b, a very flexible region of the channel peptide, and is
mostly buried and inaccessible to the aqueous solvent. Phe-443 is
located in the extended loop structure between helices 5b
and 6 and is close enough to Lys-470, which is a candidate for the
source of the internal quenching seen in the F443W single Trp mutant
(32), and the Trp-443 residue is highly exposed to the aqueous solvent (39). Trp-460 is located in a moderately polar region within the
channel peptide, at the NH2 terminus of helix 7. Tyr-478 is located at the NH2 terminus of nonpolar helix 8 and is
packed inside the protein in a largely nonpolar pocket that has Lys-470 at one end. Phe-484 is located in the middle of helix 8 and is well
sequestered from solvent but may become exposed by any movement of
helice 1 or 2. Leu-492 is located in the loop region between hydrophobic helices 8 and 9 and is located in a nonpolar environment. Trp-495 is situated at the NH2 terminus of helix 9 and is
only slightly exposed to the aqueous solvent. Moreover, Ile-499 is located in the middle of nonpolar helix 9 and is sandwiched between helices 4 and 6 in a hydrocarbon milieu. Finally, Tyr-507 is located near the COOH terminus of nonpolar helix 9 with its phenolic group largely buried and facing toward the center of the peptide core; it is
the most inaccessible Tyr residue to the aqueous solvent (39).
Furthermore, Trp-478, 484, 492, 495, 499, and 507 are located within
the hydrophobic domain of the channel peptide, a nonpolar segment of
the peptide consisting of a stretch of 35 amino acid residues;
consequently, these Trp residues should report peptide structural
changes involving the purported membrane anchor domain.
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Fig. 1.
Panel A, primary sequence of the
thermolytic colicin E1 channel peptide. Residue numbering corresponds
to the numbering of whole colicin E1 (54). The locations of the
-helices assigned from the crystal structure of the P-190 peptide
(3) are indicated as shaded cylinders, and the hydrophobic
-helical domain is underscored. The three naturally
occurring Trp residues are identified with a filled circle
above the residue number, and the sites where a Trp residue has been
substituted into the peptide by site-directed mutagenesis to generate
single Trp mutants are indicated by indole rings.
Panel B, alignment of the helical segments in the
(i) soluble peptide and (ii) the proposed
transmembrane and translocating peptides for the membrane-bound state
of the colicin E1 channel domain. The helices are designated as
H1-H10, the trans-membrane segments as TM1-TM4, and the
translocating segment as TLP. Helix 5 is shown as consisting of helices
5a and 5b, and the disposition and structure of
helix 10 are not known in the membrane-bound peptide.
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Helical Alignment of Water-soluble and Membrane-bound Channel
Peptide--
The comparison of the helices in the water-soluble and
the transmembrane (TM) and translocating peptides (TLP) of
the membrane-bound channel peptide is shown in Fig. 1B,
i-ii. The assignment of the helical segments for the
water-soluble structure is based on the x-ray data (3), whereas the
assignment of the TM and TLP segments within the membrane-associated
channel domain is much less straightforward. The segments designated
TM1, 2, 3, and 4 represent TM segments whose existence has been implied
from various topological mapping experiments (see "Discussion").
The segment TLP refers to the translocating segment that has been
located on the trans side of the membrane by biotinylation
and epitope-mapping experiments (41-43). The membrane disposition of
this peptide segment in the open channel state is highly controversial
and is currently being investigated. Furthermore, the structure and
disposition of helix 10 are currently not known in the membrane-bound
peptide; consequently, it is shown as a single helix in Fig.
1Bii. A summary of the data presented in Fig. 1B
is as follows. The open state of the colicin E1 channel is proposed to
consist of four trans-membrane segments: TM1 (helix 1 and
part of helix 2), TM2 (helices 6 and 7), TM3 (helix 8), and TM4 (helix
9). In addition, an unusually long segment of the peptide has been
identified on the trans side of the membrane; this segment,
TLP, is composed of helices 3, 4, 5a, and
5b.
Colicin E1 Channel Aggregate State--
Representative
fluorescence emission spectra for the WT colicin E1 channel peptide in
the presence of LUVs at various concentrations of acceptor are
presented in Fig. 2, A and
B. Titration of LUV-incorporated WT colicin E1 channel
peptide with AEDANS-labeled Trp channel peptide did not
cause appreciable quenching of the fluorescence emission of WT colicin
E1 channel peptide as a result of intermolecular Trp-AEDANS FRET. While
the fluorescence emission signal arising from the three naturally
occurring Trp residues of WT channel peptide remained essentially
unchanged during the titration, the signal arising from the AEDANS
moiety of AEDANS-labeled Trp channel peptide increased as
the titration progressed. This increase correlated with the change in
concentration of AEDANS (as AEDANS-labeled Trp channel
peptide), and consequently, the increase in AEDANS fluorescence emission signal (Fig. 2A) is not caused by FRET from the
three Trp residues of WT channel peptide.
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Fig. 2.
Panel A, titration of WT colicin E1
channel peptide in LUVs with AEDANS-labeled Trp-deficient channel
peptide. The WT peptide (0.24 µM) was titrated with
AEDANS-Trp peptide (0-0.25 µM) in 218 µM lipid and 100 mM NaCl, 10 mM
dimethylglutarate buffer, pH 4.0 (AEDANS labeling stoichiometry of
Trp peptide  1:1). The numbers
(1-7) indicated on the figure refer to the following
concentrations of AEDANS-Trp peptide
(AEDANS:Trp peptide, 1:1 molar ratio): 1, 0 µM; 2, 0.042 µM; 3,
0.084 µM; 4, 0.13 µM,
5, 0.17 µM; 6, 0.21 µM; and 7, 0.25 µM. The donor
Trp residues of the WT peptide were excited selectively using 295-nm
light. In the case of an oligomeric channel, energy transfer from the
donor (Trp) to the acceptor (AEDANS) chromophore is expected to be
manifested as a decrease in the Trp fluorescence emission with a
corresponding enhancement of the AEDANS fluorescence emission. The Trp
fluorescence emission, however, remained relatively constant,
indicating that the colicin E1 channel formed in LUVs is monomeric.
Panel B, titration of AEDANS-labeled Trp-deficient colicin
E1 channel peptide in LUVs with WT channel peptide. AEDANS-labeled
Trp peptide (0.125 µM) was titrated with WT
channel peptide (0-0.25 µM) in 105 µM
lipid and 100 mM NaCl, 10 mM dimethylglutarate,
pH 4.0, buffer. 11 spectra are overlaid in the figure with each
spectrum representing a 0.025 µM increment increase in
the WT peptide concentration. In the case of an oligomeric channel,
energy transfer from the donor (Trp) to the acceptor (AEDANS)
chromophore is expected to be manifested as an increase in the AEDANS
fluorescence emission. The AEDANS fluorescence, however, remained
largely unchanged, indicating that the colicin E1 channel formed in
LUVs is monomeric.
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The degree of quenching of the intrinsic fluorescence emission of WT
channel peptide was assessed by comparing the area of the fluorescence
emission peak arising from the donor species (Trp) in the presence of
the highest concentration of acceptor (AEDANS) to the area of the donor
fluorescence peak in the absence of acceptor. The mean quenching of
donor fluorescence was only 6.4 ± 13% and did not show an
increasing trend with the addition of acceptor.
In contrast, Steer and Merrill (36) demonstrated that FRET occurred
within a dimeric unfolding intermediate of the colicin E1 channel
peptide, using the same donor-acceptor pair. The observation of FRET
between the Trp-AEDANS donor-acceptor pair (36) serves as a convenient
positive control for this investigation. The absence of intermolecular
Trp-AEDANS FRET under conditions where the channel peptide readily
associates with membrane bilayers indicates that adjacent channel
peptides are greater than 50 Å apart. A separation distance as large
as 50 Å is not consistent with the formation of a multimeric channel.
To confirm that the absence of energy transfer was indicative of a
monomeric colicin E1 channel, the FRET data were plotted according to
the channel peptide association model (Equation 1). The data did not
fit a model with greater than one subunit (data not shown). The absence
of FRET in this series of experiments supports previous evidence for a
monomeric colicin E1 channel.
Results from the titration of AEDANS-labeled Trp channel
peptide in LUVs with WT channel peptide also support a monomeric colicin E1 channel. The fluorescence emission arising from
LUV-associated AEDANS-labeled Trp channel peptide
remained unchanged as additional donor (WT) channel peptide was added
to the system (Fig. 2B). The absence of Trp-AEDANS FRET
indicates that the colicin E1 channel is composed of a single channel
peptide within the membrane bilayer.
emmax Values of Membrane-bound Channel
Domain--
The emmax values for membrane-associated WT
and 14 single Trp mutant proteins of the colicin E1 channel peptide are
shown in Table I. The
emmax values for the WT and a subset of the soluble
single Trp mutant proteins (11 in total) were reported previously by
our laboratory (39). The emmax values shown in Table I
range from 325.1 nm (W-495) to 337.5 nm (W-404). Eight of the 14 membrane-associated single Trp mutant proteins, along with the WT
protein, possessed emmax values that were greater than
330 nm. The remaining six mutant proteins exhibited
emmax values that were less than 330 nm but greater than
325 nm. These data are in contrast with the emmax values
for the soluble mutant proteins; in the latter situation, the values
were generally more blue-shifted, i.e. shifted to shorter
wavelength values. This indicates that the channel peptide is in a more
polar (with mobile polar groups) environment upon membrane association
(closed channel state in the absence of a membrane potential). This
notion can be illustrated further by considering the average
emmax for the WT and 11 single Trp mutant proteins in
the soluble as opposed to the membrane-bound forms (mean
emmax of 330.9 ± 3.7 and 327.3 ± 5.8 nm for
membrane and soluble forms, respectively). Interestingly, the WT
protein showed an 8-nm red shift in its emmax value upon membrane association, reflecting a change in the chemical environment surrounding the bound peptide (Table I, Ref. 39, and Fig.
3).
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Fig. 3.
Change in the fluorescence emission maximum
upon membrane association of the colicin E1 channel peptide. The
 emmax parameter was determined by the difference
between the emmaxmem (membrane-bound
protein) and the emmaxsol (soluble protein)
for the Trp fluorescence emission of the corresponding protein. A
positive value indicates a red shift in the fluorescence emission
maximum upon membrane association, and a negative value indicates a
blue shift. The emmax values for the membrane-bound
peptides were taken from the data in Table I, and the
emmax values for the soluble peptides were
determined previously (39). The emmax data were acquired
as described under "Experimental Procedures" and in the legend
to Table I.
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The WT peptide possesses three Trp residues at 424, 460, and 495 and
thus reports on more global changes occurring within the COOH-terminal
two-thirds of the protein. A plot showing the divergence of the
emmax values (emmax = emmaxmem values 
emmaxsol values) between the soluble and
membrane-bound channel peptide states is shown in Fig. 3. There are six
proteins that show large red shifts in emmax values upon
membrane binding (emmax values between 5 and 11 nm).
Each of these proteins (WT, W-367, W-413, W-460, W-492, and W-507)
contains a single Trp residue (except for the WT protein) at various
sites within the primary sequence of the channel peptide (Fig.
1A). In the soluble peptide, Trp-367 is located in the
middle of helix 2 in a relatively polar environment surrounded by
Gly-364, Lys-366, Ser-368, and Met-379. It has a blue-shifted
emmax value (320 nm, Ref. 39) and an intermediate
quantum yield (0.18, Table I). The former can be explained by its
relative inaccessibility to the aqueous solvent (39), whereas the
latter is likely caused by the close proximity of this residue to the
sulfur atom of Met-370. Trp-413 is found in the center of helix 4 in a
nonpolar pocket lined with Ile-412, Phe-413, and Leu-416. This residue
is part of the pH-activated trigger mechanism of the channel peptide
proposed recently by our laboratory (14). Trp-460 is a naturally
occurring residue that is found in a loop region between helices 6 and
7, possesses a relatively high QF,sol (0.27, Ref. 14), and is enveloped in a nonpolar niche within the soluble
peptide. Trp-492 is also found in a loop region between hydrophobic
helices 8 and 9 and is mostly buried in a nonpolar region of the
soluble peptide. Trp-507 is situated at the COOH terminus of nonpolar
helix 9 where it is buried in the core of the peptide (39) but
possesses a low quantum yield (0.05, Table I) probably because of the
proximity of the thiol group of Cys-505. Mutant proteins W-355, W-404,
W-424, W-478, W-484, and W-495 show smaller, less significant red
shifts in emmax values upon membrane binding (Fig. 3).
Two mutant proteins, W-431 and W-499, exhibit significant blue shifts
in the emmax values upon membrane association; both of
these Trp residues are located in the center of helices (helices
5b and 9, respectively; Fig. 1A).
Trp Quantum Yields of Membrane-bound Channel Peptides--
The
fluorescence quantum yields for the soluble forms of nine of the single
Trp mutant (and WT) proteins were measured previously using
N-acetyl-L-tryptophanamide as the quantum
standard (14). The quantum yield for the membrane-associated mutant
proteins are shown in Table I. The QF value for
the soluble WT protein, which possesses Trp-424, 460, and 495, represents the average of the QF values for the
three single Trp mutant proteins (14). However, this is clearly not the
case for the membrane-bound proteins. The WT has a
QF value of 0.19, whereas the average of W-424,
W-460, and W-495 is 0.36. This indicates that there is significant
structural rearrangement of the WT peptide upon membrane association
which is only observed for the single Trp mutants because Trp to Trp FRET can still occur within the WT peptide. It would appear that the
QF value of the membrane-bound WT peptide
reflects this possibility for the W-424 bound protein, suggesting that
Trp to Trp FRET occurs from Trp-460 and 495 to Trp-424. The
membrane-bound QF values for the WT and single
Trp mutants ranged from 0.17 to 0.51. Trp residues exhibiting high
QF values include:
(QF,mem 
0.30) Trp-355, 404, 413, 431, 443, 460, 495, and 507. In addition, the RQF values were calculated for the WT and 12 of the membrane-associated single Trp
mutant proteins, and these are listed in Table I. Mutant proteins
W-355, W-404, W-443, and W-507 showed a large increase (>2 fold) in
RQF upon membrane binding, whereas only W-484
exhibited a significant decrease in its RQF
value (0.7 ± 0.03, Table I). The remaining seven mutant proteins
(W-367, W-413, W-424, W-431, W-460, W-478, and W-495; Table I) showed
little or no change in their RQF values.
Consequently, from the data in Table I, the Trp residues within the
membrane-bound mutant channel peptides which may be considered as being
located within a hydrophobic environment include Trp-355, 404, 413, 431, 443, 460, 495, and 507. From this subset of Trp residues, a few
can be discounted based on the following observations. Trp-495 is
located within a nonpolar environment in the soluble peptide, and its
QF does not change upon membrane binding; hence,
it is difficult to assess whether any change in environment has
occurred for Trp-495. The increase in RQF values
for Trp-404, 443, and 507 may partly be the result of alleviation of
internal quenching mechanisms upon the transition of the soluble
peptide to the membrane-bound state based on previous observations for
the x-ray structure of the soluble peptide (3, 14, 32, 35, 38, 39).
However, all of these Trp residues possess relatively high
QF values. Therefore, these data imply that
Trp-355, 404, 413, 431, 443, 460, and 507 are at least partially
embedded within the membrane bilayer.
Acrylamide Quenching of Trp Fluorescence--
The Stern-Volmer
quenching data for the WT,
N-acetyl-L-tryptophanamide standard, and 14 single Trp mutant proteins are shown in Table I. The Stern-Volmer
quench constant (KSV) values ranged from 0.93 M1 (W-507) to 3.95 M1 (W-413). However, because the
KSV values are composite constants that possess
the parameter of the <f> (where
KSV = kq<f>) a better measure of the
collisional rate between the Trp chromophore in the channel peptide and
the aqueous quencher, acrylamide, is the bimolecular quench constant,
kq. The second-order bimolecular quench constant
(kq) values ranged from 0.24 M1 ns1 (W-507) to 0.95 M1 ns1 (W-413). Consequently,
the channel proteins were divided into two classes according to their
accessibility to acrylamide as a water-soluble, fluorescence quencher.
Group I consisted of WT, W-413, W-424, W-478, W-484, and W-495 proteins
that showed kq values greater than 0.5 M1 ns1. Group II is comprised
of W-355, W-367, W-404, W-431, W-443, W-460, W-492, W-499, and W-507
proteins that were less accessible to the aqueous quencher and, hence,
had kq values that ranged from 0.24 to 0.46 M1 ns1 (Table I). The
acrylamide quenching data for the soluble form of 11 single Trp mutant
proteins was reported previously (39). Generally, as expected, both of
these quench parameters, KSV and kq, were lower for the membrane-bound compared
with the soluble form of the proteins (Table I, Fig.
4, and Ref. 39), although changes in
<f> upon the transition from solution to
membrane-bound form may result in a change in
KSV without a concomitant alteration in
kq (this was observed for W-418, W-424, and
W-484 proteins; Table I, Figs. 4 and 5,
and Ref. 39).
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Fig. 4.
Change in the average bimolecular quench
constant, kq, upon
membrane binding of the colicin E1 channel peptide. The
kq value represents the difference between
the kq,sol (soluble protein) and
kq,mem (membrane-bound protein). A positive
kq value indicates a reduced accessibility of
the Trp residue in the protein upon membrane association, and a
negative value represents an enhanced accessibility. The
kq data for the membrane-bound peptides were
taken from Table I; the kq data for the soluble
proteins were taken from previous data (39). The
kq data were acquired as described under
"Experimental Procedures" and in the Table I legend.
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Fig. 5.
Change in the amplitude average fluorescence
lifetime upon membrane binding of the colicin E1 channel peptide.
The <a> parameter was determined by subtracting the
<a>sol value for the soluble peptide from
the <a>mem value for the membrane-bound
peptide. A positive <a> value indicates that the
<a> increased for the membrane-bound peptide relative
to the soluble peptide. The <a> data for the
membrane-bound peptides were taken from Table I, and the
<a> data for the soluble proteins were taken from
previous data (39). The <a> data were obtained as
described under "Experimental Procedures" and in the Table I
legend.
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The change in kq values of the various single
Trp channel peptides, and hence the extent of solvent accessibility,
upon the binding of the channel peptides to a LUV membrane surface is
shown in Fig. 4 (kq = kq,sol kq,mem).
Notably, a positive kq indicates a reduction
in the solvent accessibility of the Trp residue(s) being probed or, in
other words, a protection of the Trp residue within the channel peptide
from the aqueous solvent as provided by the membrane bilayer structure.
It is obvious from the data in Fig. 4 that generally there is a
reduction in aqueous solvent exposure of the Trp residues upon
association of the channel protein with membranes
(kq > 0 for the WT and all single Trp mutant
peptides except W-413, W-424, W-484, and W-495). The largest degree of protection of the channel peptide provided by the membrane bilayer upon
the solution to membrane transition occurred with Trp residues 367, 404, and 443. This indicated that at these sites along the channel
peptide there is significant interaction, and possibly immersion of Trp
residues, within the membrane bilayer. Trp residues at 355, 431, 460, 492, and 499 were protected to a lesser extent (Fig. 4). Trp residues
at 413, 478, 484, 495, and 507 showed little or no significant change
in solvent exposure upon channel protein associating with a membrane
surface. Trp-424 was the only site that showed a significant increase
in solvent exposure upon peptide binding to LUVs. Notably, the WT
protein kq value was equal to the average of the
three single Trp mutant protein kq values and
showed a modest decrease in solvent accessibility upon membrane binding, reflecting the average of W-424, 460, and 495 (Fig. 4).
Fluorescence Lifetimes--
The amplitude average fluorescence
lifetime (<a>) and the intensity average lifetime
(<f>) were calculated from the measured decay time
components (37) for the membrane-associated channel peptides and are
shown in Table I. The intensity weighted fluorescence lifetimes
<f> were used for the calculation of the
kq values only. The <a> values
can be more readily correlated with Trp photophysical processes and
thus were used to aid in the interpretation of the topological data for
the channel peptide. The <a> values ranged from 2.54 ns (W-355 and 507) to 4.24 ns (W-460) and exhibited changes within a
large number of peptides upon the soluble to membrane transition (14, 39; Fig. 5) but generally did not show the range reported previously
for the soluble channel peptides (39). The change in decay times
(<a>) upon peptide binding to membrane LUVs is
plotted in Fig. 5 (<a> = <a>mem <a>sol). A positive
<a> reflects an increase in the Trp fluorescence lifetime of the respective channel peptide upon binding to the membrane
surface. Upon membrane association, seven peptides showed a significant
increase in <a> values (<a> 0;
WT, W-355, W-404, W-413, W-431, W-460, and W-507). Five single Trp
mutant proteins, W-367, W-424, W-443, W-484, and W-492, showed a large
decrease in <a> values upon binding to a bilayer
surface (Fig. 5), whereas W-495 and W-499 showed modest decreases in
<a> values when membrane-bound. W-478 exhibited
little or no significant change in <a> when converting from the water-soluble state to the membrane-bound form.
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DISCUSSION |
Channel Aggregate State by FRET Analysis--
The absence of FRET
in this series of experiments is further evidence that the colicin E1
channel formed by the channel-forming domain in LUVs is monomeric.
Other experiments that support a monomeric channel model include the
original cytotoxicity experiments on colicin E1 in which colicin E1 was
observed to act according to a "one-hit" mechanism, demonstrating
that a single protein molecule could kill a cell (15). In addition,
kinetic data indicate that channel formation is first-order with
respect to either colicin E1 channel peptide or colicin E1
concentration (12), suggesting that a single molecule forms each channel.
One piece of evidence which stands in opposition of a monomeric colicin
E1 channel is the observation that large organic cations, including
NAD+, can pass through the channel (21) and that the
hydrated channel dimensions include a 7 Å constriction (23) as
determined with nonelectrolytes that fill the channel lumen. It is
presently not clear how a 178-190-residue monomeric peptide can form a
channel with a lumen of such large dimensions unless most, if not all, of the polypeptide chain is involved in forming the channel structure.
It is important to recognize that multimeric channels that are composed
either exclusively of donor channel peptides or exclusively of acceptor
channel peptides may be formed. Multimeric channels of this type would
be spectroscopically silent, yielding FRET data that would be
indistinguishable from FRET data that corresponded to the formation of
monomeric channels. The model of Veatch and Stryer (38) (Equation 1)
accounts for the probability of forming oligomers composed either
exclusively of donor channel peptides or exclusively of acceptor
channel peptides. It is highly improbable that the absence of FRET in
this experiment was the result of every one of the channels formed
arising from a combination of monomers of the same population.
The absence of FRET between the donor- and acceptor-labeled peptides
indicates that the distance separating the two chromophores was greater
than the distance over which significant FRET occurs, approximately 50 Å in this system (Ro = 22 Å for the Trp and
AEDANS pair (35, 44). Notably, this dimension is the distance that separates the donor and acceptor chromophores and not the distance separating the peptides. The three Trp residues of WT channel peptide
serve as the donor chromophores, and so we would expect, based on the
x-ray structure for the soluble peptide (3), that there is a reasonable
distribution of the three donor chromophores throughout the peptide.
The acceptor chromophore, however, is an extrinsic fluorescent probe
(AEDANS) that is covalently attached to the lone Cys residue, Cys-505,
of the Trp channel peptide. Theoretically, it is possible
for an oligomeric colicin E1 channel to form such that the AEDANS label
of the Trp channel peptide is directed outward from the
rest of the channel complex. In an arrangement of that type, the
distance between the donor Trp residues and the acceptor AEDANS moiety
could, conceivably, be large enough to prevent efficient FRET,
resulting in a second type of spectroscopically silent oligomer. The
magnitude of the Stokes radius of soluble colicin E1 channel peptide,
21-23 Å (20), however, essentially precludes an arrangement of
monomers, within an oligomeric colicin E1 channel, which would have a
donor-acceptor distance sufficient to prevent energy transfer.
In conclusion, Trp-AEDANS FRET was not observed for WT channel peptide
and AEDANS-labeled Trp channel peptide incorporated
within membrane bilayers. Previously, FRET had been observed (36) for a
dimeric unfolding intermediate of colicin E1 channel peptide,
indicating that the donor-acceptor pair was suitable for measuring the
association of colicin E1 channel peptides. The absence of FRET for the
membrane-associated channel peptides provides further evidence that the
colicin E1 membrane-bound closed channel is monomeric.
Topology of the Membrane-bound Closed Channel State--
It is
generally believed that the Trp emmax parameter is the
best gauge of the chemical environment of a Trp residue within a
protein (14, 44-46). The emmax data for the contingent
of single Trp mutant and WT proteins strongly suggest that the polarity of the Trp residues (at 11 of the 14 different Trp sites within the
peptide) increases when the protein associates with the membrane surface. This suggests that the protein unfolds and spreads out on the
surface of the membrane, which is in agreement with the previous
proposals for colicin A (26-28) E1 (29, 31, 47-49) and Ia
(25, 41-43). The emmax values for W-355 and W-367
single Trp proteins indicate that both helices 1 and 2 are
membrane-associated under the conditions used in this study but that
the corresponding Trp residues are in a more polar environment than
those found within the hydrophobic anchor domain of the channel protein
(Table I). Previously, Trp-355 and 367 were determined by
depth-dependent fluorescence quenching analysis to reside
at moderately buried positions within the membrane bilayer (29, 31).
Upon membrane binding, W-367, 413, 460, 492, and 507 exhibit a red
shift in their emmax values, indicating that these
residues are likely interfacial and are not buried deeply within the
membrane bilayer. It appears that both Trp-495 and 499, found on the
same side of helix 9, are located in highly nonpolar environments,
probably within the bilayer hydrocarbon core, when the channel domain
is membrane-bound (Table I). However, Trp-495 is more accessible to
aqueous quencher than Trp-499. This may reflect the orientation of the
hydrophobic anchor within the membrane because Trp-499 is in the middle
of helix 9, whereas Trp-495 is near the amino terminus of that helix.
This implies that helix 9 is relatively deeply embedded within the
membrane in the closed state of the colicin channel. Trp residues at
positions 492 and 507 define the ends of helix 9, and both Trps report
a more polar environment. Helix 8 is also submerged within the bilayer
as reported by Trp-478 and 484, which exhibit relatively blue-shifted
emmax values (Table I) and are located on opposite faces
of this nonpolar helix. A complication to the interpretation of Trp
locations within the closed state of the colicin channel may entail a
consideration of the lumen geography of the pore or partial pore
structure in the absence of a membrane potential. It is conceivable
that a given Trp residue may be deeply embedded within the membrane but be relatively accessible to the solvent via the channel lumen through
the indole polar N-H group.
Acrylamide quenching experiments reported in this paper provide a means
to probe the solvent accessibility of the Trp residues found at various
sites throughout the closed channel structure. Acrylamide is believed
to be superior to KI as a probe of the solvent exposure for a
lipid-associated fluorophore because the former does not partition to a
significant degree into the membrane bilayer and acts as a collisional
fluorescence quencher (50). Notably, this approach does not simply
provide a dipstick measure of the distance below the surface of a given
Trp residue, as in the case where spin-labeled phospholipids were used
as quenchers of indole fluorescence (29, 31), but also provides a means to probe any vestiges or nuclei of an embryonic channel lumen that
exists as a precursor to the open channel state. Based on the
overwhelming abundance of accumulated evidence, it seems most likely
that the NH2-terminal half of the channel peptide (closed channel state) is simply floating on the membrane surface with the
amino-terminal helices (helices 1, 2, 3, and 4) oriented parallel to
the lipid bilayer. However, the kq data for
helices 1 and 2 suggest that these two helices are appressed close to
the surface of the membrane and are not fully exposed to the solvent
(Table I and Fig. 4). An earlier report (47) indicated that there are a
few protease accessible sites within this region of the membrane-associated peptide. This may simply reflect the dynamic nature
of this association given that the digestion conditions used (47) were
relatively harsh (1 h at 35 °C, high protease:protein mass ratio)
and that not all of the protein was digested. Previous reports (20, 27,
28, 31, 41-43, 48) involving completely different approaches to probe
the closed channel structure led to similar conclusions as to the
dynamic nature and the existence of multiple states for membrane-bound
colicin channel domains. In summary, the fluorescence quench data
indicate that Trp-355, 367, 404, 431, 443, 460, 495, and 499 are
partially embedded in the membrane bilayer and are protected from the
aqueous solvent.
Trp-495 is deeply entrenched within the bilayer (29, 31), possesses a
blue-shifted emmax value, yet it is relatively accessible to acrylamide (kq = 0.75 M1 ns1, Table I) and hence the
aqueous solvent. This implies that the N-H group of its indole ring is
partially exposed to the solvent in the closed channel structure.
Unfortunately, it is not possible to discriminate between the presence
of a membrane defect caused by protein insertion or whether this
moderate level of solvent accessibility represents a primordial feature
of the channel. However, the Trp-495 residue is not required for normal
channel function because its replacement with Phe does not alter its
channel properties (51).
The plots shown in Figs. 3-5 indicate that there are significant
structural changes within the channel peptide upon the transition from
the water-soluble structure to the membrane-bound state. The data in
these figures indicate that the changes are not localized to a few
sites within the peptide structure but are global in nature.
Interestingly, the largest changes in the RQF
values (membrane-bound peptides) were observed for single Trp mutant
proteins that exhibited low QF values in
solution (Table I and Ref. 39). This may indicate that the structural
changes induced within the channel peptide upon membrane association
cause an alleviation of the internal quenching mechanism(s) inherent to
the soluble protein. However, the QF,mem values
do indicate that the following Trp residues are likely membrane
embedded: Trp-355, 404, 413, 431, 443, 460, 495, and 507. Unfortunately, the QF data were not determined
for two additional Trp mutants that possess a Trp residue within the hydrophobic anchor domain of the channel peptide, W-492 and W-499. It
is anticipated that these residues would also possess high QF,mem values similar to Trp-495 and 507.
Presently, there is no theoretical basis for assigning membrane
location and/or depths to Trp residues based on the
<a> values for the single Trp mutant peptides. It is
known that the Trp fluorescence lifetime is sensitive to a number of
factors and that it provides a "fingerprint" for a given Trp
residue in a specific chemical environment (14, 44, 46, 52). There is a
correlation between the solvent dielectric constant and the magnitude
of the <a>; as the dielectric constant decreases the
fluorescence lifetime generally increases. However, there are several
interacting factors within protein structure, and also membrane bilayer
structure, which complicate the interpretation of changes in
<a> values. It is curious that no Trp residue for any
of the mutant proteins exhibited a high (5-6 ns) <a>
value, which might have been expected for a Trp residue buried in the
hydrocarbon core of the membrane bilayer. This may indicate that no Trp
residue for the closed channel state of the WT or any of the single Trp
mutant proteins is completely buried within the membrane bilayer or
that the direct interpretation of the <a> value is
complicated by the heterogeneous solvent system consisting of numerous
interfacial regions that is part of a protein-liposome complex.
Although at present it is difficult to correlate membrane location of
Trp residues within proteins from the measured <a> values, it is clear that changes invoked in the <a>
values for the various mutant peptides reflect changes in the chemical
environment around the indole chromophore (52). Therefore, the data in
Fig. 5 indicate that the Trp residues in the WT, W-355, W-404, W-413, W-431, W-460, and W-507 peptides are likely located in a more nonpolar
environment, whereas W-367, W-424, W-443, W-484, and W-492 are found in
a less polar environment upon the solution to membrane transition of
the channel peptide. Small changes (decreases) in <a>
values were observed for W-495 and W-499 peptides, and no change was
seen for W-478 channel peptide (Fig. 5). Although not definitive
concerning the disposition of the Trp residues at or below the membrane
bilayer surface, these data strongly suggest that there are significant
structural rearrangements occurring within the channel peptide when it
binds to the membrane surface.
Topological Models of Colicin E1 Channel--
Simplified models of
the closed channel structure as well as the more speculative open
channel state are shown in Fig. 6. The
former model (Fig. 6A) incorporates previous data from a
large number of studies involving a wide array of experimental
techniques and approaches to the elucidation of membrane protein
topology. Important components of the model for the closed state
include a monomeric species that features the partial immersion of
helices 1 and 2 into the bilayer and the flickering of the hydrophobic domain (helices 8 and 9) between states 1 and 2 as suggested by Kienker
et al. (41) except that state 2 is not fully
trans-membrane. Depth-dependent quenching data
suggest that state 2 is the preferred orientation and is the most
heavily populated state for the closed channel structure (29, 31). The
imposition of a membrane potential induces the formation of a mature,
open channel by the translocation of the gating peptide (42, 51, 53)
comprised of helices 5-7 from the soluble structure. However, recent
data employing engineered epitopes suggest that colicin Ia is capable
of translocating a much larger segment of hydrophilic peptide than
previously believed possible and that the COOH-terminal 85 residues
(residues 542-626 of Ia) functions as a protein translocator (43). It
now seems likely that the gating peptide is part of a larger protein
segment that moves across the membrane bilayer in response to an
imposed membrane potential (41, 43). How this "dragon's tail"
arranges itself to form an ion-conducting pathway through the membrane bilayer is currently beyond comprehension. However, considering that
each channel entity is composed of a single colicin polypeptide, it
seems reasonable to suggest that the channel makes use of nearly all of
the polypeptide sequence. This channel (helix bundle) structure undoubtedly must involve sufficient polypeptide length to form an ion
channel with a lumen of the physical dimensions as shown previously
(21) with a 7 Å pore (23). Notably, amid a blurred picture for the
open state of the colicin channels, one issue is clear, that the open
channel model for the colicins, including the E1 protein, requires more
detailed experimental evidence on the specific topological arrangement
of the protein in the membrane bilayer in the presence of a membrane
potential. Presently, this model provides a working hypothesis that can
be subjected to the rigors of membrane biochemical/biophysical
experiments.
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Fig. 6.
Models for the membrane-bound states of the
colicin E1 channel peptide. Panel A, the closed state
of the colicin E1 channel and (panel B) a
four-helix bundle in the membrane in the presence of a membrane
potential. The models are based on data from photolabeling (51);
biotinylation (41, 42, 53); epitope mapping (43); saturation
mutagenesis (55); protease accessibility (28, 47);
depth-dependent fluorescence quenching (29, 31);
cysteine-scanning mutagenesis (56); FRET (27); CD, Fourier transform
infrared radiation, and differential scanning calorimetry (48, 49);
time-resolved spin labeling (57); and acrylamide quenching and other
fluorescence data presented in this work. The initial surface-bound
structure adopts at least two states with state 2 being the most
populated. The channel is fully assembled in the presence of a membrane
potential, which involves insertion of helices 1 and 2 (TM1), the
translocating peptide (TLP, helices 3-5b), and the
amphipathic TM2 (helices 6 and 7), with the hydrophobic domain (helices
8 and 9) providing the anchor for the peptide to the membrane. This
results in an unusual channel structure that must form a lumen with the
dimensions suggested by previous experiments (21, 23). The predicted
trans-membrane helices in the channel domain, formed upon
membrane association, are designated as TM, and the
circles indicate helical segments lying on the surface of
the bilayer parallel to the plane of the membrane.
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TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 519-824-4120, ext. 3806; Fax: 519-766-1499; E-mail:
merrill@chembio.uoguelph.ca.
