From the Wadsworth Center, New York State Department
of Health, Albany, New York 12201-0509, the § Department of
Biomedical Sciences, State University of New York, Albany, New York
12222, and the ¶ Vollum Institute, Oregon Health Sciences
University, Portland, Oregon 97201
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ABSTRACT |
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Several forms of the
voltage-dependent anion-selective channel (VDAC) have been
expressed at high yield in Escherichia coli. Full-length
constructs of the proteins of Neurospora crassa and Saccharomyces cerevisiae (ncVDAC and scVDAC) have been made
with 20-residue-long, thrombin-cleavable, His6-containing
N-terminal extensions. ncVDAC purified from bacteria or mitochondria
displays a far-UV CD spectrum (in 1% lauryl dimethylamine oxide at pH
6-8) similar to that of bacterial porins, indicating extensive
-sheet structure. Under the same conditions, the CD spectrum of
bacterially expressed scVDAC indicates lower
-sheet content, albeit
higher than that of mitochondrial scVDAC under the same conditions. In phospholipid bilayers, the bacterially expressed proteins (with or
without N-terminal extensions) form typical VDAC-like channels with
stable, large conductance open states (4-4.5 nanosiemens in 1 M KCl) and voltage-dependent transitions to a
predominant substate (about 2 nanosiemens). A variant of scVDAC missing
the first eight residues and having no N-terminal extension also has been expressed in E. coli. The truncated protein has a CD
spectrum similar to that of mitochondrial scVDAC, but its channel
activity is abnormal, exhibiting an unstable open state and rapid
transitions between multiple subconductance levels.
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INTRODUCTION |
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VDAC1 or mitochondrial
porin is the most abundant transport protein in the mitochondrial outer
membrane. Although VDAC proteins from different species have only weak
sequence homology, their functional properties are highly conserved (1,
2). In artificial lipid bilayers, the channel occupies a high
conductance, anion-selective open state (4-4.5 nS in 1 M
KCl) at small membrane potentials and switches, at potential amplitudes
above 30-40 mV, to lower conductance substates (2-2.5 nS) that are
cation-selective and less permeable to ATP and ADP (3-5). The critical
potential at which gating occurs is decreased in the presence of
polyanions and a soluble mitochondrial protein fraction (6, 7). VDAC sequences from a number of species contain numerous stretches of
alternating hydrophobic and hydrophilic residues (e.g. see Refs. 8 and 9), suggesting an amphipathic -barrel motif like that of
the bacterial porins (10, 11). Analysis of VDAC sequences with the
Gibbs sampler indicates the presence of numerous matches to a
residue-frequency motif associated with transmembrane
-strands in
bacterial porins (12). Circular dichroism studies of VDAC purified from
Neurospora crassa in both lipids and nondenaturing detergents (13) suggest that ncVDAC has a high
-sheet content, consistent with a bacterial porin-like
-barrel structure
(e.g. see Ref. 14).
The main focus of our research is to determine the molecular basis for VDAC's functional properties, in particular, its permeability and mechanism of gating. In this report, we describe the bacterial expression of functional VDAC from Saccharomyces cerevisiae and N. crassa and compare the effects of N-terminal extension and truncation on the properties of this channel protein.
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EXPERIMENTAL PROCEDURES |
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Isolation of Mitochondria-- Mitochondria were isolated from liquid cultures of wall-less N. crassa (FGSC 326) and from blocks of dry S. cerevisiae (Red Star) (15, 16). Chemicals were obtained from Sigma unless indicated otherwise, and solutions were made with reagent-grade deionized water (Milli-Q system, Millipore Corp., Bedford, MA).
Mitochondrial VDAC Isolation--
VDAC was purified using a
procedure modified from that of Shao et al. (13).
Mitochondrial suspensions were solubilized at room temperature in 20 ml
of 2× Buffer A (4% LDAO (Calbiochem, La Jolla, CA), 20 mM
Tris·HCl, 2 mM EDTA, pH 7.0) and centrifuged (27,000 × g, 30 min). (This and subsequent steps were performed at
4 °C.) The supernatant was diluted to a final concentration of 2%
LDAO with TE buffer (10 mM Tris·HCl, 1 mM
EDTA, pH 7.0) and loaded onto a prewashed (Buffer A) ceramic
hydroxylapatite column (Bio-Rad). VDAC was eluted with Buffer A
containing 50 mM KCl and 5 mM potassium
phosphate (pH 6.8), and pooled fractions were concentrated by
centrifugation in Centricon 30 tubes (Amicon, Beverly, MA) to 100-200
µg of protein/ml and stored at 70 °C.
Construction of Expression Plasmids-- Four different VDAC constructs were generated using two different IPTG-inducible expression systems. Two constructs were made with the pMAL system (New England Biolabs, Beverly, MA), which yields fusion proteins containing maltose-binding protein (MBP) at the N terminus, followed by a Factor Xa cleavage site. scVDAC cDNA was partially digested to make an EcoRV-HindIII fragment that was incorporated into the pMAL-c expression plasmid between StuI and HindIII sites (nucleases obtained from New England Biolabs). Factor Xa cleavage would yield scVDAC lacking the first 8 residues. Polymerase chain reaction was used to generate a 165-base pair fragment of ncVDAC cDNA encompassing residues 3-51, and silent mutations were made at nucleotide 9 to create a SnaBI site and at nucleotide 132 to remove a second BstEII site. This cDNA fragment and a BstEII-NsiI fragment containing residues 52-283 were cloned between the XmnI and PstI sites of pMAL-c2 (New England Biolabs). Factor Xa cleavage of the resulting protein would be missing the first 2 residues. The pMAL-based plasmids were transformed into a competent Escherichia coli host strain, NM522, and grown in the presence of 100 µg/ml ampicillin. Full-length constructs of ncVDAC and scVDAC were made by polymerase chain reaction amplification and in-frame insertion of the cDNAs into the pET-15b expression vector (Novagen, Milwaukee, WI). These proteins contain a 20-residue N-terminal extension MGSSHHHHHHSSGLVPRGSH that ends with a thrombin cleavage site. DNA sequences of the entire reading frames were determined and found to be identical to published sequences (8, 17). The pET-15b based plasmids were transformed into the E. coli host strain BL21(DE3) and grown in the presence of 100 µg/ml ampicillin.
VDAC Expression and Purification from Inclusion Bodies-- Bacterial growth and induction were carried out essentially according to plasmid manufacturers' instructions, and inclusion bodies were collected using a method based on that of Kaplan et al. (18). Briefly, overnight cultures were diluted 50-fold (1 liter final volume) into LB media (containing 100 µg/ml ampicillin) and grown to a final A600 of 0.5-0.6. After addition of 0.5 mM IPTG, the cells were grown for an additional 2.5 h, placed on ice for 5 min, and centrifuged (4080 × g, 10 min). The pellets were resuspended with 100 ml of 50 mM Tris·HCl (pH 8.0), 2 mM EDTA, 20% sucrose, and incubated with lysozyme (12.5 µg/ml, 22 °C, 10 min) and Triton X-100 (0.6%, 4 °C, 10 min). This lysate was sonicated (two 30-s pulses at a setting of 4 using a model 50 sonic dismembranator, Fisher Scientific, Pittsburgh PA), and centrifuged (12,000 × g, 15 min). Pellets were washed with 20 mM Tris·HCl, pH 8.0 (containing 2 mM CaCl2 for the pET-based constructs), repelleted (12,000 × g, 15 min), and dissolved in 75 ml of solubilization buffer (100 mM NaCl, 20 mM Tris·HCl, pH 8.0, and 6 M GdnHCl). After stirring for 1 h, insoluble material was removed by centrifugation (12,000 × g, 15 min).
Purification of MBP Fusion Proteins-- LDAO (2% final concentration) was added to the solubilized inclusion bodies containing MBP-VDAC fusion proteins and GdnHCl was removed by dialysis against loading buffer (2% LDAO, 20 mM Tris·HCl, pH 8.0). The protein suspension was loaded onto a prewashed ceramic hydroxylapatite column, and bacterial porins were eluted with a KCl gradient (100-500 mM) containing 5 mM potassium phosphate (pH 6.8). MBP-VDAC was eluted with 300 mM potassium phosphate (pH 6.8) and treated with factor Xa (20 µg of enzyme/mg of protein in loading buffer containing 100 mM NaCl and 2 mM CaCl2) to cleave the MBP and VDAC polypeptides. Following dialysis against loading buffer, the sample was loaded onto a second hydroxylapatite column and VDAC was eluted with 100 mM KCl, 5 mM potassium phosphate (pH 6.8). Pooled fractions were concentrated to 2 mg/ml by centrifugation in Centricon 30 tubes.
Purification of His6-containing VDAC
Constructs--
Solubilized inclusion bodies (75 ml) containing
His6-VDAC proteins were diluted with 25 ml of
solubilization buffer without GdnHCl and added to 20 ml (bed volume) of
prepared TALONTM metal affinity resin (CLONTECH,
Palo Alto, CA). After 30 min, the matrix was poured into a column (16 mm diameter) and washed with three volumes of column buffer (4.5 M GdnHCl, 100 mM NaCl, 20 mM
Tris·HCl, pH 8.0) containing 10 mM imidazole.
His6-VDAC was eluted with two volumes of column buffer
containing 50 mM imidazole. Protein fractions were
concentrated to 6-8 mg/ml by centrifugation in Centricon 30 tubes.
LDAO was added to a final concentration of 2% and GdnHCl was removed
by dialysis against loading buffer. Proteins were stored at 70 °C
at a concentration of 1-5 mg/ml.
Gel Electrophoresis and Western Blots-- Proteins in LDAO-containing fractions were precipitated with cold acetone and solubilized in 1% SDS-containing buffer prior to electrophoresis on either 10% or 12% polyacrylamide slab gels (Mini-Protein II, Bio-Rad) as described by Stanley et al. (19). The proteins were either visualized by Coomassie staining or electrotransferred (Mini-Trans-Blot, Bio-Rad) to nitrocellulose membranes (0.45 mm, Bio-Rad). Electrotransfer and immunoblotting were done as described previously (19) using polyclonal antipeptide antibodies that react with the N- and C-terminal regions of ncVDAC and scVDAC (19), an anti-MBP antibody (New England Biolabs), or an anti-His6 antibody (San Diego Bio, San Diego, CA).
Mass Spectroscopy-- MALDI-TOF mass spectroscopic data were obtained with a Bruker (Billerica, MA) REFLEX II instrument using a matrix of 2,5-dihydroxybenzoic acid (Aldrich Chemical, Milwaukee, WI). Spectra, typically the average of 50 shots, were acquired in linear mode and calibrated with carbonic anhydrase (molecular mass: 28,834 daltons) as both an external and internal standard.
Planar Lipid Bilayer Experiments--
Electrophysiological
measurements were made using planar bilayers composed of soybean
L- lecithin phosphatidylcholine (Sigma) or synthetic
diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL).
The method of Labarca and Latorre (20) was used to generate bilayers
across a 0.15-mm hole separating two chambers containing 1 M KCl, 5 mM CaCl2, 5 mM
Hepes, pH 7.4. Bacterially expressed proteins were incubated 30 min on
ice with 10 volumes of an ergosterol suspension (1 mg/ml chamber buffer
containing 2% LDAO) before being added to the bilayer system (21).
Channel activity was observed after adding 6-13 pmol of VDAC to one
chamber, and resulting current traces were recorded and analyzed as
described previously (13). Bilayers were voltage-clamped using a Dagan 3900A amplifier (Dagan, Minneapolis, MN) in the "inside-out" mode. Mean open times were determined at
25 mV from current traces usually
40-60 s in duration, which were low pass-filtered to 2 kHz and stored
at 5 kHz using Strathclyde Electrophysiological Data Analysis Software
(courtesy of J. Dempster, University of Strathclyde, Strathclyde,
United Kingdom).
Far-UV Circular Dichroism Spectropolarimetry--
CD
measurements were carried out on a J-720 spectropolarimeter (Jasco,
Easton, MD). Measurements were taken from VDAC suspensions (approximately 1 mg/ml) at 20 °C using path lengths of 0.5-0.05 mm.
Spectra were recorded from 260 to 180 nm in 0.2-nm increments with a
1-s time constant. Each spectrum shown is the average of eight scans
corrected for background by subtraction of the spectrum corresponding
to the appropriate solvent minus protein. Mean residue molar
ellipticities, (degrees·cm2·dmol
1),
were based on protein concentrations measured using the bicinchoninic acid assay (Sigma); a mean residue molecular weight of 106 was used
(13). Solutions were buffered with either 20 mM Tris·HCl (pH 7.0-8.0) or 20 mM sodium citrate (pH 3.8-7.0).
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RESULTS |
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Purification of Bacterially Expressed VDAC--
First attempts to
overexpress VDAC in E. coli employed the pMAL expression
system, which offered, in principle, easy purification. Proteins are
expressed as fusion proteins with MBP, fractionated over an
amylose-containing matrix, and cleaved with factor Xa. Both MBP-scVDAC
and MBP-ncVDAC were obtained in large quantities (20-30 mg/liter of
cell culture) in inclusion body fractions. The MBP-ncVDAC fraction
yielded two strong bands with SDS-PAGE at Mr 70,000 and 66,000 (Fig. 1, lane
2). Both bands reacted with antibodies against both the N- and C
termini of VDAC (data not shown), indicating that any significant
truncation due to proteolysis probably occurred in the MBP part of the
protein. Expression of MBP-scVDAC yielded a single strong band at
Mr
66,000 (Fig. 1, lane 3).
Unfortunately, neither fusion protein bound to the amylose affinity
matrix in the presence of LDAO or other detergents tested
(e.g. octyl
-glucoside, Triton X-100, or SDS). Therefore,
an alternative purification scheme was devised involving sequential
fractionation of solubilized inclusion body fractions on
hydroxylapatite columns before and after cleavage with factor Xa.
The yeast protein, designated scVDAC
1-8 since it lacked
residues 1-8, gave a single band at Mr
30,000 (Fig. 1, lane 5) and was labeled by both N- and
C-terminal antibodies on Western blots (data not shown). The final
yield of purified scVDAC
1-8 obtained by this method was
1-2 mg/liter of cell culture, which represents less than 10% of the
VDAC in the inclusion bodies but still a 10-fold increase over the
yield of mitochondrial VDAC from fungal cultures (13). Conditions could
not be found for quantitative cleavage of MBP-ncVDAC with factor
Xa.
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Detergent Solubility of Bacterially Expressed Proteins--
Of
several nondenaturing detergents screened, including octyl
-glucoside, the only one in which the bacterially expressed VDAC
proteins are soluble at concentrations above 1 mg/ml is LDAO. ncVDAC(His6) is soluble at 4-20 °C in 1% LDAO at
concentrations above 5 mg/ml, while scVDAC(His6) has a
solubility limit of around 2 mg/ml.
Circular Dichroism and Channel Characteristics of Mitochondrial
VDAC--
Far-UV CD spectra of VDAC isolated from N. crassa
and S. cerevisiae mitochondria are shown in Fig.
3. The spectrum of ncVDAC in 1% LDAO at
pH 6-8 (Fig. 3A, solid curve) closely resembles those of the same protein (13) and of bacterial porins like OmpF (14)
in nondenaturing detergents. The CD spectrum of ncVDAC, containing a
single broad minimum at 214 nm and a crossover to positive ellipticity
at 205 nm, is consistent with high -sheet content (45%) and low
-helical content (12%) (Table I). The far-UV CD spectrum of mitochondrial scVDAC in 1% LDAO (Fig.
3A, dashed curve) is considerably different,
displaying two minima, at 220 and 208 nm, and a crossover to positive
ellipticity at 200 nm. This CD spectrum is similar to that of ncVDAC in
2% octyl
-glucoside at pH < 5 (13) and corresponds to lower
-sheet content (30%) and higher
-helical content (27%) relative
to ncVDAC at pH 6-8 (Table I).
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Circular Dichroism of Bacterially Expressed VDAC--
The far-UV
CD spectra of ncVDAC(His6) (Fig. 3B,
dashed curve) and of scVDAC1-8 (Fig.
3A, dotted curve) in 1% LDAO at pH 7 were found
to be very similar to those of the corresponding mitochondrial VDAC
proteins. In contrast, the CD spectrum of scVDAC(His6) in
1% LDAO (Fig. 3B, dotted curve) differs
significantly from that of mitochondrial scVDAC under the same
conditions. In the region above 205 nm, the spectrum of
scVDAC(His6) more closely resembles that of ncVDAC, having
a single broad minimum at 216 nm. However, the crossover to positive
ellipticity occurs at 202 nm, in between that of ncVDAC and scVDAC, and
the ellipticity maximum is somewhat reduced compared with mitochondrial
ncVDAC. The secondary structure content predicted from this spectrum is 35%
-sheet and 20%
-helix (Table I), indicating secondary
structure intermediate between that of mitochondrial scVDAC and
ncVDAC.
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Effects of SDS and pH on CD Spectra--
As shown in Fig.
5A, the far-UV CD spectra of
the three bacterially expressed VDAC proteins in 1% SDS are very
similar, closely resembling that of mitochondrial ncVDAC in this
detergent (13). The spectra, containing two minima (206-207 and 224 nm) and a crossover to positive ellipticity at 200 nm, are similar (but not identical) to those of ncVDAC in 1% LDAO at pH < 5 (13) and
scVDAC in 1% LDAO at pH 7 (Fig. 3A). Analysis of the CD
spectrum of ncVDAC(His6) in SDS indicates a correspondingly
low -sheet content (22%, Table I).
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Electrophysiological Characteristics of Bacterially Expressed
VDAC--
Both His6-containing proteins insert from LDAO
suspension into phospholipid bilayers and form stable channels when the
protein suspensions were preincubated with 0.1% ergosterol (see
"Experimental Procedures"). Differences in channel-forming
activities per mg of bacterially expressed and mitochondrial proteins
fall within the normal variability of the bilayer reconstitution
experiments. Typical current traces and amplitude histograms at 25 mV
are shown in Fig. 6 and single-channel
parameters are summarized in Table II. The results indicate that the
ion channels formed by the full-length bacterially expressed VDAC
proteins are comparable to those formed by the mitochondrial proteins
in terms of conductance levels (around 4.3 and 2.1 nS in 1 M KCl) and mean open times (around 100 ms).
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DISCUSSION |
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Using the inducible pET expression system and metal affinity chromatography, we have been able to obtain and purify multi-milligram quantities of VDAC protein from bacterial inclusion bodies. In the case of ncVDAC, CD spectra and channel properties of the mitochondrial protein and His6-containing bacterial construct are virtually indistinguishable, with one exception. The latter appears to resist the low pH-induced conformational change in nondenaturing detergent previously reported for ncVDAC isolated from mitochondria (13). This bacterially expressed protein is a suitable candidate for large scale crystallization trials, which are in progress.
Full-length scVDAC, both mitochondrial and bacterially expressed, forms
typical voltage-dependent ion channels in phospholipid bilayers, although the CD spectra in nondenaturing detergents indicate
considerably reduced -sheet structure relative to ncVDAC at pH 7. In
fact, the CD spectrum of mitochondrial scVDAC in LDAO at pH 7 is like
that of ncVDAC at pH < 5. Shao et al. (13) have shown
that the high
-sheet conformation of ncVDAC at pH 7 corresponds to
an "open" state in liposomes and that lowering the pH below 5 causes full (but reversible) loss of permeability to sucrose.
The CD spectrum of the His6-containing scVDAC construct in
LDAO at pH 7 is intermediate between that of mitochondrial ncVDAC and
scVDAC. This is borne out by the predicted -sheet content for
scVDAC(His6) of 35%, which is between that of
mitochondrial ncVDAC (45%) and scVDAC (30%). This raises the
possibility that the scVDAC(His6) fraction may contain a
mixture of high
and low
conformers of VDAC. The same appears to
be true for ncVDAC(His6) at low pH, for which the CD
spectrum is very similar to that of scVDAC(His6) at pH 7. After thrombin-cleavage of the His6-containing N-terminal
extensions, bacterially expressed ncVDAC and scVDAC still do not assume
the low
conformation of their mitochondrial counterparts (at pH 4 and 7, respectively). It may be that the N-terminal extensions induce
folding differences in VDAC that persist even after their removal, or
that the N-terminal extensions are not released by the proteins after
thrombin cleavage.
That ncVDAC and scVDAC assume distinctly different secondary structures
in nondenaturing detergents at pH 7 is an unexpected and potentially
useful finding. Xu and Colombini (25) have shown that ncVDAC channels
form more rapidly in phospholipid bilayers in the presence of urea and
GdnHCl, suggesting that partial unfolding of the polypeptide may
expedite the insertion process. Shao et al. (13) have
speculated that the low form of VDAC (reversibly induced by low pH
in ncVDAC) might represent a folding intermediate in the membrane
insertion process. It should be possible to test the hypothesis of Shao
et al. by comparing the insertion kinetics of scVDAC and
ncVDAC under conditions (e.g. nondenaturing detergent at pH
7) in which the former is in the low
conformation and the latter is
in the high
conformation. (In the bilayer experiments reported in
this paper, no attempt was made to compare initial rates of channel
insertion for the different proteins.)
The N-terminal domain of VDAC has been implicated as a voltage-sensing
region (26), and as a region that undergoes large scale motion in the
course of channel gating (27-30). It may form part of the lumen wall
in one or more states of the channel (30, 31), as does the N-terminal
domain of bacterial porins that H-bonds to the C-terminal transmembrane
-strand (10, 11). There is evidence that the N-terminal region of
VDAC tends to fold as an amphipathic
-helix (32) and not as a
transmembrane
-strand as it does in the bacterial porins. Table
III is a composite of the N-terminal
sequences and channel characteristics of the three bacterially
expressed constructs of scVDAC and ncVDAC reported in this paper and
those of three other ncVDAC constructs previously reported by Popp
et al. (21). Several important inferences may be drawn from
the table.
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First, lengthening the VDAC polypeptide by 12-20 residues at the N terminus has no obvious effect on channel stability, pore size, or voltage dependence. This finding is not inconsistent with the purported involvement of the N-terminal domain in forming part of the open-state lumen or in serving as a voltage sensor, since the extensions carry little or no net charge at the pH (7) at which the determinations were made. However, it may be useful to compare the effects of varying pH and multivalent metal ion concentration on the gating characteristics of VDACs with and without the His6-containing N-terminal extension. Additionally, since movement of the N terminus has been implicated in the mechanism of gating of VDAC, the gating kinetics of the channels with and without N-terminal extensions should be compared.
Second, while lengthening the N-terminal domain of VDAC has no
significant effects on channel properties, other types of alterations in this region have marked effects. For example, substituting 11 of the
first 20 amino acids of ncVDAC and extending the overall length of the
N-terminal region by 1 residue (variant nc N(2-12) of Popp et
al. (Ref. 21)) destabilizes the open state of the channel. The
same degree of channel instability is exhibited by nc
N(3-20) (21),
which retains only two of the first 20 residues and is 7 residues
shorter than wild-type VDAC. However, the variant that forms the most
atypical channels is the one with the shortest N-terminal domain,
scVDAC
1-8. This protein retains more N-terminal
residues (12) than either truncated variant of Popp et al.
but is 8 residues shorter than wild-type VDAC, 9 residues shorter than
nc
N(2-12), and 2 residues shorter than nc
N(3-20). Unlike the
longer VDAC variants, which display voltage-induced partial closures
from a long-lived open state (21), scVDAC
1-8 has a
seldom-observed fully open state and displays rapid transitions between
multiple lower conductance substates. Apparently shortening of the VDAC
polypeptide at the N terminus by more than 6-7 residues makes the
normal fully open state of the pore essentially inaccessible to the
polypeptide. This observation strongly supports those structural models
that have the N-terminal domain forming an integral part of the lumen
wall in VDAC's open state (30, 31). It is possible that the highest
conductance substate of scVDAC
1-8
(S2 in Fig. 6) may correspond to the partially
closed substate (S in Fig. 6) occupied by wild-type VDAC at
large transmembrane potentials, both of which are 2-2.5 nS. If so, the
fact that the open probability of substate S2 increases
with voltage (Fig. 7) would explain why occupancy of this particular
substate in wild-type VDAC normally predominates at high voltages.
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ACKNOWLEDGEMENTS |
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We thank Tailiang Guo, who first constructed
MBP-scVDAC1-8 in the laboratory of one of the authors
(P. M.), and Scott Stanley (laboratory of C. A. M.), who
raised the anti-peptide VDAC antibodies. We also gratefully acknowledge
the assistance of the directors of the Wadsworth Center's Biochemistry
Instrumentation and Biological Mass Spectroscopy core facilities, Drs.
Robert MacColl and Charles Hauer.
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FOOTNOTES |
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* This work was supported by Research Grant MCB-9506113 from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Wadsworth Center,
New York State Dept. of Health, Empire State Plaza, Albany, NY
12201-0509. Tel.: 518-474-2462; Fax: 518-486-4901; E-mail: carmen{at}wadsworth.org.
1
The abbreviations used in this paper: VDAC,
voltage-dependent anion-selective channel; LDAO, lauryl
dimethylamine oxide; PAGE, polyacrylamide gel electrophoresis; MBP,
maltose-binding protein; IPTG,
isopropyl-1-thio--D-galactopyranoside; GdnHCl, guanidine hydrochloride; MALDI-TOF, matrix-assisted laser desorption
ionization-time of flight; nS, nanosiemen(s); ncVDAC, N. crassa VDAC; scVDAC, S. cerevisiae VDAC.
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REFERENCES |
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