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Biophys J, December 2002, p. 3202-3210, Vol. 83, No. 6
*Department of Medical Biochemistry and Genetics, The Texas A&M
University System Health Science Center, College Station, Texas
77843-1114; and Department of Chemistry, Texas A&M
University, College Station, Texas 77843-3255 USA
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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DNA molecules tethered inside a protein pore can be used
as a tool to probe distance and electrical potential. The approach and
its limitations were tested with 
-hemolysin, a pore of known structure. A single oligonucleotide was attached to an engineered cysteine to allow the binding of complementary DNA strands inside the
wide internal cavity of the extramembranous domain of the pore. The
reversible binding of individual oligonucleotides produced transient
current blockades in single channel current recordings. To probe the
internal structure of the pore, oligonucleotides with 5' overhangs of
deoxyadenosines and deoxythymidines up to nine bases in length were
used. The characteristics of the blockades produced by the
oligonucleotides indicated that single-stranded overhangs of increasing
length first approach and then thread into the transmembrane
-barrel. The distance from the point at which the DNA was attached
and the internal entrance to the barrel is 43 Å, consistent with the
lengths of the DNA probes and the signals produced by them. In
addition, the tethered DNAs were used to probe the electrical potential
within the protein pore. Binding events of oligonucleotides with an
overhang of five bases or more, which threaded into the -barrel,
exhibited shorter residence times at higher applied potentials. This
finding is consistent with the idea that the main potential drop is
across the -hemolysin transmembrane -barrel, rather than the
entire length of the lumen of the pore. It therefore explains why the
kinetics and thermodynamics of formation of short duplexes within the
extramembranous cavity of the pore are similar to those measured in
solution, and bolsters the idea that a "DNA nanopore" provides a
useful means for examining duplex formation at the single molecule level.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The shape of the electrostatic profile within the
lumen of transmembrane channels and pores is an important unsettled
problem that remains the subject of experimental and theoretical
investigation (Hille, 2001
). Electrostatics can, for example, make a
major contribution to ion selectivity (Imoto et al., 1988; Cheung and
Akabas, 1997; Roux and MacKinnon, 1999; Corringer et al., 1999; Wilson
et al., 2000). The potential at a point within a pore at a fractional distance "d" from the entrance can be separated into two
components, 
M(d) and
S(d) (Pascual and Karlin, 1998). In the
simplest analysis, M(d), the electrostatic
potential arising from the applied transmembrane potential
(M), is assumed to be a linear function of
"d" such that M(d) = (1 
d) · M (Fig.
1 A). Of course, the actual
profile is related to the shape of the lumen. For example, in a channel with a closed gate, almost the entire transmembrane potential drops off
across the high resistance constriction. For this reason, the concept
of electrical distance (
) is often used, where (1 )
· M is the electrostatic potential at
"" arising from the applied transmembrane potential. When the
relationship between M(d) and "d" is
linear, d = . In other cases, "" increases with "d,"
but in a more complex manner. The intrinsic electrostatic potential at
a point "d," S(d), arises primarily from
permanent charges and dipoles on the protein, together with which
factors such as the local dielectric constant and screening by ions
within the lumen must be considered (Roux and MacKinnon, 1999).
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The large number of adjustable parameters that must be considered make
the computation of electrostatic profiles in channels and pores, and
the interpretation of related experimental work, a profoundly difficult
task. Nevertheless, progress has been made both in computation (Adcock
et al., 1998; Roux and MacKinnon, 1999) and measurement (Stauffer and
Karlin, 1994; Chiamvimonvat et al., 1996; Cheung and Akabas, 1997;
Pascual and Karlin, 1998; Wilson et al., 2000). In the case of narrow
ligand- or voltage-gated channels the contribution of
S() to the potential at a point in the
lumen is comparable with or greater than the contribution of (1 ) · M. However, in pores of large
diameter, particularly at high salt concentrations, (1 ) · M is likely to dominate. Here,
we study the dynamics of a DNA probe tethered within the staphylococcal
-hemolysin (HL) pore. Our results are consistent with the notion
that the applied transmembrane potential drops off slowly in the
internal cis cavity of the pore and more rapidly in the
transmembrane -barrel (Fig. 1 B).
The mushroom-shaped heptameric transmembrane pore formed by HL, a
293-residue bacterial toxin, has interesting internal dimensions (Song
et al., 1996). The mushroom cap, which is found on the cis side of the lipid bilayer (Fig. 1 B), contains a large
cavity, which measures ~46 Å in internal diameter and is
entirely located within the extramembranous domain. In the
transmembrane domain, the pore lumen narrows to form a 14-stranded
-barrel with an average internal diameter of ~20 Å. The two
domains are separated by a constriction of diameter ~14 Å (Fig. 1
B). In previous studies on other proteins, detailed
electrostatic profiles have been obtained by mutating the target
protein so that reactive cysteine residues are located at strategic
positions within the lumen. The rate of reaction with charged reagents
is then measured for each mutant (Chiamvimonvat et al., 1996; Pascual
and Karlin, 1998) and a profile can be obtained in terms of the
electrical distance . Here, we take on a simpler task and ask how
the applied potential falls off in the extramembranous domain of the
HL pore as compared with the transmembrane barrel. A DNA
oligonucleotide is tethered at a fixed position near the cis
entrance of the pore (Fig. 1 B). Probe nucleotides of
different lengths can then be attached by duplex formation and used to
sense the potential at defined depths within the lumen. While the
approach is limited by the large dimensions of the DNA probe, in the
case of a pore such as HL, it does provide useful information.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Oligonucleotides
Unless otherwise stated, DNA oligonucleotides were purchased
from Integrated DNA Technologies (Coralville, IA). The purity of the
oligonucleotides was checked by non-denaturing polyacrylamide gel
electrophoresis (Chory and Pollard, 1999). Instead of TBE buffer, TAE
(40 mM Tris acetate, 2 mM EDTA, pH 8.0) was used to prepare the gel and
the running buffer. After electrophoresis, the gels were stained for 2 min in a 0.2% aqueous solution of 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide (Stains-all; Sigma, St. Louis, MO, E-9379) containing 50% formamide, and destained in water for 20 min. Destained gels were
scanned and analyzed using the software Scion Image. DNA oligonucleotides of up to 10 nucleotides contained <2% of truncated products, and oligonucleotides of between 10 and 17 nucleotides <6%.
HL heptamers modified with single DNA oligonucleotides
HL heptamers modified with single DNA oligonucleotides
were generated as described (Howorka et al., 2001a,b). Briefly,
5'-thiol-modified DNA oligonucleotides with a hexamethylene linker
(Research Genetics, Huntsville, AL) were activated with
2,2'-dithiodipyridine to yield 5'-S-thiopyridyl oligonucleotides (Corey
et al., 1995). The activated oligonucleotides were coupled to the in
vitro expressed (Cheley et al., 1999) mutant, HL-17C-D4 (Howorka et
al., 2001a,b). Modified HL-17C-D4 and unmodified wild-type monomers
(H) were then co-assembled on erythrocyte membranes and the
resultant heptamers were purified by SDS-polyacrylamide gel
electrophoresis (Howorka et al., 2000). Heptamers with different
subunit compositions migrated in separate bands due to a gel shift
caused by the C-terminal polypeptide extension of four aspartates (D4)
(Howorka et al., 2001b). Heptameric H617C-oligo1 was extracted
from gel slices as described (Howorka et al., 2000).
Bilayer recordings
Electrical recordings were carried out with a planar lipid
bilayer apparatus at 22 ± 1.5°C (Montal and Mueller, 1972;
Hanke and Schlue, 1993; Braha et al., 1997). A bilayer of
1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPPC) was
formed on a 100-µm orifice in a Teflon septum (25 µm thick;
Goodfellow Corporation, Malvern, PA), which separated the two
compartments of the apparatus. The chambers contained 1.3 ml of 2 M
KCl, 12 mM MgCl2, 5 mM Tris-HCl, pH 7.4. Bilayers
were formed by first treating the surface around the orifice with a solution of hexadecane in n-pentane (10 mg/ml). Then, DPPC dissolved in
n-pentane (10 mg/ml) was allowed to spread on top of the electrolyte in
both compartments. After the solvent had evaporated, a bilayer was
formed by lowering and raising the electrolyte level once with respect
to the orifice. Heptameric protein was then added to the cis
compartment to a final concentration of 0.01 to 0.1 ng/ml, and the
electrolyte was stirred until a channel inserted. Recordings were
performed at an applied potential of +100 mV, unless otherwise stated,
with the cis chamber grounded. In general, the currents were
low-pass filtered with a 4-pole Bessel filter at 10 kHz, sampled at 50 kHz by computer with a Digidata 1200 A/D converter (Axon Instruments,
Union City, CA) and analyzed as described (Movileanu et al., 2000). For
the analysis of the lifetimes of the binding events and their
dependence on the length of the single-stranded extension (Fig. 4),
signals were filtered at 1 kHz and sampled at 5 kHz. In general,
current traces displayed in the figures were filtered at 10 kHz and
sampled at 50 kHz. Current traces in Fig. 5 A were filtered
at 1 kHz and sampled at 5 kHz.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The current signatures of covalently attached DNA duplexes depend on the lengths of their single-stranded extensions
An HL heptamer,
H617C-oligo-A1, with an
oligonucleotide, oligo-A, attached covalently at position 17 of the
single derivatized subunit (Fig. 1 B) was prepared by a
procedure devised previously (Howorka et al., 2001b). The attachment of
the single-stranded oligonucleotide decreases the conductance of the
HL pore by ~7% in 2 M KCl, the salt concentration used in this
work, and by ~17% in 1 M KCl and ~4% in 3.5 M KCl. Oligo-A served
to anchor a series of oligonucleotides based on oligo-B near the
cis mouth of the pore. Oligo-B is fully complementary to
oligo-A, and the other nucleotides in the series consisted of oligo-B
with 5'-oligo dA extensions, for example,
dA8-oligo-B (Fig. 1 C). Oligo-B
presented from the cis chamber binds to the immobilized
oligo-A to produce a transient current blockade at an applied potential
of +100 mV (Fig. 1 C-2 and Fig.
2 A-0) (Howorka et al.,
2001a,b). The block was characterized by an amplitude reduction
(Ramp) of 0.32, where Ramp is defined as the ratio of the
event amplitude to the mean current through the modified pore before
the oligonucleotide binding event (Fig. 1 C-2 and Fig. 2
A-0).
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The remaining oligonucleotides in the series,
dA1-oligo-B through
dA8-oligo-B, also bound, each giving a
characteristic Ramp value and current
signature (Fig. 2 A, panels 1 through 8-1). The
Ramp value for
dA1-oligo-B was slightly increased over oligo-B, with no extension. For dAN-oligo-B with
N 
2, two types of events were observed. Type 1 events comprised ~15% of the total number of events and had an
Ramp value that was independent of the
length of the extension and similar to
Ramp for oligo-B, the oligonucleotide without an extension (Fig. 2 A, panel 8-2; Fig. 2
B, open circles). Ramp of the type 2 events (~85% of
the total) increased with the length of the extension (Fig. 2
A, panels 2 through 8-1; Fig. 2 B, black
squares). In the case of dA2-oligo-B, the
type 2 events exhibited slight excess noise when compared with the
oligo-B and dA1-oligo-B events. In the case of
dA3-oligo-B, the excess noise was readily
apparent. For N 4, the signatures of the events indicate the presence of two current levels and transitions between them: state A (higher conductance), state B (lower conductance). The
midpoint of the transition to the lower conductance state was at
N = 5, at an applied potential of +100 mV (Fig. 2,
B and C).
The events arising from the shorter extensions, oligo-B and
dA1-oligo-B through
dA3-oligo-B (Fig. 2 A, panels 0 through 3), exhibit an "exit spike." It is likely that the spike
arises as the oligonucleotide dissociates to the trans side
in the positive applied potential, and in doing so passes through the
inner constriction of the pore. A similar signal was seen by Vercoutere
and colleagues when DNA hairpins unfolded at the cis
entrance and passed through the HL pore (Vercoutere et al., 2001).
We also studied the binding of oligo-B with 5'-dT extensions. The first oligonucleotide in the series, dT1-oligo-B, bound to immobilized oligo-A (Fig. 3 A-1) with an Ramp value of 0.36 that was slightly higher than Ramp for the oligonucleotide without the extension, oligo-B. The other oligonucleotides in the series, dT2-oligo-B through dT9-oligo-B, also bound to oligo-A, each giving a characteristic Ramp and current signature (Fig. 3 A, panels 2-9). As noted for the 5'-dA extensions, type 1 events of the dTN-oligo-B series had an amplitude reduction that was independent of the length of the extension (Fig. 3 B, open circles). Type 2 events comprised ~90% of the total number of events, with the exception of dT9-oligo-B (66% of the events). In the case of dT2-oligo-B, the type 2 events exhibited slight excess noise when compared with the oligo-B and dT1-oligo-B events. In the case of dT3-oligo-B, the excess noise was readily apparent. For dT4-oligo-B and longer oligonucleotides, two current levels were again observed, although the occupancy of state B was low for dT4-oligo-B. The midpoint of the transition to the lower conductance state B was between N = 6 and N = 7, at an applied potential of +100 mV (Fig. 3, B and C).
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The lifetimes of the DNA duplexes are dependent on the lengths of the single-stranded extensions
The lifetimes of the binding events were determined for oligo-B,
dA1-oligo-B through
dA8-oligo-B, and
dT1-oligo-B through dT9-oligo-B. The average duration of the type 1 events did not vary greatly with the length of the extension. For
dA2-oligo-B through
dA8-oligo-B the mean duration was 1090 ms, and
for dT2-oligo-B through
dT9-oligo-B, 840 ms. By contrast, the lifetimes
of the type 2 binding events for dAN-oligo-B
oligonucleotides were dependent on the length of the extension. The
lifetimes were obtained from dwell-time histograms, which could be
fitted with single exponentials (data not shown). As seen in a plot of
lifetime versus the number of bases, N, in the extension
(Fig. 4 A), the longest
lifetime was observed for N = 1. For N 2, the event duration decreased with increasing length up to
N = 7. The lifetime for N = 8 was slightly higher than N = 7. Similar behavior was
observed for the type 2 events associated with oligonucleotides with
5'-dT extensions (Fig. 4 B). Again, the lifetime increased
for N = 1 over N = 0, and dropped from
this maximum for extensions of N 2.
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The current signatures and lifetimes of the DNA duplexes are voltage-dependent
We studied the influence of the applied potential on the amplitude reduction and the fine structure of dA5-oligo-B binding events (Fig. 5 A). At +70 mV, the amplitude reduction, Ramp, derived from the major conductance level was 0.57. Higher potentials led to a shift in the amplitude reduction: +100 mV, Ramp = 0.56 and 0.77; +130 mV, Ramp = 0.79; and +160 mV, Ramp = 0.82. Once more, the signatures of the events suggest two current levels. In this case, the occupancy of the lowest conducting state, state B, increases with voltage rather than chain length: +70 mV, pB = 0.12; +100 mV, pB = 0.51; +130 mV, pB = 0.75; and +160 mV, pB = 0.92.
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Next, the influence of the applied potential on the lifetimes of the
oligonucleotide binding events was investigated. The voltage-dependence
of the lifetime was determined for two extreme cases: oligonucleotides
with the longest extensions, dA8-oligo-B and
dT9-oligo-B, and oligonucleotides without
extensions. The durations of dA8-oligo-B and
dT9-oligo-B binding events were determined by
fitting single exponentials to lifetime histograms (not shown) and were
found to decrease with increasing potential (Fig. 5 B). The
event lifetimes (
) for dA8-oligo-B (Fig. 5
B, filled diamonds) exhibit a subexponential
voltage dependence indicating that tends toward a constant value at
high potentials. By comparison, the lifetimes for
dT9-oligo-B events (Fig. 5 B,
open squares) were shorter, at a given voltage, than those
for dA8-oligo-B, but also exhibited a
subexponential dependence on applied potential.
In contrast to the negative voltage dependencies of the lifetimes of
duplexes formed by oligo-B with dA8 or
dT9 extensions, the mean event durations for
unextended oligo-B increased linearly with voltage (Fig. 5
C) with a slope of 10 ms mV
1. A
positive voltage dependence of the lifetimes was also observed for
another oligonucleotide without an extension. Oligo-D, an 8-mer
like oligo-B but with a different sequence (5'-TACGTGGA-3'), also forms
a duplex with a complementary oligonucleotide tethered within the pore
(Howorka et al., 2001b). The lifetimes for oligo-D show a linear
voltage dependence with a slope of 3.5 ms mV1.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the engineered HL heptamer,
H617C-oligo-A1, the
attached oligonucleotide, the 8-mer oligo-A, is capable of hybridizing to a complementary oligonucleotide, oligo-B, as shown previously (Howorka et al., 2001a,b) and here (Fig. 1 C-2). The binding
events were manifested as a sudden jump to a lower conductance state. A
spike of even lower conductance appeared upon dissociation as the
detached oligo-B made its way through the constriction in the lumen of
the pore (Fig. 1 B) (Vercoutere et al., 2001; Howorka et
al., 2001a). Interestingly, the covalent attachment of a
single-stranded oligonucleotide decreases the conductance of the HL
pore by ~7%, while the formation of a duplex with the same attached
DNA strand gives a much larger reduction in conductance of ~38%
compared to the unmodified pore. The reason for this is not certain,
but perhaps it is related to the greater stiffness of double-stranded DNA compared with single-stranded DNA.
When the hybridizing oligonucleotide was extended by one dA base, the
general appearance of the binding events was unchanged. However,
further increases in the length of the extension first produced events
that were noisy, indicating that the oligonucleotide approaches the
internal constriction (dA2-oligo-B to
dA3-oligo-B), and then a signal characteristic of
two states (dA4-oligo-B to dA8-oligo-B), indicating that the oligonucleotide
enters the transmembrane -barrel. We assign the higher conductance
state A to a conformation in which dAN-oligo-B is
entirely in the cavity, and the lower conductance state B to a
conformation in which the 5'-end of dAN-oligo-B extends through the inner constriction into the barrel (Fig. 1 C-3).
This behavior is similar to that of the highly flexible polymer
poly(ethylene glycol), PEG, when it is tethered by one end within the
cavity (Howorka et al., 2000; Movileanu et al., 2000). The presence of
PEG in the cavity leads to a relatively small conductance decrease, but
the occasional threading of the free PEG end into the -barrel is
accompanied by a larger drop in conductance. For the DNA-modified pore,
the duplex in state A is thought to be localized in the cavity and
exhibits a current blockade that increases slightly with the length of
the extension (Fig. 2 B). The extent of block in state B is
higher than in state A, and also increases with the length of the
extension for dA4 and higher. The amplitude of
state B approximates the amplitude of the exit spike, which is clearly
visible in the traces for dAN-oligo-B with
N 
3, but not for N 4 (Fig. 2
B). This finding supports the idea that state B arises
from the penetration of the oligonucleotide extension into the
-barrel.
In many cases, the binding events are initiated by a transient increase
in conductance of unknown origin, which was more readily apparent with
a lower frequency filter cutoff (but see Fig. 2 A, traces 4, 5, and 81; and Fig. 3 A, traces 6 and 8). As noted in
Results, a small fraction of events (type 1, Fig. 1 C-4)
were featureless (e.g., Fig. 2 A, panel 82) and most
likely arise from another state, distinct from state A, in which the
duplex is confined to the cavity. This interpretation is supported by the finding that the duration of type 1 events is voltage-independent. Therefore, in this case, it is unlikely that the extension penetrates into the -barrel, where it would "feel" the transmembrane
potential (see below). Transitions between the type 1 and type 2 states were rare (data not shown).
As expected, the occupancy of state B in the type 2 events for
dAN-oligo-B with N 4 increases with the
length of the 5'-extension, most likely because, in going from state A
to state B, the movement (z · 
) of the
negatively charged backbone of the DNA strand in the transmembrane
potential of +100 mV increases with length. The data, however, do not
support the notion that dA3 extensions penetrate
into the -barrel to give rise to state B. The maximum amplitude of
the current block of dA3 events falls short of
the amplitude of the exit spike, suggesting that during the existence of the duplex either the three nucleotides produce spikes that are so
short-lived they are filtered at 10 kHz, or that full penetration of
the barrel does not occur. We favor the latter interpretation because
of the relatively large amplitude of the exit spike, which can be taken
to be characteristic of penetration of the barrel. However, the form of
the exit spike is not a completely reliable argument against state B
for dA3-oligo-B, because the transient spikes may
be shorter than the exit spikes, reflecting their slightly different
molecular nature. Only the exit spikes include the separation of the
duplex and the complete passage of an oligonucleotide to the
trans side of the pore.
The data indicate that dA4-oligo-B and longer
oligonucleotides are capable of penetrating the inner constriction,
which is in keeping with the known dimensions of the pore (Song et al., 1996). The shorter dA2-oligo-B approaches the
constriction, as evidenced by increased current noise while the duplex
is present. The length of dA2-oligo-B including
the linker is ~42 Å, as estimated by using previous determinations
of the dimensions of nucleic acids ((Saenger, 1983), the duplex was
assumed to be a double helix) and the expected bond lengths and angles
for the linker. It should be noted that this approach yields an actual
measurement of "d" and not the electrical distance "," which
is the outcome of several other approaches. The
C
-C distance between residue 17, the point of attachment of oligo-A, and residue 111, in the
constriction, is 45 Å, while a surface-to-surface measurement between
these sites yields ~43 Å.
When another series of oligonucleotides,
dTN-oligo-B, was hybridized to
H617C-oligo-A1, the binding
events were similar to those observed with
dAN-oligo-B (Fig. 3). Similarly, additional noise
was first perceptible with the dT2-oligo-B.
Transitions between states A and B were apparent with
dT4-oligo-B, and two states were approximately
equally populated for dT7-oligo-B. For dAN-oligo-B the states are equally populated for
dA5-oligo-B. It is possible that this difference
is related to the lower propensity of oligo-dT to form secondary
structure and the greater flexibility of oligo-dT chains (Bonnet et
al., 1998; Meller et al., 2000; Bar-Ziv and Libchaber, 2001), which
might make them less likely to thread through the inner constriction.
The lifetimes of the type 2 binding events are dependent on the length
of the 5'-extension (Fig. 4). The findings are consistent with a model
in which the voltage-dependent step is a preequilibrium between states
A and B, and in which dissociation from state B is faster than
dissociation from state A. We have no ready explanation for the
decrease in the dissociation rate for dA1-oligo-B
compared to oligo-B itself. The increased rate of dissociation in state B might arise from the fact that the oligonucleotide is already threaded into the -barrel (the exit route) or because of a voltage dependence of koff. The latter is not
expected to be large because the movement in the potential gradient to
attain the transition state is small, but its existence is supported by
measurements of the voltage-dependence of the lifetime of
dA8-oligo-B and dT9-oligo-B type 2 binding events (Fig. 5 B). At +100 mV, both bound
dA8-oligo-B and dT9-oligo-B
are predominantly in state B. Therefore, assuming that simple
transition state theory applies, the lifetime of this state should
exhibit an exponential dependence on applied potential (Moczydlowski,
1986). While that is not the case, the negative slope of ln versus
V (Fig. 5 B) suggests that the
oligonucleotides "feel" the transmembrane potential. By contrast,
in the case of oligo B, which does not reach into the transmembrane
-barrel, the slope is weakly positive (Fig. 5 C).
Whatever the detailed mechanism of dissociation might be, the results
suggest that the duplexed oligonucleotide does not sense a strong field
in the cavity in the range of applied potentials we have examined. This
fact has proved useful in measurements of the kinetics and
thermodynamics of duplex formation, in which the results were
comparable with those determined in solution (Howorka et al., 2001b).
The weak strength of the field (d/dd) in the cavity is reasonable on
geometric and structural grounds (Fig.
6). The cavity is the widest part of the
lumen and is expected to be of low resistance (Fig. 6 A).
Furthermore, the cavity appears to contain holes that connect the lumen
to the aqueous phase on the cis side of the bilayer (Fig. 6
B). In either case, the voltage drop in the cavity is
expected to be <10 mV in an applied potential of +100 mV, while the
drop across the constriction is ~20 mV followed by a drop of ~1.3
mV Å1 in the barrel. This simple description
is complicated by the fact that the potential profile will change
during duplex formation, as implied by the large drop in conductance.
Finally, the contributions of the local electrostatic potential,
S, are likely to be small given the high salt
concentration (2 M KCl) used in our experiments and the wide
cross-section of the cavity, and this was supported by calculations
using the software package Spock (Christopher, 1998) (data not shown).
This situation contrasts with the case of narrow ion channels, such as
the acetylcholine receptor, at lower salt concentrations, where the
magnitude of M and S
can be comparable (Adcock et al., 1998; Pascual and Karlin, 1998; Wilson et al., 2000).
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In summary, the method we have proposed is a convenient way to probe
distance and potential within a channel or pore. Only one engineered
construct need be made and the subsequent experiments can be executed
with a series of synthetic oligonucleotides. However, the duplex that
is formed takes up a large space and the procedure would not be
expected to work in narrow channels and cannot be used for measuring
short distances. This problem might be circumvented by the direct
attachment of different probes, although this requires additional work
(Blaustein et al., 2000). A second problem is that formation of a DNA
duplex must alter the potential profile across the pore. Finally,
because the dissociation step for a duplex is not expected to be highly
voltage-dependent, we cannot determine the magnitude of the potential
drop in the cavity. Nevertheless, the increase in the duplex lifetime
for dA1-oligo-B and
dT1-oligo-B in the presence of a strong positive
applied potential suggests that the drop is small, in agreement with
structural considerations (Fig. 6).
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ACKNOWLEDGMENTS |
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We thank Orit Braha for her advice, and Katerina Kouri and Rosa Lemmens-Gruber for their assistance with reacquiring data.
This work was supported by the U.S. Department of Energy, the National Institutes of Health, the Office of Naval Research (Multidisciplinary University Research Initiative 1999), and the Texas Advanced Technology Program. S.H. held fellowships from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung) and the Max-Kade Foundation.
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FOOTNOTES |
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Address reprint requests to Hagan Bayley, Ph.D., Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, 440 Reynolds Medical Building, College Station, TX 77843-1114. Tel.: 979-845-7047; Fax: 979-862-2416; E-mail: bayley{at}tamu.edu.
Submitted November 21, 2001, and accepted for publication August 5, 2002.
Dr. Howorka's present address is Upper Austrian Research, Scharitzerstrasse 6-8, A-4020, Linz, Austria.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-hemolysin, a heptameric transmembrane pore.
Science.
274:1859-1865
Biophys J, December 2002, p. 3202-3210, Vol. 83, No. 6
© 2002 by the Biophysical Society 0006-3495/02/12/3202/09 $2.00
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l/d2, where l
is the length of the segment and d is the diameter. The
following values were used (l, d in Å):
entrance 19, 25; cavity 28, 45; constriction 8, 15; barrel 45, 20. (B) The fractional resistance of segments of the pore
incorporating the "side exits" in the cap of the pore. The
dimensions of the side exits were taken to be l = 15 Å, d = 5 Å).