U.S. patent application number 10/946802 was filed with the patent office on 2005-06-16 for designed protein pores as components for biosensors.
Invention is credited to Bayley, Hagan, Braha, Orit, Gouaux, Eric, Kasianowicz, John.
Application Number | 20050131211 10/946802 |
Document ID | / |
Family ID | 21986220 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050131211 |
Kind Code |
A1 |
Bayley, Hagan ; et
al. |
June 16, 2005 |
Designed protein pores as components for biosensors
Abstract
A mutant staphylococcal alpha hemolysin polypeptide containing a
heterologous analyte-binding amino acid which assembles into an
analyte-responsive heptameric pore assembly in the presence of a
wild type staphylococcal alpha hemolysin polypeptide, digital
biosensors, and methods of detecting, identifying, and quantifying
analytes are described.
Inventors: |
Bayley, Hagan; (College
Station, TX) ; Braha, Orit; (College Station, TX)
; Kasianowicz, John; (Darnestown, MD) ; Gouaux,
Eric; (New York, NY) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
21986220 |
Appl. No.: |
10/946802 |
Filed: |
September 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10946802 |
Sep 21, 2004 |
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09784985 |
Feb 15, 2001 |
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6824659 |
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09784985 |
Feb 15, 2001 |
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09122583 |
Jul 24, 1998 |
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60053737 |
Jul 25, 1997 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
G01N 33/48721 20130101;
C07K 14/31 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C12P 021/06; C07K
001/00; C07K 014/00; C07K 017/00 |
Goverment Interests
[0002] This invention was made with U.S. Government support under
the Office of Naval Research grant No. N00014-93-1-0962. The
government has certain rights in the invention.
Claims
What is claimed is:
1. A mutant staphylococcal alpha hemolysin polypeptide comprising a
heterologous amino acid, wherein said heterologous amino acid binds
an analyte and wherein said polypeptide assembles into a
heteroheptameric pore assembly in the presence of a plurality of
wild type staphylococcal alpha hemolysin polypeptides.
2. The polypeptide of claim 1, wherein said heterologous amino acid
occupies a position in a transmembrane channel of said heptameric
pore assembly.
3. The polypeptide of claim 2, wherein said heterologous amino acid
projects into the lumen of said transmembrane channel.
4. The polypeptide of claim 2, wherein said heterologous amino acid
occupies a position in a stem domain of said polypeptide.
5-20. (canceled)
21. A heptomeric pore assembly comprising a mutated staphylococcal
.alpha.HL polypeptide (MUT), wherein said MUT is an analyte-binding
.alpha.HL polypeptide.
22. The pore assembly of claim 21, wherein said pore assembly is a
heptamer having the formula WT7-nMUTn, wherein n is greater than
zero and less than seven.
23. The pore assembly of claim 21, wherein said analyte-binding
.alpha.HL polypeptide comprises a heterologous amino acid at a
position in a transmembrane channel of said pore assembly, wherein
said heterologous amino acid binds a metal.
24. The pore assembly of claim 21, wherein said pore assembly is a
heptamer having the formula WT7-nMn, wherein n is greater than zero
and less than seven.
25. A staphylococcal alpha hemolysin (.alpha.HL) polypeptide
comprising at least two non-consecutive heterologous amino acids in
a stem domain of said polypeptide, wherein each of said
heterologous amino acids binds an organic molecule, wherein the
amino acids occupy two or more of the following positions of SEQ ID
NO: 1: 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,
135, 137, 139, 141, 143, 145, 147 or 149, and wherein said
analyte-binding .alpha.HL polypeptide is 4H.
26. (canceled)
27. The pore assembly of claim 25, wherein the pore assembly is a
heptamer having the formula WT7-n4Hn.
28. The pore assembly of claim 27, wherein the pore assembly is a
heteroheptamer having the formula WT64H1.
29. A digital biosensor device comprising the pore assembly of
claim 21.
30-34. (canceled)
35. A method of detecting the presence of an analyte in a test
sample, comprising (a) contacting said sample with the pore
assembly of claim 21; and (b) detecting an electrical current in a
digital mode through two or more channels, wherein a modulation in
current compared to a current measurement in a control sample
lacking said analyte indicates the presence of said analyte in said
test sample.
36. A method of detecting the presence of an analyte in a test
sample, comprising (a) contacting said sample with the pore
assembly of claim 21; (b) detecting an electrical current in a
digital mode through a single channel, wherein a modulation in
current compared to a current measurement in a control sample
lacking said analyte indicates the presence of said analyte in said
test sample.
37. The method of claim 36, wherein said analyte is a metal
ion.
38. The method of claim 37, wherein said metal ion is Zn(II).
39. The method of claim 37, wherein said metal ion is Co(II),
Cu(II), Ni(II), or Cd(II).
40. A method of identifying an unknown analyte in a mixture of
analytes comprising, (a) contacting said mixture with the pore
assembly of claim 21; (b) detecting an electrical current in a
digital mode through two or more channels to determine a mixture
current signature; (c) comparing said mixture current signature to
a standard current signature of a known analyte, wherein a
concurrence of said mixture current signature and said standard
current signature indicates the identity of said unknown analyte in
said mixture.
41. The method of claim 40, wherein each of said known and unknown
analytes is a metal ion.
42. A method of identifying an analyte in a mixture of analytes
comprising, (a) contacting said mixture with the pore assembly of
claim 21; (b) detecting a single channel current in a digital mode
to determine a mixture current signature; (c) comparing said
mixture current signature to a standard current signature of a
known analyte, wherein a concurrence of said mixture current
signature and said standard current signature indicates the
identity of said unknown analyte in said mixture.
43. The method of claim 42, wherein each of said unknown and known
analytes is a metal ion.
44. The method of claim 43, wherein said metal ion is Zn(II).
45. The method of claim 43, wherein said metal ion is Co(II),
Cu(II), Ni(II), or Cd(II).
Description
[0001] This application claims priority from provisional
application 60/053,737, filed Jul. 25, 1997, which is incorporated
herein by reference in full.
BACKGROUND OF THE INVENTION
[0003] The field of the invention is metal detection.
[0004] Biosensors are analytical devices that convert the
concentration of an analyte into a detectable signal by means of a
biologically-derived sensing element. Well-known biosensors include
commercial devices for sensing glucose. In addition, true
biosensors, biomimetric devices, and devices that use living cells
have recently been developed. For example, to detect divalent metal
cations, true biosensors have been made using the enzyme carbonic
anhydrase (Thompson et al., 1993, Anal. Chem. 65:730-734), the
metal binding site of which has been altered (Ippolito et al.,
1995, Proc. Natl. Acad. Sci. USA 92:5017-5020). To monitor HIV
antibody levels, the enzyme alkaline phosphatase into which an HIV
epitope has been inserted has been utilized (Brennan et al., 1995,
Proc. Natl. Acad. Sci. USA 92:5783-5787).
SUMMARY OF THE INVENTION
[0005] The invention features a mutant staphylococcal alpha
hemolysin (.alpha.HL) polypeptide containing a heterologous
metal-binding amino acid. The polypeptide assembles into a
heteroheptameric pore assembly in the presence of a wild type (WT)
.alpha.HL polypeptide. Preferably, the metal-binding amino acid
occupies a position in a transmembrane channel of the
heteroheptameric pore assembly, e.g., an amino acid in the stem
domain of WT .alpha.HL is substituted with a heterologous
metal-binding amino acid. More preferably, the metal-binding amino
acid projects into the lumen of the transmembrane channel.
[0006] By the term "heterologous amino acid" is meant an amino that
differs from the amino acid at the corresponding site in the amino
acid sequence of WT .alpha.HL. By "analyte-binding amino acid" is
meant any amino acid having a functional group which covalently or
non-covalently binds to an analyte. By "transmembrane channel" is
meant the portion of an .alpha.HL polypeptide that creates a lumen
through a lipid bilayer. The transmembrane channel of an .alpha.HL
pore assembly is composed of 14 anti-parallel .beta. strands (the
".beta. barrel") two of which are contributed by the stem domain of
each .alpha.HL polypeptide of the pore. By "stem domain" is meant
the portion of an .alpha.HL polypeptide which spans approximately
amino acids 110 to 150 of SEQ ID NO:1 (see, e.g., FIG. 1F).
[0007] An .alpha.HL polypeptide containing at least two
non-consecutive heterologous metal-binding amino acids in a stem
domain of .alpha.HL is also within the invention. By "metal-binding
amino acid" is meant any amino acid which covalently or
noncovalently binds to a metal ion, e.g., Ser, Thr, Met, Tyr, Glu,
Asp, Cys, or His. Unnatural amino acids, such as 1,2,3
triazole-3-alanine and 2-methyl histidine, which have altered
pK.sub.a values, steric properties, and arrangement of N atoms
resulting in different abilities to bind metal ions, can also be
introduced to confer metal-responsiveness. Preferably, the
heterologous amino acids project into the lumen of the
transmembrane channel, i.e., the amino acids occupy two or more of
the following positions of SEQ ID NO:1: 111, 113, 115, 117, 119,
121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,
147 or 149. Alternatively, the heterologous amino acids are located
on the outside of the transmembrane channel, i.e., the amino acids
occupy two or more of the following positions of SEQ ID NO:1: 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,
138, 140, 142, 144, 146, 148. The polypeptide may contain at least
three non-consecutive heterologous metal-binding amino acids in the
stem domain. Preferably, the polypeptide contains at least 4
non-consecutive heterologous metal-binding amino acids in the stem
domain; more preferably, the amino acids occupy positions 123, 125,
133, and 135 of SEQ ID NO:1; more preferably, each these positions
are occupied by the heterologous metal-binding amino acid His; and
most preferably, the polypeptide is the .alpha.HL mutant 4H, as
described below.
[0008] To facilitate separation and purification of mutant
analyte-responsive .alpha.HL polypeptides, the polypeptide may also
contain a heterologous amino acid, e.g., a Cys residue, at a site
distant from the stem domain, e.g., at position 292 of SEQ ID
NO:1.
[0009] The invention also features a heteromeric pore assembly
containing a metal-responsive (M) .alpha.HL polypeptide, e.g., a
pore assembly which contains a wild type (WT) staphylococcal
.alpha.HL polypeptide and a metal-responsive .alpha.HL polypeptide
in which a heterologous metal-binding amino acid of the
metal-responsive .alpha.HL polypeptide occupies a position in a
transmembrane channel of the pore structure. For example, the ratio
of WT and M .alpha.HL polypeptides is expressed by the formula
WT.sub.7-nM.sub.n, where n is 1, 2, 3, 4, 5, 6, or 7; preferably
the ratio of .alpha.HL polypeptides in the heteroheptamer is
WT.sub.7-n4H.sub.n; most preferably, the ratio is WT.sub.64H.sub.1.
Homomeric pores in which each subunit of the heptomer is a mutated
.alpha.HL polypeptide (i.e., where n=7) are also encompassed by the
invention.
[0010] Also within the invention is a digital biosensor device
comprising a heteromeric .alpha.HL pore assembly. The device
detects binding of a metal ion to a heterologous amino acid through
a single channel (single current) or through two or more channels
(macroscopic current). Rather than containing a heterologous amino
acid substitution, the metal-responsive .alpha.HL polypeptide in
the device may contain a chelating molecule associated with an
amino acid in the stem domain.
[0011] The analyte-responsive .alpha.HL polypeptides (and pore
assemblies containing such polypeptides) can be used in a method of
detecting the presence of an analyte, e.g., a metal such as a
divalent Group IIB and transition metal. Zn(II), Co(II), Cu(II),
Ni(II), or Cd(II) can be detected using the methods described
herein. For example, a detection method may include the steps of
(a) contacting the sample to be analyzed with an analyte-responsive
.alpha.HL pore assembly, and (b) detecting an electrical current in
a digital mode through a single channel (single current) or two or
more channels (macroscopic current). A modulation or perturbation
in the current detected compared to a control current measurement,
i.e., a current detected in the absence of the analyte indicates
the presence (and concentration) of the analyte.
[0012] The invention also includes a method of identifying an
unknown analyte in a mixture of analytes which includes the
following steps: (a) contacting the mixture with an
analyte-responsive .alpha.HL pore assembly; (b) detecting an
electrical current in a digital mode through a single channel (or
through two or more channels) to determine a mixture current
signature; and (c) comparing the mixture current signature to a
standard current signature of a known analyte. A concurrence of the
mixture current signature with the standard current signature
indicates the identity of the unknown analyte in the mixture.
[0013] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims. All references cited
herein are incorporated by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A is a diagram showing the interpretation of a
digital/stochastic response of a single channel (patch clamp)
recording using an analyte-responsive .alpha.HL pore assembly (the
average upspike time durations .fwdarw.K.sub.1, the analyte
identity; the average downspike durations .fwdarw.[.circle-solid.],
the analyte concentration).
[0015] FIG. 1B is a series of graphs of digital single channel
recordings showing metal-responsiveness of an .alpha.HL pore
assembly at various concentrations of Zn(II).
[0016] FIG. 1C is a diagram of the structure of a heteromeric
.alpha.HL pore (WT.sub.64H.sub.1) assembly showing a Zn(II) binding
site with a view of the heptamer perpendicular to the seven-fold
axis of the pore. The top of the structure is on the cis side of
the membrane in bilayer experiments. The 14-strand .beta. barrel at
the base of the structure opens the lipid bilayer. In the 4H
subunit, residues Asn123, Thr125, Gly133, and Leu135 were replaced
with histidine and Thr292 with cysteine. A close-up view of the
antiparallel .beta. strands that contribute to the lower part of
the barrel is shown in FIG. 1E below.
[0017] FIG. 1D is a diagram of the structure of a heteromeric
.alpha.HL pore (WT.sub.64H.sub.1) assembly showing a Zn(II) binding
site with a view of the heptamer down the seven-fold axis from the
top (cis side) of the pore. The four heterologous histidinyl
residues project into the lumen of the channel, while Cys292 is
distant from the channel mouth.
[0018] FIG. 1E is a diagram of the structure of the transmembrane
channel portion of a heteromeric .alpha.HL pore assembly containing
the Zn(II)-responsive .alpha.HL polypeptide 4H. Zn(II) is shown
bound to the polypeptide at a binding site created by a
heterologous metal-binding amino acid substitution.
[0019] FIG. 1F is a diagram of the structure of an .alpha.HL
polypeptide showing the stem domain spanning approximately amino
acids 110-150.
[0020] FIG. 2A is a diagram of heteromeric combinations resulting
from the assembly of mixtures of wild-type (WT) and mutant (MUT)
.alpha.HL monomers showing the assembly of heteromeric .alpha.HL
pores. The 20 different heteromers (WT.sub.n-mMUT.sub.m; n=7, the
total number of subunits; WT, open circles; mutant, closed circles)
fall into n+1 classes categorized by the number of mutant subunits
(m) in the heptamer. The proportions of heptamers (%) in each class
is shown for three starting ratios of monomers (WT:MUT, 5:1; 1:1;
1:5). The values were calculated assuming that the oligomerization
process does not distinguish between WT and MUT monomers, by using
100.P.sub.m=100.[n!/m! (n-m!)].f.sub.MUT.sup.m- .f.sub.WT.sup.n-m,
where f.sub.MUT and f.sub.WT are the fractions of mutant and WT
subunits, respectively, in the starting monomer mix.
[0021] FIG. 2B is a diagram showing the procedure for assembly and
separation of heteroheptameric .alpha.HL pore assembly. Heteromers
were formed from the desired ratio of WT and MUT subunits on either
rabbit red blood cell membranes (rRBCM) or liposomes. The
heteromers were then derivatized with IASD, which introduced two
negative charges for each mutant (Cys292-containing) subunit. The
eight classes of heptomer were then separated by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE). The members of a particular class
were obtained by elution from the polyacrylamide.
[0022] FIG. 2C is a photograph of an electrophoretic gel showing
separation of different classes of .alpha. heptamers. WT .alpha.HL
and the mutant 4H, both [.sup.35]-labeled, were mixed in the ratios
indicated, allowed to assemble on rRBCM and then treated as shown
in FIG. 2B. The membranes were solubilized in gel loading buffer
containing SDS and, without heating, subjected to electrophoresis
in a 7% gel. A phosphorimager display of the molecules migrating
near the 200 kDa marker (myosin heavy chain) is shown. The observed
ratios of oligomer classes seen in each lane approximate those
shown in FIG. 2A. The lane marked "All" contained a mixture of the
solubilized samples at all five WT:4H ratios.
[0023] FIG. 3A is a photograph of an electrophoretic gel showing
purified .alpha.HL heteroheptamers. Heptamers were stable in SDS
and the subunits did not interchange. All eight radiolabeled
WT.sub.n-m4H.sub.m heptamers were purified by SDS-PAGE, rerun on a
40 cm long 8% SDS-polyacrylamide gel and visualized by
autoradiography. The individual heteromer species (lanes 1-8)
retained their relative mobilities, resulting in the staircase
appearance of the image.
[0024] FIG. 3B is an electrophoretic gel showing that WT.sub.7 and
4H.sub.7 did not become scrambled under the conditions used for
extraction, storage and reconstitution. An excised WT.sub.7 band
was mixed and coeluted with an excised 4H.sub.7 band. The sample
was kept at 4.degree. C. for 24 h and then stored at -20.degree. C.
The thawed sample was run on a 40 cm long 8% SDS polyacrylamide
gel. The bands retained their integrity (i.e. there is no ladder of
species to suggest subunit interchange).
[0025] FIG. 3C is a photograph of an electrophoretic gel showing
the ratio of the WT and 4H subunits in each purified heptamer.
Heptamers were made as described in the legend to FIG. 3A. Half of
each sample was subjected to electrophoresis without heating (top
panel), while the other half was dissociated by heating to
95.degree. C. (bottom panel). The mutant .alpha.HL monomers,
modified with IASD, were separated from the more rapidly migrating
WT polypeptides in a 40 cm long 10% SDS-polyacrylamide gel,
allowing the quantitation of the two monomer species contained in
each heptamer by phosphorimager analysis (ImageQuant, Molecular
Dynamics) The expected and measured ratios are shown below each
lane.
[0026] FIG. 4A is an autoradiogram of the SDS-PAGE separation of
approximately 5:1 mixture of WT and 4H, from which WT.sub.64H.sub.1
was eluted and used for single channel studies. Unlabeled WT was
used, so the first detectable band is WT.sub.64H.sub.1. This band
appears relatively weak here because it contains a single
.sup.35S-labeled 4H subunit.
[0027] FIGS. 4B, 4C, and 4D are a series of graphs of digital
single-channel recordings from a heteromeric .alpha.HL channel
containing a Zn(II) binding site. Single-channel recordings were
made using purified WT.sub.7 and WT.sub.64H.sub.1 pore assemblies
in planar lipid bilayers. Both cis and trans chambers of the device
contained 1 M NaCl, 50 mM MOPS, pH 7.5. Four consecutive traces of
a single-channel current at -40 mV are shown for each species.
Left, currents in the presence of 100 .mu.M EDTA; right, currents
after the addition of 150 .mu.M ZnSO.sub.4 to the trans side of the
membrane (approximately 50 .mu.M free Zn(II)).
[0028] FIG. 4B is a series of graphs showing digital single channel
recordings using a WT.sub.7 pore assembly (band 1 in FIG. 4A). The
channel is open with an amplitude of -26.7 pA (mean=27.0.+-.2.5 pA,
n=B). Zn(II) had no effect on the current, even when increased to
500 .mu.M.
[0029] FIG. 4C is a series of graphs showing digital single channel
recordings using a WT.sub.64H.sub.1 pore assembly (band 2 in FIG.
4A). In the presence of 100M EDTA the channel is open with an
amplitude of -28.4 pA at -40 mV (mean=26.3.+-.1.6 pA, n=7). The
addition of 150 .mu.M Zn(II) to the trans chamber results in
discrete fluctuations between two open states, the original state
(-28.4 pA) and another of -25.7 pA (mean=-24.4.+-.1.8 pA, n=7). The
ratio of the conductance of the new state to the conductance of the
original state (g/g.sub.o) was 0.93.+-.0.01 (n=7).
[0030] FIG. 4D is a series of graphs of digital single channel
recordings showing the dependence of the partial channel block of
the heteromeric pore WT.sub.64H.sub.1 on Zn(II) concentration.
Single-channel current recordings were made at various trans free
Zn(II) concentrations. A solution containing 1 M NaCl, 50 mM MOPS,
pH 7.5, Zn(II) was buffered with 100 .mu.M
pyridine-2.6-dicarboxylic acid and 10 .mu.M EDTA. All points
amplitude histograms are shown below each graph. The histograms can
be fitted to the sum of two Gaussian functions, suggesting two
distinct states: (i) the fully open channel as seen in the absence
of Zn(II), (ii) the partly closed, g/g.sub.o-0.93, Zn(II) dependent
substrate. The normalized areas of the Gaussian functions represent
the occupancy of each state at the displayed Zn(II) concentration.
When the openings or closing are short, the amplitudes of the
transitions are underestimated, resulting in shifts of the peaks to
lower values, for example, for 190 nM Zn(II).
[0031] FIG. 5A is a series of graphs of digital single channel
recordings from WT.sub.64H.sub.1 in the presence of 5 .mu.M free
Zn(II) or 5 .mu.M free Co(II) showing response of the heteromeric
pores to different M(II)s and tuning of the sensitivity to M(II)s
by adjustment of subunit composition. Top, transmembrane potential
-40 MV; bottom, transmembrane potential +40 mV.
[0032] FIG. 5B is a series of graphs showing the response of pores
containing more than one 4H subunit to Zn(II). WT.sub.54H.sub.2
(concentration of free Zn(II)=50 .mu.M), WT.sub.74H.sub.3 (20
.mu.M) and 4H, (10M). Left, digital single channel recordings of
currents in the absence (EDTA) and presence of Zn(II). The zero
current level is indicated (i=o). Right, the corresponding all
points histograms (light line, EDTA; dark line, Zn(II)).
[0033] FIG. 6A is a graph of a digital single channel recording
from WT.sub.64H.sub.1 in the presence of 150 nM Zn(II)
[0034] FIG. 6B is a graph showing an expanded view of a portion of
the graph in FIG. 6A.
[0035] FIGS. 7A and 7B are graphs of digital single channel
recordings from WT.sub.64H.sub.1 in the presence of a solution
containing 40 nM Zn(II) and 40 nM Ni(II) at a transmembrane
potential of -40 MV.
[0036] FIGS. 8A and 8B are graphs of digital single channel
recordings using a pore assembly containing a 123W/135W subunit in
the presence and absence of a solution containing the explosive
trinitrotoluene (TNT). FIG. 5A is a recording from pores in the
absence of TNT, and FIG. 8B is a recording from pores in the
presence of 1M TNT.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] Analyte-Responsive .alpha.HL Polypeptides as Components of
Biosensors
[0038] Biosensors generally have three elements: a) a binding site
to recognize a target analyte (e.g., introduced by engineering
metal-binding amino acid into an .alpha.HL polypeptide to create a
metal binding site in the transmembrane channel of an .alpha.HL
pore assembly), b) a transduction mechanism that signals the
fractional occupancy of the binding site by the analyte (e.g., salt
ions flowing through the .alpha.HL pore assembly/channel at a rate
of 100 million/sec for the open channel compared to an altered
rates when an analyte is bound), and c) a method of measurement
(and processing) of the transduction signal (e.g., pA, electrical
measurements of the ion flux through the .alpha.HL pore
assembly/channel in a membrane separating two liquid phases).
[0039] The compositions, devices and methods described herein can
be used to track diverse analytes of interest in spatio-temporal
gradients in water, in sediments and in the air. Such a capability
would permit, for example, gradiometer-directed locomotion of
robots. Other uses include detection, identification, and
quantification of analytes in the environment, e.g., Cu, Zn, or Ni
in effluents from underwater and dry dock hull cleaning operations,
in shipboard waste processing, and an ocean micronutrient
analyses.
[0040] Biosensors which incorporate protein pores as sensing
components have several advantages over existing biosensors. In
particular, bacterial pore-forming proteins, e.g., .alpha.HL, which
are relatively robust molecules, offer all the advantages of
protein-based receptor sites together with an information-rich
signal obtained by single-channel recording.
[0041] .alpha.HL is a 293 amino acid polypeptide secreted by
Staphylococcus aureus as a water-soluble monomer that assembles
into lipid bilayers to form a heptameric pore. The heptamer is
stable in sodium dodecyl sulfate (SDS) at up to 65.degree. C. The
biophysical properties of .alpha.HL altered in the central
glycine-rich sequence, by mutagenesis or targeted chemical
modification, demonstrate that this part of the molecule penetrates
the lipid bilayer and lines the lumen of the transmembrane channel.
The channel through the heptamer is a 14-strand .beta. barrel with
two strands per subunit contributed by the central stem domain
sequence (spanning approximately amino acids 110-150 of SEQ ID
NO:1).
1TABLE 1 WT .alpha.HL amino acid sequence (SEQ ID NO:1) ADSDINIKTG
TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK KLLVIRTKGT IAGQYRVYSE
EGANKSGLAW PSAFKVQLQL PDNEVAQISD YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG
KIGGLIGANV SIGHTLKYVQ PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV
YGNQLFMKTR NGSMKAADNFL DPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE
RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN
[0042] There is a need for biosensors that can detect a variety of
analytes, ranging from simple ions to complex compounds and even
microorganisms. Protein pores made from .alpha.HL polypeptides have
been remodeled so that their transmembrane conductances are
modulated by the association of analytes, e.g., divalent metal
ions, M(II)s. The lumen of the transmembrane channel was altered to
form different analyte-binding sites by design, e.g., by using
site-directed mutagenesis to insert heterologous metal-binding
amino acids. An analyte-binding .alpha.HL polypeptide is one that
contains an engineered analyte-binding site not present in the WT
.alpha.HL polypeptide. An analyte-binding site can be created by
the introduction of as few as one heterologous analyte-binding
amino acid, i.e., native residues may participate in forming a
binding site. M(II)-binding sites can also be formed by the
attachment of chelating molecule's by targeted chemical
modification. Combinatorial assembly is another way to generate
diversity (see FIG. 2A). M(II) detection is rapid (e.g., single
channel conductance is approximately 10.sup.8 ions sec.sup.-1),
reversible and sensitive. With digital single-channel recording for
analyte detection, the binding sites need not be fully selective
because the kinetics, extent and voltage-dependence of channel
block provide a distinctive analyte signature. The voltage is
gateable to further tune the biosensor. More than one analyte can
be assayed simultaneously using the compositions and biosensor
devices described herein. Selectivity is not a problem because a
single analyte binding site can only be occupied by a single
analyte at one time. Analyte-responsive .alpha.HL pores have been
successfully used to detect an analyte of interest, e.g., a metal
ion, in a solution containing a mixture of analytes as well as in
solutions containing various concentrations of a single
analyte.
[0043] Digital/Stochastic Single Channel Biosensors Using
Analyte-Responsive .alpha.HL Polypeptides
[0044] The attainment of sensitivity and selectivity is a major
problem with most known biosensors as they are based on an
integrated signal from numerous sensor molecules. The resulting
signal is analogue/steady state and contains limited information
about analyte identity(ies) and concentration(s). Analogue/steady
state detection data is extremely difficult to extract reliably,
even by modern processing hardware and software. For example,
simultaneous competition for an analyte-binding site by many
different analytes is a major problem. This problem is solved by
the analyte-responsive .alpha.HL pores described herein.
[0045] The disclosed analyte-responsive .alpha.HL compositions are
unique. A biosensor using an analyte-responsive .alpha.HL as the
sensing component is tunable to, any analyte target of interest by
introducing an analyte-binding site directly into a measurable
channel. Biosensors which incorporate an analyte-responsive
.alpha.HL pore assembly reliably detect analytes in single channel
mode, i.e., an individual analyte is detected as it randomly
(stochastically) hops on and off a single binding site. These
events are detected as modifications or perturbations of the ion
conductance in the single channel.
[0046] A digital/stochastic biosensor device incorporating an
.alpha.HL pore assembly as a sensing component has several
important advantages over analogue/steady state biosensors. For
example, the quality of the digital signal is independent of site
occupancy; therefore, the dynamic range is orders of magnitude
greater. Also, rate and equilibrium constants are read directly
from the averages of a few spikes providing fundamental signature
information about analyte identity and concentration. Simultaneous
occupancy of a single binding site by different analytes cannot
occur. Instead, competing analytes appear separated in time on the
signal trace, each with it's own characteristic current
signature.
[0047] FIG. 1A shows the interpretation of a digital/stochastic
response of a single channel (patch clamp) recording using an
analyte-responsive .alpha.HL pore assembly (FIGS. 6A-B show an
expanded view of a recording). Digital detection reports stochastic
behavior of a single analyte in real chemical time. The dynamic
range of a biosensor incorporating an analyte-responsive .alpha.HL
pore assembly is greater than 10,000 fold compared to approximately
20 fold for other known biosensors.
[0048] Structure-based design and a separation method that employs
targeted chemical modification have been used to obtain a
heteromeric form of the bacterial pore-forming protein .alpha.-HL,
in which at least one of the seven subunits contains a binding site
for a divalent metal ion, M(II), which serves as a prototypic
analyte. The single-channel current of the heteromer in planar
bilayers is also modulated by nanomolar Zn(II). Other M(II)s (e.g.,
Co, Cu, Ni, and Cd) modulate the current and produce characteristic
signatures. In addition, heteromers containing more than one mutant
subunit exhibit distinct responses to M(II)s. Analyte-responsive
.alpha.HL pores were generated through subunit diversity and
combinatorial assembly.
[0049] Sensor arrays with components with overlapping analyte
specificity, i.e., pore assemblies made from .alpha.HL polypeptides
which respond to a variety of analytes, e.g., metal ions, provide a
yet more powerful means for the simultaneous determination of
multiple analytes and to expand the dynamic range. By using the
design principles disclosed herein, binding sites for diverse
analytes, e.g., different metal ions, can be engineered into the
lumen of the transmembrane channel of an heteromeric .alpha.HL pore
assembly or near an entrance to the transmembrane channel, e.g.,
near the cis entrance of the channel. The digital/stochastic
detection mode can be generalized to classes of proteins other than
pore-forming proteins, e.g., receptors, antibodies, and enzymes,
with attached fluorescent probes to monitor individual binding
events using imaging technology directly analogous to single
channel recording. For example, analyte binding and dissociation
from an active site (e.g., naturally-occurring or re-engineered
analyte-binding site) of a remodeled fluorescent-tagged antibody,
lectin, or enzyme is detected using the detection methods described
above to determine the presence and/or concentration of an antigen,
carbohydrate moiety, or enzyme ligand, respectively.
[0050] The compositions and biosensor devices described herein
offer sensitivity, speed, reversibility, a wide dynamic range, and
selectivity in detecting and determining the identity and
concentration of analytes such as metal ions. .alpha.HL pores,
remodeled so that their transmembrane conductances are modulated by
the association of specific analytes, make excellent components of
biosensors.
[0051] Engineered pores have several advantages over existing
biological components of biosensors, e.g., sensitivity is in the
nanomolar range; analyte binding a rapid (diffusion limited in some
cases) and reversible; strictly selective binding is not required
because single-channel recordings are rich in information; and for
a particular analyte, the dissociation rate constant, the extent of
channel block and the voltage-dependence of these parameters are
distinguishing. A single sensor element can, therefore, be used to
quantitate more than one analyte at once. Furthermore, the
biosensor is essentially reagentless and internally calibrated. The
approach described herein can be generalized for additional
analytes, e.g., small cations and anions, organic molecules,
macromolecules and even entire bacteria or viruses, by introducing
a binding site for any given analyte into a portion of the
.alpha.HL polypeptide, e.g, the stem domain, which participated in
forming the transmembrane channel of the .alpha.HL pore assembly.
For example, a heterologous aromatic amino acid substitution can be
engineered into an .alpha.HL polypeptide, e.g., in the
transmembrane channel portion of an .alpha.HL pore assembly or at
the mouth of the channel, to confer responsiveness to a variety of
organic molecules. Furthermore, combinatorial pore assembly of
metal-responsive .alpha.HL polypeptides and WT .alpha.HL
polypeptides generate pores with diverse detection capabilities
(see FIG. 2A).
[0052] An analyte-responsive .alpha.HL pore containing a subunit in
which amino acids positions 123 and 125 of SEQ ID NO:1 were
substituted with tryptophan (123W/135W) was made. This mutant
.alpha.HL polypeptide was used to discern the presence and/or
concentration of organic molecules. For example, 123W/125W binds
the explosive TNT. Single-channel recordings using pore assemblies
containing a 123W/125W subunit detected TNT (FIGS. 8A-B).
[0053] .alpha.HL Pore Assemblies
[0054] WT .alpha.HL pores are homomeric; that is, all seven
subunits are the same. The analyte-responsive pores described
herein may be homomeric or heteromeric and contain at least one
mutated .alpha.HL polypeptide subunit. For example, a pore
assembled from seven subunits has the formula WT.sub.7-nMUT.sub.7,
where MUT is a mutant .alpha.HL polypeptide and where n=1, 2, 3, 4,
5, 6, or 7. Preferably, the MUT subunit is an analyte-binding
.alpha.HL polypeptide. The amino acid sequence of MUT differs from
that of WT in that MUT may be longer or shorter in length compared
to the WT subunit (e.g., MUT may be truncated, contain internal
deletions, contain amino acid insertions, or be elongated by the
addition terminal amino acids, compared to the WT sequence);
alternatively, MUT may contain one or more amino acid substitutions
in the WT sequence (or MUT may differ from WT both in length and by
virtue of amino acid sequence substitutions). The engineered
changes in the MUT subunit preserve the ability of MUT to associate
with other .alpha.HL polypeptides to form a pore structure.
[0055] A heteromeric pore was made that binds the prototypic
analyte Zn(II) at a single site in the lumen of the transmembrane
channel, thereby modulating the single-channel current. In
addition, M(II)s other than Zn(II) modulate the current and produce
characteristic signatures. Heteromers containing more than one
mutant subunit exhibit distinct responses to M(II)s. The invention
therefore provides an extensive collection of heteromeric
responsive pores suitable as components for biosensors.
[0056] Molecular Modeling of .alpha.HL Pore Assemblies
[0057] The three-dimensional structure of an .alpha.HL pore
assembly was determined using known methods, e.g., those described
in Song et al., 1996, Science 274:1859-1865. Using the modeling
techniques described below, the position of amino acids which
occupy the transmembrane channel portion of an .alpha.HL pore
assembly and/or protrude into the lumen of the transmembrane
channel can be determined. For example, to analyze the structures
of .alpha.HL polypeptides described herein, the coordinates of
carbonic anhydrase 11 (Eriksson et al., 1988, Proteins: Struct.
Funct. Genet. 4:283-293) were obtained (PDB accession number 1CA3).
Two strands (residues 91-98 and 116-121), containing the histidines
that bind Zn(II), were isolated and fitted by a blast square
procedure to the .beta. strands in the stem of protomer A of the
.alpha.HL structure (Song et al., 1996, Science 274:1859-1865).
Residues 123-126 and 132-135 of .alpha.HL were then replaced with
117-120 and 93-96 of carbonic anhydrase. The .alpha.HL sidechains
were substituted back into the structure, with the exception of the
histidines at positions 123, 125, 133, and 135. The Zn(II) ion and
the attached water molecule from carbonic anhydrase were left in
place. In addition, Thr292 was replaced with a cysteine residue.
The new molecule was drawn with Molscript (Kraulis, P. J., 1991, J.
Appl. Cryst. 24:946-949) and a final version rendered with Raster3D
(Merritt et al., 1994, Act Cryst. D50:869-873).
[0058] Mutagenesis
[0059] Recombinant .alpha.HL polypeptides, e.g., metal-responsive
.alpha.HL polypeptides, were made using methods well known in the
art of molecular biology. For example, the metal-responsive
.alpha.HL polypeptide, 4H, was made using DNA encoding a
full-length .alpha.HL (.alpha.HL-RL) that had been partly
reconstructed from the native S. aureus .alpha.HL gene (Walker et
al., 1992, J. Biol. Chem. 267: 10902-10909) with synthetic
oligonucleotides to introduce unique restriction sites in the
central region (residues 116-147). Four conservative amino acid
replacements are present in .alpha.HL-RL: Val124.fwdarw.Leu,
Gly130.fwdarw.Ser, Asn139.fwdarw.Gln and Ile142.fwdarw.Leu. The
region encoding amino acids 118-138 was removed by digestion with
BsiWI and Apal and replaced with two synthetic duplexes (BsiWi-Spel
and Spel-Apal) encoding the replacements Asn123.fwdarw.His,
Val124.fwdarw.Leu, Thr125.fwdarw.His, Gly130.fwdarw.Ser,
Gly133.fwdarw.His, Leu135.fwdarw.His. A 700 base pair fragment of
the resulting construct, encompassing the four new histidines, was
removed with Ndel and Mfel and used to replace the corresponding
sequence in .alpha.HL-Thr292.fwdarw.Cys. The entire coding region
of the resulting .alpha.HL-4H/Thr292.fwdarw.Cys construct was
verified by sequence analysis.
[0060] Expression and Purification of .alpha.HL Polypeptides
[0061] Monomeric WT-.alpha.HL was purified from the supernatants of
S. aureus cultures using known methods, e.g., the method described
in Walker et al., 1992, J. Biol. Chem. 267: 10902-10909.
[.sup.35S]-Methionine-labe- led WT-.alpha.HL and .alpha.HL-4H were
obtained by coupled in vitro transcription and translation (IVTT).
Separate reactions conducted with a complete amino acid premix and
the premix without unlabeled methionine were mixed to yield a
solution containing .alpha.HL at >10 .mu.g/ml. .alpha.HL in the
IVTT mix was partially purified by (i) treatment with 1% (w/v)
polyethyleneimine (PEI) to precipitate nucleic acids, (ii)
treatment with SP Sephadex C50, pH 8.0 (to remove the residual,
PEI), and (iii) binding to S-Sepharose Fast Flow at pH 5.2,
followed by elution with 10 mM sodium acetate, pH 5.2, 800 mM NaCl.
The concentration of .alpha.HL (in the IVTT mix or after the
purification) was estimated by a standard quantitative hemolytic
assay.
[0062] Oligomerization of .alpha.HL Polypeptides
[0063] WT and .alpha.HL-4H were mixed in various molar ratios (6:0,
5:1, 1:1, 1:5, and 0:6) and allowed to oligomerize on rabbit
erythrocyte membranes, liposomes, and other planar bilayers. The
.alpha.HL polypeptides self-assemble into heteroheptameric pore
assemblies in bilayers. For rabbit erythrocytes membranes,
oligomerization was carried out as follows. Mixtures were incubated
for 1 h at room temperature in 10 mM MOPS, pH 7.4, 150 mM NaCl. The
membrane were washed and resuspended in 200 mM TAPS, pH 9.5,
treated with 0.5 mM DTT for 5 min and then with 10 mM
4-acetamido-4'-[(iodoacetyl)amino]stilbene-2.2.degree.-disulfonate
(IASD, Molecular Probes, Eugene, Oreg., USA) for 1 h at room
temperature to modify the Cys292 residue on the 4H polypeptide
chain. The membranes were recovered by centrifugation, taken up in
gel loading buffer, without heating, and loaded onto a 7%
SDS-polyacrylamide gel (40 cm long, 1.5 mm thick). Electrophoresis
was carried out for 16 h at 120 V at 4.degree. C. with 0.1 mM
thioglycolate in the cathode buffer. The dried gel was subjected to
phosphorimager or audioradiographic analysis.
[0064] Heteroheptamer Formation and Purification
[0065] Heteromeric pore assembly by .alpha.HL polypeptides in
membranes and other planar bilayers suitable for use in biosensor
devices was carried out using known methods, e.g., those described
by Hanke et al., 1993, Planar Lipid Bilayers, Academic Press,
London, UK; Gutfreund, H., 1995, Kinetics for the Life Sciences,
Cambridge University Press, Cambridge, UK). Rugged planar bilayers
are described in Cornell et al., 1997, Nature 387:580-583.
[0066] For example, to generate 4H heteroheptamers, unlabeled
WT-.alpha.HL and .sup.35S-labeled 4H were mixed in a 5:1 ratio
(WT-.alpha.HL; 2.5 .mu.l of 0.5 mg/ml in 20 mM sodium acetate, pH
5.2, 150 mM NaCl; .sup.35S-labeled 4H; 50 .mu.l of 5 .mu.g/ml). The
mixed subunits were allowed to oligomerize on liposomes for 60 min
at room temperature by incubation with 10 mM MOPS, pH 7.4, 150 mM
NaCl (26 .mu.l) and egg yolk phosphatidylcholine (Avanti Polar
Lipide, Birmingham, Ala., USA; 1.5 .mu.l of 10 mg/ml). The latter
had been bathed sonicated at room temperature until clear (30 min)
in 10 mM MOPS, pH7.4, 160 mM NaCl. The mixture (60 .mu.l) was then
treated with 2 M TAPS, pH 8.5 (10 .mu.l), and 10 mM DTT (6 .mu.l)
for 10 min at room temperature, followed by 100 mM IASD (5 .mu.l in
water) for 60 min at room temperature. Gel loading buffer
(5.times., 25 .mu.l) was then added, without heating, and a portion
(50 .mu.l) was loaded into an 8 mm wide lane of a 40 cm long, 1.5
mm thick 6% SDS-polyacrylamide gel, which was run at 4.degree. C.
at 120 V for 16 h, with 0.1 mM thioglycolate in the cathode buffer.
The unfixed gel was vacuum dried without heating onto Whatman 3MM
chromatography paper (#3030917).
[0067] Each of the eight heptamer bands was cut from the gel, using
an autoradiogram as a guide. The excised pieces were rehydrated
with water (100 .mu.l). After removal of the paper, each gel strip
was thoroughly crushed in the water and the protein was allowed to
elute over 18 h at 4.degree. C. The solvable eluted protein was
separated from the gel by centrifugation through a 0.2 .mu.m
cellulose acetate filter (#7016-024, Rainin, Woburn, Mass., USA). A
portion (20 .mu.l) was saved for single channel studies. Sample
buffer (5.times., 20 .mu.l) was added to the rest of each sample.
Half was analyzed, without heating, in a 40 cm long 8%
SDS-polyacrylamide gel. The other half was dissociated at
95.degree. C. for 5 min for analysis of the monomer composition in
a 10% gel.
[0068] Biosensor: Planar Bilayer Recordings
[0069] Detection of analytes using heteroheptameric .alpha.HL pore
assemblies in planar bilayers was carried out as follows. A bilayer
of 1,2-diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids)
was formed on a 100-200 .mu.m orifice in a 25 .mu.m thick teflon
film (Goodfellow Corporation, Malvern, Pa., USA), using standard
methods, e.g., the method of Montal and Mueller (Montal et al.,
1972, Proc. Natl. Acad. Sci. USA 69:3561-3566). Both chambers of
the device contained 1 M NaCl, 50 mM MOPS, pH 7.5, and other
solutes as described in the figure legends. Two to 10 .mu.l of the
eluted protein were added to the cis chamber to a final
concentration of 0.01-0.1 ng/ml. The bilayer was held at -10 mV
with respect to the trans side. The solution was stirred until a
channel inserted. The analyte Zn(II) was added with stirring, to
the trans chamber from a stock solution of 100 mm ZnSO.sub.4 in
water. Where Zn(II) was buffered, the concentration of free Zn(II)
was calculated using the program Alex (Vivadou et al, 1981, J.
Membrane Biol. 122:155-175). Currents were recorded by using a
patch clamp amplifier (Dagan 3900A with the 3910 Expander module),
filtered at 5 kHz (four-pole internal Bessel filter) and stored
with a digital audio tape recorder (DAS-75; Dagan Corporation,
Minneapolis, Minn., USA). For example, the data were filtered at
1-2 kHz (eight-pole Bessel filter, Model 900, Frequency Devices)
and acquired at 5 kHz onto a personal computer with a Digidata 1200
D/A board (Axon Instruments). The traces were filtered at 100-200
Hz for display and analysis with the Fetchan and pSTAT programs,
both of pCLAMP 6. Negative current [downward deflection] represents
positive charge moving from the cis to the trans chamber.
[0070] Molecular Design of Heteromeric .alpha.HL Pores
[0071] A Zn(II)-binding .alpha.HL polypeptide was made by
substituting one or more amino acids in the stem domain of WT
.alpha.HL with a heterologous metal-binding amino acid. One example
of such a Zn(II)-binding polypeptide is 4H which contains the
following amino acid substitutions in the stem domain of .alpha.HL:
Asn123.fwdarw.His, Thr125.fwdarw.His, Gly133.fwdarw.His,
Leu135.fwdarw.His, Thr292.fwdarw.Cys. Four histidines were
introduced by mutagenesis to project into the lumen of the channel
(e.g., at odd numbered positions of the stem domain) to form a
cluster of imidazole sidechains. .alpha.HL polypeptides in which
heterologous metal-binding amino acids have been introduced such
that they are located on the outside of the barrel (e.g., at even
numbered positions of the stem domain) of the pore assembly also
confer responsiveness to metal ions. In addition, amino acid
substitutions in regions of the .alpha.HL polypeptide outside the
stem domain but which are close to the lumen of the transmembrane
channel, e.g., at the mouth of the channel, also confer metal
responsiveness.
[0072] The channel through the heptamer is a 14-strand .beta.
barrel with two strands per subunit (see FIGS. 1C-F) contributed by
the central stem domain sequence which spans approximately amino
acids 110-150 of SEQ ID NO:1: EYMSTLTYGF NGNVTGDDTG KIGGLIGANV
SIGHTLKYVQ (SEQ ID NO:2). Structural data indicates that the .beta.
barrel is sufficiently flexible for at least three sidechains to
act as ligands to Zn(II) in the preferred tetrahedral
configuration.
[0073] To facilitate separation of polypeptides, the 4H polypeptide
was also cogged by chemical modification of the single cysteine (at
position 292) with
4-acetamido-4'-[(iodoacety)amino]stilbene-Z,Z'-disulfonate (IASD).
The Cys-cogged .alpha.HL (Thr292.fwdarw.Cys; without amino acid
substitutions in the stem domain) modified with IASD forms fully
active homomers. This modification caused an incremental increase
in the electrophoretic mobility of heptamers in SDS-polyacrylamide
gels allowing heteromers to be easily separated from each other and
from wild-type (WT) heptamers. Each disulfonate made an
approximately equal contribution to the mobility, which is
independent of the arrangement of the subunits about the seven-fold
axis. The chemical modification was distant from the stem domain of
the polypeptide which lines the channel of the heteromeric pore
assembly.
[0074] Assembly and Separation of .alpha.HL Metal-Responsive
Heteromeric Pores
[0075] There is only one possible arrangement of heteromers
containing six WT and one 4H subunit (WT.sub.64H.sub.1; FIG. 2A).
Therefore, the WT.sub.64H.sub.1 pore assembly is consistently and
reliably formed. The 4H mutant of .alpha.HL was prepared using
known methods for making recombinant proteins, e.g., in vitro
transcription and translation (IVTT). In some cases, .alpha.HL was
radiolabeled with [.sup.35S]methionine. WT .alpha.HL was also
prepared by IVTT when labeling was desired. Alternatively, WT
.alpha.HL was purified from S. aureus. WT and 4H were mixed in a
molar ratio of 5:1 and allowed to assemble on lipid bilayers, e.g.,
rabbit red blood cell membranes (rRBCM) or on liposomes made from
egg yolk phosphatidylcholine (FIG. 2B). After assembly, the 4H
subunits were modified at Cys292 with IASD. The membranes were
solubilized in SDS and the heteromers separate by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE; FIG. 2C).
Heptamers were eluted passively from the polyacrylamide with water,
for reconstitution into bilayers for biophysical characterization.
The eluted heteromers remained intact as shown by
re-electrophoresis (FIG. 3A). These data demonstrated that the
polypeptides did not become scrambled, thus indicating the
predictability of pore assembly. For example, WT.sub.7, and
WT.sub.54H.sub.2 were not formed from WT.sub.64H.sub.1.
[0076] In two out of five such runs, small amounts of monomer
(<5%) were detected. Such breakdown was probably due to the
storage conditions that the two samples experienced (e.g. for the
sample displayed in FIG. 3A, several freeze/thaw cycles, followed
by storage at 4.degree. C. for ten days). In the other three runs,
where freshly eluted heptamers were examined, monomers were not
detected at all. In a definitive experiment, gel slices containing
the homomers WT, and IASD-modified 4H.sub.7 were mixed and taken
through the elution and storage procedures before
re-electrophoresis, which again indicated no scrambling (FIG. 3B).
Furthermore, the eluted heptamers were free of residual proteins
from the IVTT mix, as determined by silver staining. Finally the
ratio of the .alpha.HL polypeptides in each of the heteromeric pore
assemblies examined was as expected, when determined by
quantitative analysis of radio-labeled polypeptides from purified
heteromers dissociated by heating to 95.degree. C. (FIG. 3C). The
electrophoretic gel shown in FIG. 4A confirms the heteromeric
channel structure of the .alpha.HL pore assembly.
[0077] Digital Single-Channel Currents from Heteromeric
Metal-Responsive Pores
[0078] The properties of WT.sub.64H.sub.1 were examined by digital
single-channel recording in a planar bilayer biosensing apparatus.
Methods for forming planar bilayers in biosensors are known in the
art, e.g., Hanke et al., 1993, Planar Lipid Bilayers, Academic
Press, London, UK or Gutfreund, H., 1995, Kinetics for the Life
Sciences, Cambridge University Press, Cambridge, UK. In this
experiment, a lipid bilayer was formed across an aperture (100-200
.mu.m diameter) in a teflon film (25 .mu.m thick) that separates
two chambers (2 ml each) containing electrolyte. With a potential
applied across the bilayer, the ion flux through single .alpha.HL
pores was measured with a sensitive, low-noise amplifier.
[0079] To obtain single-channel currents, the eluted heptamers were
added at high dilution (typically 1:1000) to the cis chamber of the
bilayer apparatus to a final concentration of 0.02-0.1 ng/ml (FIGS.
4B-D). WT.sub.64H.sub.1 exhibited a partial and reversible channel
block (g/g.sub.0-0.93.+-.0.01.sub.1; n=7) in the presence of 50
.mu.M Zn(II) in the trans compartment with the transmembrane
potential held at -40 mV (FIG. 4C).
[0080] The behavior of heteromeric WT.sub.64H.sub.1 pores were
compared to two different control pores. WT.sub.7 control pores
were not sensitive under the conditions described above (see FIG.
4B) and were unaffected by up to 500 .mu.M Zn(II). Heteromeric
WT.sub.64H.sub.1 pores were also compared to control pores made
with WT.sub.64H.sub.1 with an additional Thr292.fwdarw.Cyr
mutation, modified with IASD. This heptamer also gave no response
with Zn(II). FIG. 4B shows data from WT, .alpha.HL pore assemblies
(i.e., control pores); control pores did not respond to the
presence of Zn(II) or EDTA (a chelating agent that complexes
M(II)).
[0081] Analysis of conductance histograms for WT.sub.64H.sub.1
obtained for a series of buffered Zn(II) concentrations (FIG. 4D)
yielded an EC.sub.50 for trans Zn(II) of 112.+-.23 nM (n=3). The
EC.sub.50 is the concentration of free M(II) that effects 50%
occupancy of the binding site of 4H. Kinetic analysis of the
current traces yielded a second-order associated rate constant
(k.sub.on) for Zn(II) of 3.2.+-.0.4.times.10.sup-
.8M.sup.-1s.sup.-1 (n=4) which approaches the diffusion limit, and
a dissociation rate constant (k.sub.off) of 33.+-.2 s.sup.-1 (n=4).
The EC.sub.50 value was lower than expected for two histidinyl
ligands and approached the values found for structures with three
histidines with favorable geometry (e.g. 36 nM for a mutated
retinol-binding protein), suggesting that a modest distortion of
the .beta. barrel can be tolerated that places at least three of
the four histidines in conformations suitable for coordination of
the bound metal. The flexibility of the barrel is supported by (1)
the three-dimensional structure of .alpha.HL, (2) the fact that for
.alpha.HL in liposomes blue shifts of the fluorescent probe
acrylodan (attached at single cysteine residues in the .beta.
barrel) do not alternate with residue number (as would be required
for nondistorted .beta. strands), and (3) the existence of mutants
with proline residues in the central domain that form pores.
[0082] The conductance of WT.sub.7 pores (675.+-.62 pS.sub.1 1M
N.sub.aCl.sub.1 50 mM MOPS, pH7.5.sub.1-40 mV.sub.1 n=8) was
similar to that of WT.sub.64H.sub.1 in the absence of Zn(II)
(660.+-.40 pS.sub.1 n=7). The conductance of WT.sub.64H.sub.1 with
Zn(II) bound was reduced to 610.+-.45 pS (n=7). A partial channel
block may be due to a simple physical blockade, distortion of the
barrel, or electrostatic effects.
[0083] FIGS. 4C and 4D show digital responses of the engineered
WT.sub.64H.sub.1 hybrid channel to various levels of Zn(II). The
digital pattern is due to the stochastic (random) effect of single
zinc ions hopping on and off the tetra-histidyl binding site
engineered into the lumen of the transmembrane channel of an
.alpha.HL pore assembly. The two channel states are open (Zn(II)
off, 100% open) and gated (Zn(II) on, 93% open). Average time in
the open state is the reciprocal of bimolecular rate constant x
[Zn(II)], from which Zn(II) is quantified, while average time in
the gated state is the reciprocal of the first order off constant
(the analyte signature or identity). Monovalent metal cations gave
no signal. These data indicate that the metal-responsive .alpha.HL
polypeptides and pore assemblies used as components of a biosensor
provide a means to achieve unambiguous analyte identity and
concentration(s). Existing chemo/bio-sensors are analog/steady
state, whereas the channel of the .alpha.HL pore assembly is
digital/stochastic. FIG. 4 also shows that .alpha.HL pore
assemblies have an wide dynamic range of analyte detection (at
least 10,000-fold in analyte concentration. Even at very low
fractional site occupancies, the signal (being digital and not
analog) is not degraded. At very low site occupancy, it simply may
take longer to collect to collect data (however, sensitivity and
selectivity is not compromised).
[0084] Metal-Responsive .alpha.HL Pores Produce Characteristic
Single-Channel Signatures in Response to Various Divalent Metal
Cations
[0085] To determine whether WT.sub.64H.sub.1 can distinguish
between different M(II)s, the effects of Co(II), Ni(II) and Cu(II)
on single-channel currents were examined. Each gave a
characteristic current signature. For example, at -40 mV 5 .mu.M,
CO(II) produced a distinctive current signature compared to, e.g.,
Zn(II) (FIG. 5A, top). At higher CO(II) concentrations, the signal
was continuous resulting from the rapid interconversion of three
states, one with higher conductance than WT.sub.64H.sub.1 in the
absence of M(II). At +40 mV, two states were seen with 5 .mu.M
CO(II) (FIG. 5A, bottom). The effect on current amplitude is
similar to that of Zn(II) at this membrane potential, but the rates
of CO(II) association and dissociation are considerably slower.
These data also show that the responses of single-channel currents
to membrane potential contain additional information about the
concentration and identity of analytes.
[0086] The data in FIG. 5A indicate that different M(II) give
different digital output patterns, i.e., spikes from one M(II),
e.g., Zn(II), are not hidden under the spikes of another, e.g.,
Co(II), because only one metal ion can occupy a single site at one
time. In a complex mixture of analytes, deconstruction of the
signal is required to isolate the current signature of an analyte
of interest. The sensitivity and precision of analyte
identification achieved by the compositions and digital/stochastic
devices of the invention vastly exceed those achieved by known
analogue/steady state biosensor devices. For example, simultaneous
competitive inhibition owing to incomplete selectivity is a
universal problem with conventional chemo/bio-sensors, requiring
extensive down-stream processing. In contrast, the identity and
concentration of analytes can easily, reliably, and accurately
determined from traces such as those in FIGS. 4A-D and 5A-B, i.e.,
analytes can be identified (as well as quantified) by the
single-channel current signature (.DELTA.g, k.sub.on, k.sub.off,
voltage dependence of these parameters). FIG. 5 also illustrates
that the channel can further be tuned by changing the transmembrane
voltage. FIGS. 7A-B show that digital output patterns corresponding
to different analytes allow the detection and quantification of
analytes, e.g., Zn (II) and Ni (II), even in solutions containing a
mixture of analytes. These data indicate that .alpha.HL biosensors
may be used to detect, identify, and quantify analytes in complex
mixtures, e.g., environmental samples or waste water samples.
[0087] Additional 4H Heteromers Exhibit Different Responses to
Divalent Cations
[0088] Structural variants of .alpha.HL pores resulting from
combinatorial assembly provide yet another means by which to tune
an .alpha.HL channel for detection of analytes. In addition to the
experiments described above, other combinations of
WT.sub.7-n4H.sub.n were tested. The extent of single-channel block
by Zn(II) increased with the number of 4H subunits. Multiple
subconductance states were observed as exemplified by the data for
WT.sub.54H.sub.2, WT.sub.44H.sub.3, and 4H.sub.7 (FIG. 5B). The
specific permutations of the WT.sub.54H.sub.2 and WT.sub.44H.sub.3
pores in these recordings was not determined, however
single-channel recording actually provides a means to "separate"
the various permutations of each combination of heteromers.
According to these data, combinatorial assembly can provide pores
with characteristic responses over a wide range of analyte
concentrations.
[0089] Other embodiments are within the following claims.
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