From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706
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ABSTRACT |
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The structure of the N-terminal transmembrane
domain (residues 1-34) of subunit b of the
Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane
mimetic solvent mixture of chloroform/methanol/H2O (4:4:1).
Residues 4-22 form an During oxidative and photo phosphorylation ATP is synthesized by a
H+-transporting F0F1-ATP synthase.
In mitochondria, chloroplasts, and eubacteria, the enzyme consists of
an H+-transporting transmembrane domain, termed
F0, and a catalytic domain bound at the membrane surface,
termed F1. Each sector is composed of multiple subunits
that vary somewhat between species (1). The simplest enzyme is found in
E. coli where the composition is
The mechanism by which proton translocation through F0 is
coupled to rotary catalysis in F1 remains to be elucidated.
Subunit c is believed to play the central role in proton
transport via protonation-deprotonation of an essential Asp-61
carboxylate from alternate access channels on either side of the
membrane (2). The structure of subunit c, the smallest
subunit in F0, has been solved by heteronuclear NMR (11),
and a ring-like organization of the c oligomer in
F0 was recently elucidated by cross-linking approaches (12,
13). Low resolution electron microscopic and atomic force microscopic
images also suggest a ring-like arrangement of the c
oligomer with subunits a and b lying at the
periphery of the ring (14-16). The placement of subunits a
and b at the outside of the ring is supported by
cross-linking studies (13, 17). To couple H+ transport to
rotary catalysis in F1, H+-flux through
F0 is proposed to drive rotation of the c
oligomeric ring relative to the stationary subunits a and
b at the periphery of the complex (9, 18-20). In such a
model, subunit b is proposed to play the role of a stator,
holding the Subunit b is an amphipathic protein of 156 residues. The
N-terminal 33-residue segment is highly hydrophobic and the presumed membrane anchor (26). Indeed, residues in this N-terminal segment were
readily labeled with
3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine,
a lipid soluble, photoactivatable carbene precursor (27). The remainder
of the protein is quite hydrophilic and thought to extend from the
membrane surface to bind F1. The cytoplasmic domain lacking
residues 1-24, termed bsol, has been expressed
and in purified, soluble form shown to bind to F1 (28). The
cytoplasmic domain has an elongated shape with high It may be possible to solve the structure of the individual domains of
subunit b by NMR methods as a means of circumventing solubility problems inherent in approaches with detergent
solubilization of the whole subunit. The aqueous solution structures of
subunit Peptide Synthesis--
A 34-residue peptide corresponding to the
N-terminal sequence of the E. coli subunit b was
synthesized at the University of Wisconsin Biotechnology Center on an
Applied Biosystems Synergy 432A instrument using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry. The C-terminal
carboxyl was amidinated. The peptide was purified from the crude
synthesis mixture by reverse phase high pressure liquid chromatography
on a Dynamax C-4 column eluted with a linear 55-72% gradient of
acetonitrile in 0.1% aqueous trifluoroacetic acid. The identity of the
purified peptide was confirmed by amino acid analysis and electrospray
mass spectrometry. The final product was judged to be NMR Spectroscopy--
Samples for NMR were 2 mM
peptide in either CDCl3:CD3OH:H2O
(4:4:1 by volume) or
CDCl3:CD3OD:D2O (4:4:1 by volume)
containing 50 mM NaCl and 1 mM dithiothreitol.
The pH of the solution was measured with a glass electrode and adjusted
to pH 6.0 without correction for the deuterium isotope effect.
DQF-COSY,1 TOCSY, and NOESY
experiments were performed on a DMX-600 spectrometer (Brueker) with a
triple axis gradient capability. Double pulse field gradient echo
solvent suppression (36) was used for recording TOCSY and NOESY in
protic solvent. Magic angle gradients (37) were used for coherence
pathway selection and water suppression in DQF-COSY experiments. TOCSY
used the DIPSI-2 spin-lock sequence (38) and a mixing time of 75 ms. A
mixing time of 160 or 80 ms was used in NOESY experiments. Data was
collected using 640 (NOESY, TOCSY) or 800 (DQF-COSY) increments in
t1. Time domain data was also extended in
t1 by linear prediction. Squared sine apodization and zero filling to 2,048 points was applied in each dimension before Fourier transformation. Spectra were processed and
analyzed using Felix 95.0 software (Molecular Simulations Inc., Palo
Alto, CA) on a Silicon Graphics O2 computer. Coupling constants (3JH Mutagenesis, Expression, and Cross-link Analysis--
A two
stage PCR-based mutagenesis procedure was used with plasmid pNOC for
the introduction of single cysteine residues between residues 2 and 20 of subunit b (12). Mutagenic primers corresponding to 21-25
base sequence of the sense strand were designed with a single codon
changed to Cys. These were combined with the antisense primer (bases
2523-2548)2 to generate the
first PCR product or mega-primer. The purified mega-primer was then
combined with a second primer, coding bases 1844-1860 of the sense
strand, in a second PCR reaction. The product was then digested with
SnaB1 and AvaI and subcloned into these restriction sites of plasmid pNOC, and the product was verified by DNA
sequencing. A chromosomal NMR Spectra of Peptide b1-34 in
Chloroform/Methanol/H2O Solvent--
Peptide
b1-34 is quite hydrophobic and it proved to be
very soluble in a mixture of chloroform/methanol/H2O (4:4:1
by volume). The DQF-COSY spectrum of b1-34 in
this solvent mixture demonstrated a good dispersion of HN
and H General Features of the b1-34 Solution
Structure--
The three-dimensional structure of
b1-34 was calculated by simulated annealing
with the DYANA package of NMR software (40). Of the initial collection
of 200 calculated structures, 180 had a similar overall folding
pattern. In the 20 remaining structures there was no apparent
clustering of structures into an alternative fold. The 10 lowest energy
structures were energy minimized using AMBER forcefield as implemented
in DISCOVER (Molecular Simulations Inc.). The atomic coordinates of the
10 final structures have been deposited as entry 1b9U at the Protein
Data Bank, Rutgers, New Jersey. The best fit superposition of the 10 final structures is shown in Fig. 3. Mean
pairwise root mean square deviation between individual structures for
residues 3-33 was 0.4 ± 0.1 Å and 0.9 ± 0.1 Å for the
backbone and all heavy atoms, respectively. There were no distance
constraint violations exceeding 0.2 Å. The distribution of angles in a
Ramachandran plot were 74% in the "most favored" region, with 23%
in the "additionally allowed" and 3% in "generously allowed"
regions. The definitions of the Ramachandran plot regions are those
used in DYANA. Statistics on the structure calculation are presented in
Table I and Fig. 2B.
The b1-34 peptide forms a well defined
Dimerization of the Transmembrane Domain of Subunit b in
Situ--
The cytoplasmic domains of subunit b forms a
functionally important homodimer in F0, which suggests a
possible proximity of transmembrane segments as well. Single Cys
residues were introduced from position 2-20 of a cysteine-free subunit
b (bC21S) to test this possibility by
cross-linking. The bC21S mutant was shown to be functionally
equivalent to wild type (12). Each of the single Cys mutants grew on a
succinate carbon source, indicating a functional oxidative
phosphorylation system. Most mutants grew similarly to wild type
(1.5-2-mm colony diameter after 3 days at 37 °C) with the exception
of bI7C (0.5 mm), bG9C (0.8 mm), and
bI12C (1 mm). Membrane vesicles from each mutant were
analyzed for b-b homodimer formation by disulfide bridge
formation following treatment with
Cu(II)-(1,10-phenanthroline)2 (Fig.
4). Relatively intense high yield
b-b dimers were observed for the bN2C,
bT6C and bQ10C substitutions. Less intense dimer
formation was observed with Cys substitutions at residues 3, 4, 8, 9, 11, 13, 14, 17, and 18. The periodicity of high yield cross-linking
seen in Fig. 4 mimics that expected for one face of an Possible Arrangement of the Transmembrane Domains of Two Subunits b
in F0--
Intersubunit distance restraints derived from
the cross-linking pattern were used to envision the orientation of the
membrane domains of the two b subunits in the native
F0 complex. Two minimized mean
b1-34 structures were docked to each other
using distance constraints from the cross-linking data. The distances
between the Before this work, Girvin et al. (11) used solution NMR
to solve the structure of F0 subunit c dissolved
in chloroform/methanol/H2O (4:4:1) solvent. Importantly,
subunit c folds in a helical hairpin, as it is predicted to
fold in the membrane, with a number of side chains interacting in
accord with the predictions of genetic and biochemical studies of
F0 in situ. The solvent mixture used may be a
good membrane mimetic, because it can organize heterogeneously around
polar and apolar surfaces of amphipathic proteins. We have shown
previously that purified subunit c, prepared in
chloroform/methanol/H2O solvent, can be reconstituted with
subunits a and b to form an F0 with
normal proton translocating function (35). The experiment indicated
that the protein was not denatured by the solvent treatment. We have
attempted similar experiments here in reconstituting peptide b1-34 with purified subunits a and
c and were unable to reconstitute proton-translocating
activity. This negative result is in agreement with earlier
observations that partial removal of small segments of the C-terminal
domain of the subunit b disrupted the assembly of an active
F0 complex (46, 47).
The structure of the transmembrane region of subunit b
derived here by NMR analysis of the protein in
chloroform/methanol/H2O solvent fits well with features of
the protein expected in a native lipid bilayer. The N-terminal
Trp and Tyr residues are preferentially found at the hydrocarbon/polar
interface of the lipid bilayer in transmembrane proteins of known
structure (49, 51-53). The Tyr-24 and Trp-26 residues in the
b1-34 structure would also be predicted to
organize in the interfacial region. In the case of the Trp-26 side
chain, the aromatic rings lie parallel to the predicted surface of the lipid bilayer but in a region of the protein devoid of other protein contacts (Fig. 3). Trp-26 can be replaced with either an acidic or a
basic residue without impairing function (54), indicating that it may
lie in a region with a few protein-protein contacts. The orientation of
the indole ring might be expected to reorient in a phospholipid bilayer
in a more perpendicular manner to optimize hydrogen bonding between the
indole NH and fatty acyl carbonyl groups
(55).3
The two copies of subunit b present in the F0
are now thought to be adjacent to each other and the interactions
between cytoplasmic domains believed critical in F1 binding
function (28-32). The cross-linking experiments presented here
indicate that the transmembrane domains are also close enough in the
membrane to dimerize. A possible model for helical-helical interaction
within the membrane is presented based upon the NMR model and distance
constraints from the cross-linking results (Fig. 5). The model is of
interest in that the transmembrane helices cross at an angle that is
typical for helix-helix packing in polytopic membrane proteins of known
structure (56). If this model represents the packing in F0,
then the role of the hinge region may be to redirect the C-terminal
helix at an angle more perpendicular to the membrane as it emerges into
the cytoplasmic. Proper positioning may be critical in facilitating
dimerization of the cytoplasmic domain. The model is also of interest
in that it suggests a possible aromatic cluster that may be important in fostering helix-helix interactions between subunits. The aromatic ring interactions of Phe-14-Phe-17', where 17' designates the second
subunit, and Phe-17'-Phe-17 is in accord with the stabilizing geometries and distances described by Burley and Petsko (57) with
centroid distances of 5.0 and 5.8 Å, respectively.
-helix, which is likely to span the
hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical
structure is interrupted by a rigid bend in the region of residues
23-26 with
-helical structure resuming at Pro-27 at an
angle offset by 20° from the transmembrane helix. In native subunit
b, the hinge region and C-terminal
-helical segment
would connect the transmembrane helix to the cytoplasmic domain. The
transmembrane domains of the two subunit b in
F0 were shown to be close to each other by cross-linking
experiments in which single Cys were substituted for residues 2-21 of
the native subunit and b-b dimer formation tested after
oxidation with Cu(II)(phenanthroline)2. Cys residues that
formed disulfide cross-links were found with a periodicity indicative
of one face of an
-helix, over the span of residues 2-18, where Cys
at positions 2, 6, and 10 formed dimers in highest yield. A model for
the dimer is presented based upon the NMR structure and distance
constraints from the cross-linking data. The transmembrane
-helices
are positioned at a 23° angle to each other with the side chains of
Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits.
The change in direction of helical packing at the hinge region may be
important in the functional interaction of the cytoplasmic domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
1
1
1
for F1 and
a1b2c12
for F0 (2, 3). The structure of much of the
3
3
segment of F1 from
bovine mitochondria has been solved by x-ray crystallography (4) and
dramatic progress made in understanding the mechanism of ATP synthesis
by a binding change mechanism involving rotary catalysis (5-7). The
subunit has been shown to rotate within a hexameric ring of
3
3 subunits to promote changes in
substrate and product binding affinities at alternating catalytic sites
within the
subunits (8-10).
3
3 subunits of F1 fixed to the stationary F0 subunits as the
c12-
subunits rotate as a unit. The
and
subunits are known to project below the
3
3 complex as a stalk making contact with
the surface of the c-oligomer in F0 (21-23).
Recent electron micrographs now indicate a second stalk at the
periphery of the F1F0 interface, which is presumed to represent a dimer of b2 subunits
extending from F0 to F1 (7, 24, 25). Little is
known about the structure of subunit b.
-helical content
and associates to form a homodimer in solution. Dimerization appears to
be a necessary prerequisite for F1 binding (29, 30). The
soluble domain binds to subunit
of F1 in solution (31), and interactions between subunit b and subunits
and
at the top of the F1 molecule have been demonstrated in
F1F0 (32). To reach the top of the
F1 molecule, subunit b is estimated to extend
110 Å from the surface of the membrane (32).
and portions of subunit
are already in hand (33, 34), and the structure of subunit c in
chloroform/methanol/H2O solvent agrees well with
predictions made from the biochemical and genetic experiments on the
protein in situ (2, 11, 13). Further, subunit c
retains its function after passage through
chloroform/methanol/H2O solvent when reconstituted into
liposomes with subunits a and b (35). Based on
its hydrophobicity, the membrane anchoring segment of subunit
b was expected to be soluble in the
chloroform/methanol/H2O mixture used in studies of subunit
c. We report here on the structure of the membrane anchoring
segment of subunit b in chloroform/methanol/H2O solvent and its possible relevance to the structural organization of
the native subunit b dimer in F0.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
99% pure based
on analytical high pressure liquid chromatography.
, HN) were
calculated from DQF-COSY and NOESY spectra essentially as described
(39). The structure was calculated from 275 NOE-derived inter- and
intra-residue distance constraints and 20 angle constraints derived
from coupling constants. Distance calibration and structure calculation
was performed with the DYANA software package (40) by simulated
annealing. The MOLMOL program (41) was used for visual analysis of the
structure and for preparing molecular graphics figures.
uncBEFH deleted strain, JWP109 (17), was transformed with pNOC and its mutant derivatives. Plasmid
complementation of the
uncBEFH deletion was tested by growth of transformant cells on succinate minimal medium (17). Membrane
vesicles were prepared, and cross-linking analysis was carried out as
described previously (17) using polyvinylidene fluoride membrane for
Western blotting. Rabbit antiserum to subunit b was a
generous gift of D. S. Perlin and A. E. Senior (42).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chemical shifts (Fig.
1). The distribution of chemical shifts
of the HN and H
protons is typical for a
protein containing
-helical and coiled segments (43). Proton
chemical shifts were assigned by standard procedures (44). Main chain
chemical shifts assignments for all residues were complete with the
exception of Asn-2, where HN was not observed. Most of the
side chains protons have been assigned except for a few aliphatic side
chains where complete assignment was not possible due to spectral
overlap and chemical shift degeneracy. A table of chemical shift
assignments has been deposited in the BMRB data bank
(http://www.bmrb.wisc.edu). NOE analysis revealed a pattern of
sequential and medium range NOEs, which is characteristic of an
-helix (Fig.
2A). No long range NOEs were
observed (Table I), indicating that the
peptide does not form tertiary folds. The
H
-HN cross-peaks of the residues 12-21 and
25 were still readily observable by DQF-COSY in completely deuterated
solvent in an experiment where data was collected from 6 to 14 h
after dissolving the peptide. These regions of the peptide must
therefore have a particularly stable hydrogen bonded secondary structure.
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Fig. 1.
The fingerprint region of the DQF-COSY
spectrum of the b1-34 peptide.
Positions of the H -HN cross-peaks of
individual residues are indicated.
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Fig. 2.
Summary of NOEs observed in the NOESY
spectrum of the b1-34 peptide.
A, sequential and medium range NOEs and slowly exchanging
amide protons; B, number of NOE constraints per residue.
Segments of the bars correspond to interresidue (white),
sequential (light shading) and other (dark
shading) NOEs, respectively.
Statistics for 10 final structural models from DYANA calculations
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Fig. 3.
Stereoview of the best-fit superposition of
the 10 lowest energy structures of the
b1-34 peptide. The trace of backbone
bonds is shown in blue and other bonds in yellow.
The position of residues discussed in the text are indicated.
-helix from residues 4-22, which is interrupted by a bend region
from residues 23-26 with resumption of the
-helix from residue 27 to the C terminus. A large stretch of the initial
-helical segment
is unusually stable as judged by the very slow HN exchange
in deuterated solvent. The uninterrupted stretch of hydrophobic side
chains from residues 11 to 20 may stabilize the hydrogen bonded
secondary structure in either a lipid bilayer or a membrane-mimetic
solvent by forming a nonpolar sheath around the protein backbone.
Sequential proline residues at positions 27 and 28, which would break
the (i,i + 4) pattern of hydrogen bonding in an
-helix, correlate with the position of the bend in the structure.
This region is well ordered in solution despite the absence of hydrogen
bonds. Such a rigid conformation probably results from the combination of restricted torsional mobility of the backbone of the two proline residues and spatial constraints imposed by the bulky side chains of
residues 22-26. The
-helical structure resumes at residue 27 with
both Pro-27 and Pro-28 in the
-helical conformation with
angles
of about
35°. As Richardson and Richardson (45) have indicated, a
proline in this conformation is actually favored as the first residue
of an
-helix and is not uncommon in the second position as well. The
-helix from residues 27 to 33 may be part of a more extended
-helical segment of the cytoplasmic domain.
-helix. The
less intense cross-linking seen over consecutive stretches of residues
suggest that the helices may be relatively mobile in the membrane.
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Fig. 4.
Subunit b dimer formation in
Cys-substituted mutants. This figure shows an immunoblot of an
SDS-gel comparing native membranes ( ), prepared in the absence of
dithiothreitol, to the same membranes after treatment with 1.5 mM Cu(II)(phenanthroline)2 for 1 h at
22 °C (+). The immunoblot was probed with antiserum to subunit
b and stained with an ECL system (Amersham Pharmacia
Biotech). The position of the Cys substitution in each mutant is
indicated. WT, wild type.
-carbons of residues 2, 6, and 10 of two different
b subunits were constrained to 4-8 Å, the distance usually
found for natural disulfide bridges in proteins (45). The distances
between
carbons of residues forming lower yield cross-links
were constrained to 4-11 Å. Backbone angles of residues 3 to 33 were
restrained to the values in the mean structure, and side chain angles
left unrestrained. A dimer structure was calculated using molecular
dynamics and energy minimization with DISCOVER (Molecular Simulations
Inc.). The fit of the cross-linking distance constraints to the model are shown in Table II. The modeling
indicates that the membrane anchoring segments of the two b
subunits are positioned at a 23° angle to each other with interacting
helical faces making Van der Waals contact at the side chains of
residues 6 and 10 (Fig. 5).
Cross-linking distance constraints used in modeling the b1-34
dimer
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Fig. 5.
Model for possible interaction of
transmembrane domains of subunit b based on the
cross-linking constraints and NMR model. Side chains of residues
discussed in text are shown in bold color, i.e. Asn-2,
Thr-6, and Gln-10 (magenta), Phe-14 and Phe-17
(green), and Trp-26 (yellow). Van der Waals
contacts are made been the pairs of Thr-6 and Gln-10 side chains. The
rings of Phe-14, Phe-17', and Phe-17 interact in an aromatic cluster.
The aromatic rings of Trp-26 and Trp-26' project toward the front and
back of the structure, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical segment (residues 4-22) is followed by hinge region from
residues 23-26 before resumption of the
-helix. Because an
-helix of 20 amino acids is required to traverse the fatty acyl
hydrocarbon interior of a phospholipid bilayer, we expect the initial
N-terminal helix to be the hydrocarbon spanning region of the protein.
This would place Asn-2 at the periplasmic hydrocarbon/polar interface
and the hinge region (residues 23-26) at the cytoplasmic
hydrocarbon/polar interface of the phospholipid bilayer. The distance
between
-carbons of Asn-2 and Trp-26 is 34 Å in the structure,
which is close to the distance of 32 Å predicted between fatty acyl
carbonyls in opposing leaflets of a palmitoyloleoylphosphatidylcholine
bilayer determined by x-ray and neutron diffraction (48, 49). The
positioning of these residues near the glycerol moiety of the
phospholipid was previously indicated by labeling studies with a
photoactivatable phospholipid analog (50).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Gary Case for synthesizing the peptide used in this study and to Dr. Mark Girvin for introducing O. Dmitriev to NMR. We thank Drs. David Perlin (Public Health Institute of New York) and Alan Senior (University of Rochester) for the gift of antiserum to subunit b.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM23105. The National Magnetic Resonance Facility at Madison, supported by National Institutes of Health Grant R02301, was used in this study. Equipment in the facility was purchased with funds from the University of Wisconsin, the National Science Foundation Biological Instrumentation Program (Grant DMB8415048), the NIH Biomedical Research Technology Program (RR02301), the NIH Shared Instrumentation Program (Grant RR02781) and the United States Department of Agriculture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 608-262-1439;
Fax: 608-262-5253; E-mail: filingam{at}macc.wisc.edu.
2 The nucleotide numbering system is from the sequence given by Walker et al. (26).
3 Such a reorientation does occur in the molecular dynamics calculation illustrated in Fig. 5, where side chain angles were left unrestrained.
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ABBREVIATIONS |
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The abbreviations used are: DQF-COSY, double-quantum filtered correlation spectroscopy; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; PCR, polymerase chain reaction.
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