From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706
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ABSTRACT |
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We have previously shown that the
E31C-substituted The H+-transporting, F1F0-ATP
synthase of Escherichia coli utilizes an H+
electrochemical gradient to drive ATP synthesis during oxidative phosphorylation (1). Similar enzymes are found in mitochondria, chloroplasts, and other bacteria. The enzymes are composed of two
sectors, termed F1 and F0. The F1
sector contains the catalytic sites for ATP synthesis, and when
released from membrane, it shows ATPase activity. The F0
sector traverses the membrane and functions as the H+
transporter. When F1 is bound to F0, the
complex acts as a reversible, H+-transporting ATP synthase
or ATPase. In E. coli, F1 is composed of five
types of subunits in an The relation of structure and mechanism in F0 is less
thoroughly understood. The largely hydrophobic subunit a
folds in the membrane with five transmembrane helices (16, 17), at
least two of which likely interact with subunit c during
proton transport (18-20). Subunit b is anchored in the
membrane via a single transmembrane helix that is connected to a polar,
elongated cytoplasmic domain that is thought to play a key role in
fixing F1 to F0 (21). Subunit c is a
protein of 79-amino acid residues that folds in the membrane in a
hairpin-like structure. The two hydrophobic transmembrane Recent experiments now indicate that the c12
oligomer of F0 is organized in a ring with transmembrane
helix-1 on the inside and transmembrane helix-2 on the outside (2, 4,
28) and with the a and b subunits associating at
the periphery of the ring (2, 20). Such an arrangement is consistent
with low resolution electron and atomic force microscopic images
(29-31). The structural data fit well with rotary models where
H+ transport at the a-c interface is
proposed to drive rotation of the oligomeric c ring as the
Asp61 carboxylate is protonated and deprotonated from
alternate access channels on each side of the membrane (10, 32-34).
The rotation of subunit c is proposed to drive the rotation
of the In this study, the interacting regions of the polar loop of subunits
c and subunit Mutant Construction and Expression--
The plasmids constructed
in this study are derivatives of plasmid pYZ201 (25), which carries the
eight structural genes of unc operon coding
F1F0 (bases 870-10172; Ref.
40).1 The Cys substitutions
in subunit c and subunit Membrane Preparations and Cross-link Analysis--
Cells were
grown and membranes prepared by the methods described (25). To catalyze
disulfide bond formation, aliquots of 200-µl membrane vesicles at 10 mg/ml in TMG buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 10% (v/v) glycerol) were treated
with 20 µl of a mixture of 15 mM CuSO4 and 45 mM 1,10-phenanthroline in 50% ethanol. After a 1-h
incubation at room temperature, the reaction was stopped by addition of
20 µl of 0.5 M Na2EDTA and 20 µl of 0.5 M N-ethylmaleimide
(NEM)2 in dimethyl sulfoxide,
and the sample was incubated for a further 60 min. The sample was then
diluted with 230 µl of 2× SDS sample buffer (125 mM
Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 0.02% bromphenol blue)
and incubated at 30 °C for 1 h. The solubilized membrane
proteins were separated by SDS-polyacrylamide gel electrophoresis using
a 7.5-15% acrylamide gradient and the Tris-Tricine buffer described
by Schägger and von Jagow (42). After electrophoretic transfer to
nitrocellulose paper (43), immunostaining was carried out using the
Enhanced Chemiluminescence System (Amersham Pharmacia Biotech). The
rabbit antiserum to subunit c used was that described by
Girvin et al. (44). Antibodies that nonspecifically
cross-reacted with E. coli membrane proteins were removed by
preabsorption with membranes prepared from a mutant strain with a
deleted unc operon (44). The mouse monoclonal antibody to
subunit Structural Modeling of Subunit-Subunit Interaction--
A model
for subunit c interaction in the c12
oligomer has been derived from the NMR model (27) using distance
constraints derived from the cross-linking data of Jones et
al. (28).3 A subunit
c dimer, taken from the oligomer model, was manually docked
to the N-terminal domain of subunit Properties of Cys-substituted Double Mutants--
All of the
mutants constructed grew on succinate minimal medium, which indicates
formation of a functional ATP synthase (Table I). Two of the Cross-linking of Cysteine in Various
The intensity of the
Other mutants also show
A survey of Cross-linking from the Opposite Side of Subunit c--
In the NMR
structure of subunit c, Gln42 lies on a
flattened face of the loop region opposite Ala40 and
Asp44. We introduced Cys into positions 40 and 44 of the
loop to see if the pattern of cross-linking to residues surrounding
The surface of subunit subunit of F1 can be
cross-linked by disulfide bond formation to the Q42C-substituted
c subunit of F0 in the Escherichia
coli F1F0-ATP synthase complex (Zhang,
Y., and Fillingame, R. H. (1995) J. Biol. Chem.
270, 24609-24614). The interactions of subunits
and c are thought to be central to the coupling of H+ transport
through F0 to ATP synthesis in F1. To further
define the domains of interaction, we have introduced additional Cys into subunit
and subunit c and tested for cross-link
formation following sulfhydryl oxidation. The results show that Cys, in a continuous stretch of residues 26-33 in subunit
, can be
cross-linked to Cys at positions 40, 42, and 44 in the polar loop
region of subunit c. The results are interpreted, and the
subunit interaction is modeled using the NMR and x-ray diffraction
structures of the monomeric subunits together with information on the
packing arrangement of subunit c in a ring of 12 subunits.
In the model, residues 26-33 form a turn of antiparallel
-sheet
which packs between the polar loop regions of adjacent subunit
c at the cytoplasmic surface of the
c12 oligomer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
stoichiometry, and F0 is composed of three types of
subunits in an
a1b2c12
stoichiometry (2-4). The structure of much of the
3
3
portion of F1 has been solved by x-ray diffraction analysis and shows subunit
extending through the center of a hexamer of the larger, alternating
and
subunits (5, 6). During catalysis, the
and
subunits have been
shown to rotate in 120° steps between the three alternating catalytic
sites in the
subunits (7-13). Subunits
and
are thought to
rotate as a unit because they can be cross-linked to each other with
minimal inhibitory effects on ATPase activity (14, 15).
-helices
are joined by a more polar loop region that is exposed to the
F1 binding side of the membrane. Aspartyl 61, lying at the
center of transmembrane helix-2, is thought to be the site of
H+ binding during transport (22). The polar loop was
proposed to play a key role in coupling H+ transport to ATP
synthesis or hydrolysis based upon the uncoupled phenotypes of mutants
with substitutions in the conserved
Arg41-Gln42-Pro43 sequence of the
polar loop (22, 23). The "uncoupled" phenotype of the
cQ42E mutant proved to be suppressed by second site
substitutions in Glu31 of F1 subunit
(24),
and this led to cross-linking studies demonstrating a physical
proximity between the polar loop and subunits
and
of
F1 (14, 25, 26). A recently determined NMR structure of
monomeric subunit c conforms well with folding predictions
made from biochemical and genetic analysis (27).
unit in F1 via a fixed linkage between
subunit c and the
and
subunits (14) although other
explanations have been proposed (22, 25). The elongated cytoplasmic
domain of subunit b is thought to extend from the membrane
surface to the top of F1 as a "second stalk", or
stator, to hold F1 fixed as subunit
rotates at the
center of the molecule (21, 35-37).
are more thoroughly defined by disulfide cross-linking of Cys introduced into the two subunits. The experimental design and interpretation was aided by a structural model for subunit
derived by NMR (38) and x-ray crystallography (39). The Cys
residues of subunit
that form cross-links with subunit c
localize to a span of residues 26-33 which fold as two strands of
antiparallel
-sheet connected by a loop. A structural model for the
subunit-subunit interaction is developed from the structures of the
monomeric subunits and information on the organization of the
c-oligomer, using distance constraints from the
cross-linking data reported here. The model indicates that the segment
of antiparallel
-sheet encompassing residues 26-33 of subunit
packs in a space between neighboring polar loops of the subunit
c oligomer.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were introduced by
oligonucleotide-directed mutagenesis using the strategy described previously (25). The plasmids were expressed in strain OM204 (41), a
strain in which the unc operon is deleted from the chromosome.
(13-A7,
II; Ref. 45) was a gift from Dr. R. Capaldi
(University of Oregon, Eugene, OR).
(residues 1-87) so that the
distances between the
-carbons of cross-linked residues were <12
Å. The range for
-carbon distances in naturally occurring disulfide
bonds in proteins is 4-7.5 Å (46). A somewhat wider distance
constraint range of 4-11 Å was used in the molecular mechanics
calculations done here to allow for possible thermal motions and
distortions of structure on cross-link formation. The shortest of the
two distances between the Cys
-carbon in each of the two subunits
c and the Cys
-carbon in
in the manually docked
structure was used to impose a distance constraint. The positions of
all the atoms of the c subunits were fixed except for
residues 39-45, which were left unrestrained. The backbone angles in
subunit
were restrained to their value in the x-ray structure (39)
using quadratic restraints. Energy minimization was performed with CVFF
(constant valence force field) using the steepest descents and then
conjugated gradient methods as implemented in DISCOVER 3.0 (Molecular
Simulations Inc.) until the maximum derivative was below 0.1 kcal
mol
1
Å
1.4
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-substituted,
cQ42C mutants showed very little membrane ATPase activity
and nondetectable amounts of
subunit on immunoblotting (Table I).
In these two cases,
V25C/cQ42C and
G27C/cQ42C, we conclude that the
F1-F0 interaction is probably stable under
in vivo conditions but that the F1-ATPase and
subunit disassociate from the membrane during membrane
preparation.
Cross-linking of cysteine-substituted subunits to Q42C subunit c
-Substituted/cQ42C
Mutants--
We had previously shown that Cys at position 31 in
subunit
cross-links with Cys in positions 40, 42, or 43 in the
polar loop of subunit c (25). In this study, Cys was
substituted in a series of positions proximal to position 31 in subunit
, and cross-link formation was tested with Cys at position 42 in
subunit c. An experiment comparing cross-linking in
cQ42C/
E29C and cQ42C/
E31C mutant membranes
illustrates a number of typical features (Fig. 1). An
-c cross-linked
product was observed in membranes prepared from both mutants. The
cross-linked product identified as
-c was found at an
identical position on blots stained with either anti-
or
anti-c antibody.5
Comigration of the product on the two types of blots was confirmed using several types of gels of varying acrylamide concentration. The
-c product seen in untreated membranes is presumed to
form by autoxidation as the membranes are isolated from the cell. The extent of cross-link formation was enhanced by
Cu(II)(phenanthroline)2 (CuP) catalyzed oxidation.
Cross-linking was largely reversed by subsequent treatment with 0.5 mM dithiothreitol.
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Fig. 1.
Immunoblots showing cross-link formation
between subunits c and in
membrane vesicles of Cys-substituted mutants. Mutations are
indicated by referring to the positions of the Cys residues of subunit
c and subunit
. Membranes, prepared in the absence of
dithiothreitol, were either treated with CuP (+Cu) or not
treated (0Cu). Following quenching of the reaction with EDTA
and NEM, the treated or untreated membrane vesicles were centrifuged
and resuspended in TMG buffer containing (+DTT) or lacking
(0DTT) 0.5 mM dithiothreitol and incubated for
30 min at 22 C. Following solubilization with SDS and electrophoresis
of 50-µg samples, acrylamide gels were blotted to nitrocellulose
paper and probed with anti-subunit
or anti-subunit c
antibodies. The positions of subunit
, the
-c dimer,
and subunit c monomer, dimer (c2),
trimer (c3), and tetramer
(c4) are indicated. Some of the dimers, trimers,
and tetramers of subunit c result from incomplete
disaggregation of the c12 oligomer in SDS sample
buffer. Most of the staining seen at the position of
c4 is because of an immunoartifact in the
membrane. The positions of molecular mass markers, with the molecular
mass given in kDa, are shown at the side of the blots. Note that
subunit c electrophoreses anomalously relative to these
markers.
-c-immunostained product that is
detected with anti-
antibody does prove to be misleading. Beginning with the anti-
blot shown in Fig. 1, note the much greater intensity of staining of the
-c product in cQ42C/
E29C
versus cQ42C/
E31C membranes despite the
loading of equal amounts of membrane protein in all lanes. Note also
for the
E29C membranes that the changes in intensity of
-c are considerably greater than the changes in intensity
of monomeric
, i.e. the changes are not in the inverse proportions expected in a precursor-product relationship. We interpret this to mean that the
-c heterodimer of the
E29C
mutant protein binds antibody better than the
E29C monomer and also
better than the
-c heterodimer of the
E31C mutant.
This interpretation is qualitatively confirmed by the relative
intensities of the bands on the anti-c blot. In the
untreated membrane samples, the intensity of the anti-c
immunostained
-c product is much greater for
E31C than
for
E29C, i.e. just the reverse of the pattern seen with anti-
antibody. We have concluded that the best way to approximate the extent of
-c formation may be to compare the amounts
of monomeric
remaining after various treatments. Using this
criteria, the
E31C mutant would appear to form at least as much and
possibly more
-c product by either autoxidation or
CuP-catalyzed oxidation.
-c products whose staining
intensities differ considerably with the two antibodies. In the
experiment shown in Fig. 2, membranes
were washed with 1 mM Tris-HCl, pH 8.0, 0.5 mM
Na2EDTA, a procedure which removes F1 and
uncross-linked subunit
and other nonspecific immunoreactive
proteins. The
S28C,
E29C and
G30C
-c products
stain much more intensely with anti-
antibody than with
anti-c antibody. Conversely, the
-c product in
the
G33C mutant stains much less intensely with anti-
body than
with anti-c antibody. Other
-c products stain
with nearly equivalent intensities in the two blots. In summary, the
blots do not provide a good quantitative means of distinguishing the extent of cross-link formation in different mutant membranes.
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Fig. 2.
CuP-catalyzed cross-linking of
Cys42 in subunit c with Cys at
various positions in subunit analyzed by
immunoblots of "stripped" membranes. The cross-linking
reaction was carried out with whole membranes, and the reaction was
quenched with EDTA and NEM. Membranes were then stripped of
F1 by incubation in 1 mM Tris-HCl, pH 8, 0.5 mM EDTA, 10% (v/v) glycerol, and the stripped membranes
were collected by centrifugation and solubilized in SDS sample buffer.
Immunoblots prepared with anti-
and anti-c antibodies are
marked as described for Fig. 1. Numbers at the
top indicate the position of the Cys in subunit
.
-c cross-linking in a series of
double-Cys-substituted pairs is shown in Fig.
3. In the presence of CuP, most or all of
the Cys-substituted subunit
was cross-linked with Cys42-substituted subunit c when Cys was
substituted as positions 26, 28, 29, 30, 32, and 33 in subunit
.
Detectable cross-linking also occurred in the absence of CuP in each of
these mutants. No cross-linking was observed with Cys substituted at
positions 24, 25, 27, 34 and 38 of subunit
. In the case of the
V25C mutant, subunit
was not incorporated into the membrane.
This was also the case for the
G27C mutant (not shown). In other
experiments, the cQ42C/
S10C pair was also shown to not
form a cross-link (Table I). Most of the
-c-cross-linked
product formed in the various membranes shown in Fig. 3 was reduced by
treatment with dithiothreitol. The results from several similar
experiments are summarized in Table I.
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Fig. 3.
CuP-catalyzed cross-linking of
Cys42 in subunit c with Cys at
various positions in subunit .
Immunoblots of whole membranes are shown after probing with anti
antibody. Whole membranes, prepared in the absence of dithiothreitol,
were resuspended in TMG buffer and treated with CuP (+) or not treated
(0), and the reaction was quenched with EDTA and NEM. Cross-linked
products in CuP-treated membranes were reduced with 25 mM
dithiothreitol (+DTT) in SDS gel sample buffer containing 4 M urea. The positions of the Cys substitutions in subunit
are indicated.
Glu31 differed from that with Cys at cQ42C.
As shown in Fig. 4, Cys substituted at
either position 40 or 42 was readily cross-linked to Cys at positions
28, 31, and 32 of subunit
. We conclude that subunit
must be
able to interact with either face of the loop region when it binds to
the oligomer of subunit c.
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Fig. 4.
Cu-phenanthroline-catalyzed cross-linking
from Cys40 and Cys44 in
subunit c to Cys at various positions in subunit
. Immunoblots of whole membranes are shown
after probing with anti-
antibody. Whole membranes, prepared in the
absence of dithiothreitol, were treated with CuP (+) or not treated
(0), and the reaction was quenched with EDTA and NEM. Cross-linked
products in CuP-treated membranes were reduced with dithiothreitol
(+DTT) as described in Fig. 3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
lying proximal to subunit c
has been mapped by cross-linking experiments to a region encompassing residues 26-33, which in the NMR and x-ray diffraction structures of
subunit
(38, 39) reside in a loop of antiparallel
-sheet (Fig.
5A). The NMR and x-ray
diffraction structures agree closely and show subunit
to be a
protein of two distinct domains. The N-terminal domain of 86 residues
folds in a 10-stranded
-sandwich and the C-terminal domain of 45 residues is formed from two
-helices arranged in an antiparallel
coiled coil. Much of the C-terminal domain appears to be nonessential
because it can be deleted without effect on ATP synthase function (47).
Cross-linking and chemical modification experiments indicate that the
surface of the
-sandwich, including residues His38 and
Ser10, neighbor the
subunit and that His38
lies close to the surface of F0 (14, 38, 39, 48). Residue 31 of
must also lie proximal to the surface of F0
because the
E31C-substituted protein can be cross-linked to Cys at
positions 40, 42, and 43 of subunit c (25). The loop
including residues 26-33 protrudes from the "bottom" of subunit
as a well defined lobe (Fig. 5A), and most Cys
replacements in this loop were cross-linked to Q42C subunit
c in the experiments described here. In addition, Cys at
positions 28, 31, and 32 in subunit
were shown to cross-link to Cys
lying on opposing flattened faces of the polar loop of subunit
c in the NMR structure (Ref. 27; Fig. 5B),
i.e. at either position 42 or at positions 40 and 44 in the
loop.
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Fig. 5.
Monomeric structures of subunit
and subunit c showing positions
of cross-linkable residues. A, ribbon depiction of the
-barrel domain of subunit
(residues 1-87) showing positions of
cysteine residues studied here (Protein Data Bank code 1aqt). Cysteine
within the yellow loop (positions 26-33) cross-link with
subunit c. Cysteine bordering the yellow loop at positions
24, 25, and 34 (indicated by magenta) do not cross-link with
subunit c. Cys at positions 10 and 38 (green) can
be cross-linked to subunit
but not to subunit c. B,
structure of loop region of monomeric subunit c showing side
chain orientation of Ala40, Gln42, and
Asp44 on opposite faces of the polar loop (Protein Data
Bank code 1a91, model 1).
The interacting surfaces of subunit and subunit c have
been modeled beginning with a model for the c12
oligomer described elsewhere.3 In the modeling, equivalent
distance constraints were imposed for each cross-link formed because of
difficulties in quantitatively distinguishing the extent of cross-link
formation. In the model, the loop of antiparallel
-sheet that is
centered around
Glu29 packs between the polar loops of
two c subunits (Fig.
6A). The model also depicts
the
Glu31 residue lying close enough to the conserved
and essential cArg41 residue to interact
electrostatically and also close to cGln42 (Fig.
6B). The positioning of these side chains in the model should be interpreted with caution because the model is derived without
use of side chain distance constraints. However, with these
precautions, the general proximity of residues in the model does
provide a reasonable explanation for the uncoupling effects of the
cQ42E mutation (23) in that charge-charge repulsion would be
expected between the cGlu42 and
Glu31 carboxylates. The charge-charge repulsion
explanation is supported by the differences in pH dependence of neutral
versus positively charged
-31 suppressor substitutions in
restoring function to the cQ42E mutant (24). The smaller
uncoupling effects of some substitutions in
Glu31 (49),
versus the cQ42E mutation, are not as easily
rationalized by the model.
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In the model shown in Fig. 6A, it is notable that the space
between loops of subunit c is essentially filled by the
packing with subunit . Because residue 205 of subunit
is also
known to cross-link with residues 42, 43, and 44 of subunit
c (14, 26), it seems likely that the region around
-205
packs between a different set of c subunits than those
interacting with
. The shielding of two separate pairs of subunit
c by the binding of subunits
and
, respectively, may
explain the observations of Watts and Capaldi (50) on the functional
effects of NEM modification of Cys42 in cQ42C
mutant membranes. Function was retained during the initial phase of
modification of approximately 60% of the subunits and then lost during
modification of the last 40%. The inhibitory phase of NEM modification
may correspond to reaction with Cys42 at the
c-
-c or c-
-c
interfaces. In the currently envisioned rotary models, subunits
and
remain fixed to a set of c subunits and turn as the
c-oligomer rotates (14, 33, 34). The binding interaction
must be of sufficient strength to withstand a considerable torque,
estimated at exceeding 40 pN nm under load, or approximately 12 kcal
mol
1 for each 120° step in which ATP is synthesized (8,
34, 51-53). From the structural model deduced here, it seems likely
that the binding energy is derived from the combined interaction of
subunit
with the loop region of one set of subunit c and
of subunit
with an adjacent set of subunit c.
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ACKNOWLEDGEMENT |
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We thank Dr. Rod Capaldi (University of
Oregon) for the gift of anti- monoclonal antibody.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM23105 from the National Institutes of Health.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: Dept. of Biomolecular
Chemistry, 587 Medical Sciences Bldg., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253.
1 The unc DNA numbering system corresponds to that used by Walker et al. (40).
3 O. Y. Dmitriev, M. E. Girvin, P. C. Jones, and R. H. Fillingame, submitted for publication.
4 A coordinate file of the final model is available by E-mail at dmitriev{at}iris.bmolchem.wisc.edu.
5
A number of minor bands were also detected with
the anti- antibody, the intensity and number of which vary from
experiment to experiment. They appear to be artifactual because similar
bands are detected in mutant membranes lacking subunit
. Most of the proteins reacting nonspecifically are removed by the stripping procedure used to remove F1, i.e. washing
membranes with 1 mM Tris-HCl and 0.5 mM EDTA,
but this treatment also removes monomeric
(see Fig. 2).
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ABBREVIATIONS |
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The abbreviations used are: NEM, N-ethylmaleimide; CuP, Cu(II) (phenanthroline)2; Tricine, N-tris(hydroxymethyl)methylglycine.
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