Gelsolin
requires activation to carry out its severing and capping
activities on F-actin. Here, we present the structure of the isolated
C-terminal half of gelsolin (G4-G6) at 2.0 Å resolution in the
presence
of Ca2+ ions. This structure completes a triptych of the
states of
activation of G4-G6 that illuminates its role in the function of
gelsolin. Activated G4-G6 displays an open conformation, with the
actin-binding site on G4 fully exposed and all three type-2 Ca2+
sites occupied. Neither actin nor the type-l Ca2+, which
normally is
sandwiched between actin and G4, is required to achieve this
conformation.
Tropomyosin
has been shown to cause annealing of gelsolin-capped actin
filaments. Here we show that tropomyosin is highly efficient in
transforming even the smallest gelsolin-actin complexes into long actin
filaments. At low concentrations of tropomyosin, the effect of
tropomyosin depends on the length of the actin oligomer, and the
cooperative nature of the process is a direct indication that
tropomyosin induces a conformational change in the gelsolin-actin
complexes, altering the structure at the actin (+) end such that
capping by gelsolin is abolished. At increased concentrations of
tropomyosin, heterodimers, trimers, and tetramers are converted to
actin filaments. In addition, evidence is presented demonstrating that
gelsolin, once removed from the (+) end of the actin, can reassociate
with the newly formed tropomyosin-decorated actin filaments.
Interestingly, the binding of gelsolin to the tropomyosin-actin
filament complexes saturates at 2 gelsolin molecules per 14 actin and 2
tropomyosins, i.e. two gelsolins per tropomyosin-regulatory unit along
the filament. These observations support the view that both tropomyosin
and gelsolin are likely to have important functions in addition to
those proposed earlier.
In
actin from many species H73 is methylated, but the function of this
rare post-translational modification is unknown. Although not within
bonding distance, it is located close to the gamma-phosphate of the
actin-bound ATP. In most crystal structures of actin, the
delta1-nitrogen of the methylated H73 forms a hydrogen bond with the
carbonyl of G158. This hydrogen bond spans the gap separating
subdomains 2 and 4, thereby contributing to the forces that close the
interdomain cleft around the ATP polyphosphate tail. A second hydrogen
bond stabilizing interdomain closure exists between R183 and Y69. In
the closed-to-open transition in beta-actin, both of these hydrogen
bonds are broken as the phosphate tail is exposed to solvent.Here we
describe the isolation and characterization of a mutant beta-actin
(H73A) expressed in the yeast Saccharomyces cerevisiae. The properties
of the mutant are compared to those of wild-type beta-actin, also
expressed in yeast. Yeast does not have the methyl transferase
necessary to methylate recombinant beta-actin. Thus, the polymerization
properties of yeast-expressed wild-type beta-actin can be compared with
normally methylated beta-actin isolated from calf thymus. Since earlier
studies of the actin ATPase almost invariably employed rabbit skeletal
alpha-actin, this isoform was included in these comparative studies on
the polymerization, ATP hydrolysis, and phosphate release of actin.It
was found that H73A-actin exchanged ATP at an increased rate, and was
less stable than yeast-expressed wild-type actin, indicating that the
mutation affects the spatial relationship between the two domains of
actin which embrace the nucleotide. At physiological concentrations of
Mg2+, the kinetics of ATP hydrolysis of the mutant actin
were
unaffected, but polymer formation was delayed. The comparison of
methylated and unmethylated beta-actin revealed that in the absence of
a methyl group on H73, ATP hydrolysis and phosphate release occurred
prior to, and seemingly independently of, filament formation. The
comparison of beta and alpha-actin revealed differences in the timing
and relative rates of ATP hydrolysis and P(i)-release.
Profilin
and b/g-actin from calf thymus were
covalently linked
using the zero-length cross-linker
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with
N-hydroxysuccinimide, yielding a single product with an apparent
molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic
investigations showed that the cross-linked residues were glutamic acid
82 of profilin and lysine 113 of actin. The cross-linked complex was
shown to bind with high affinity to deoxyribonuclease I and
poly(L-proline). It also bound and exchanged ATP with kinetics close to
that of unmodified profilin-actin and inhibited the intrinsic ATPase
activity of actin. This inhibition occurred even in conditions where
actin normally forms filaments. By these criteria the cross-linked
profilin-actin complex retains the characteristics of unmodified
profilin-actin. However, the cross-linked complex did not form
filaments nor copolymerized with unmodified actin, but did interfere
with elongation of actin filaments in a concentration-dependent manner.
These results support a polymerization mechanism where the
profilin-actin heterodimer binds to the (+)-end of actin filaments,
followed by dissociation of profilin, and ATP hydrolysis and P(i)
release from the actin subunit as it assumes its stable conformation in
the helical filament.Nucl. Acids Res. 2001; 29
(11): 2377-81.
The Enhancement of PCR Amplification by Low
Molecular Weight Amides.
Chakrabarti R, Schutt CE
Amplification of a DNA target by the polymerase chain reaction (PCR)
often requires laborious optimization efforts. In this regard, the use
of certain organic chemicals such as dimethyl sulfoxide, polyethylene
glycol, betaine and formamide as cosolvents has been found to be very
helpful. Unfortunately, very little is known about the precise
structural features that make these additives effective and,
accordingly, the number of such chemicals currently known to enhance
PCR is limited. In order to address these issues, we decided to focus
on formamide and undertook an extensive study of low molecular weight
amides as a class to see how changing the substituents in the amide
structure influences its effect on PCR. We describe here the results of
this study, which involved 11 different amides, and present
observations that provide a cohesive picture of structure-activity
relations in this group of additives. We found several of these amides
to be exceptionally effective and introduce them as novel PCR enhancers.
J. Mol. Biol. 2000; 304 (5):861-871.
Crystal Structure of the Oligomerization Domain of NSP4 from Rotavirus Reveals a Core Metal-Binding Site.
Bowman GD, Nodelman IM, Levy O, Lin SL, Tian P, Zamb TJ, Udem SA, Venkataraghavan B, Schutt CE
Anat. Rec. (New Anat.) 2000; 261:198-216.
The New Architectonics: An Invitation To Structural Biology.
Schutt CE and Lindberg U
The philosophy of art might offer an epistemological basis for talking
about the complexity of biological molecules in
a meaningful way. The analysis of artistic compositions requires the
resolution of intrinsic tensions between disparate
sensory categories-color, line and form-not unlike those encountered in
looking at the surfaces of protein
molecules, where charge, polarity, hydrophobicity, and shape compete
for
our attentions. Complex living systems
exhibit behaviors such as contraction waves moving along muscle fibers,
or
shivers passing through the growth
cones of migrating neurons, that are easy to describe with common
words,
but difficult to explain in terms of the
language of chemistry. The problem follows from a lack of everyday
experience with processes that move towards
equilibrium by switching between crystalline order and chain-like
disorder, a commonplace occurrence in the
submicroscopic world of proteins. Since most of what is understood
about
protein function comes from studies of
isolated macromolecules in solution, a serious gap exists between what
we
know and what we would like to know
about organized biological systems. Closing this gap can be achieved by
recognizing that protein molecules reside
in gradients of Gibbs free energy, where local forces and movements can
be
large compared with Brownian motion.
Architectonics, a term borrowed from the philosophical literature,
symbolizes the eventual union of the structure of
theories-how our minds construct the world-with the theory of
structures-or how stability is maintained in the
chaotic world of microsystems.
Proteins 2000; 41:374-384.
A Comparative Structural Analysis of the ADF/Cofilin Family.
Bowman GD, Nodelman IM, Chua NH, Lindberg U, Schutt CE
Actin-depolymerizing factor (ADF)
and cofilin define a family of actin-binding proteins
essential for the rapid turnover of filamentous actin
in vivo. Here we present the 2.0 Å crystal structure
of Arabidopsis thaliana ADF1 (AtADF1), the first
plant crystal structure from the ADF/cofilin (AC)
family. Superposition of the four AC isoform structures
permits an accurate sequence alignment that
differs from previously reported data for the location
of vertebrate-specific inserts and reveals a
contiguous, vertebrate-specific surface opposite the
putative actin-binding surface. Extending the structure-based sequence
alignment to include 30 additional
isoforms indicates three major groups: vertebrates,
plants, and other eukaryotes. Within these
groups, several structurally conserved residues that
are not conserved throughout the entire AC family
have been identified. Residues that are highly conserved
among all isoforms tend to cluster around
the tryptophan at position 90 and a structurally
conserved kink in a-helix 3.
Analysis of surface
character shows the presence of a hydrophobic
patch and a highly conserved acidic cluster, both of
which include several residues previously implicated
in actin binding.
FEBS Lett. 2000; 476 (3):155-159.
Covalent Binding of ATPgS to the Nucleotide-Binding Site in S14C-actin.
Schuler H, Schutt CE, Lindberg U, Karlsson R
We have recently reported on the
characterization of beta-actin carrying the mutation S14C in one of the
phosphate-binding loops. The present paper describes the attachment of
the adenosine
5'-[g-thio]-triphosphate (ATPgS) to actin containing this
mutation. Treatment of S14C-actin with ATPgS blocked further
nucleotide exchange and raised the thermal stability of the protein,
suggesting the formation of a covalent bond between the
sulfhydryl on the terminal phosphate of ATPgammaS and cysteine-14 of
the mutant actin. The affinity of the derivatized G-actin for DNase I
as compared to wild-type ATP-actin was lowered to a similar extent as
that of ADP.AlF(4)-actin. The derivatized actin polymerized
slower than ATP-actin but faster than ADP-actin. Under these conditions
the bound ATPgammaS was hydrolyzed, suggesting the formation of
a state corresponding to the transient ADP.P(i)-state. ATPgS-actin
interacted normally with profilin, whereas the interaction
with actin depolymerizing factor (ADF) was disturbed, as judged on the
effects of these proteins on actin polymerization.
Eur. J. Biochem. 2000; 267 (13): 4054-4062.
Mutational Analysis of Arginine 177 in the Nucleotide Binding Site of b-Actin.
Schuler H, Nyakern M, Schutt CE, Lindberg U, Karlsson R
Actin
ADP-ribosylated at arginine 177
is unable to hydrolyze ATP, and the R177 side chain is in a position
similar to that of the catalytically essential lysine 71 in heat shock
cognate protein Hsc70,
another member of the actin-fold family of proteins. Therefore, actin
residue R177 has been implicated in the mechanism of ATP
hydrolysis. This paper compares wild-type beta-actin with a mutant in
which R177 has been replaced by aspartic acid. The mutant beta-actin
was expressed in Saccharomyces cerevisiae and purified by DNase
I-affinity chromatography. The mutant protein exhibited a reduced
thermal stability and an increased nucleotide exchange rate, suggesting
a weakened interdomain connection. The ATPase activity of
G-actin and the ATPase activity expressed during polymerization were
unaffected by the R177D replacement, showing that
this residue is not involved in catalysis. In the presence of
polymerizing salts, ATP hydrolysis by both wild-type Mg-beta-actin and
the mutant protein preceded filament formation. With the mutant actin,
the initial rate of ATP hydrolysis was as high as with
wild-type actin, but polymer formation was slower, reached lower
steady-state levels, and the polymers formed exhibited much lower
viscosity. The critical concentration of polymerization (Acc) of the
mutant actin was increased 10-fold as compared to wild-type actin.
Filaments formed from the R177D mutant beta-actin bound phalloidin.
Eur. J. Biochem. 2000; 267 (2): 476-486.
Thermal Unfolding of G-actin Monitored with the DNase I-inhibition Assay; Stabilities of Actin Isoforms.
Schuler H, Lindberg U, Schutt CE, Karlsson R
Actin
is one of the proteins that rely
on chaperonins for proper folding. This paper shows that the thermal
unfolding of G-actin, as studied by CD and ultraviolet difference
spectrometry, coincides with a
loss in DNase I-inhibiting activity of the protein. Thus, the DNase I
inhibition assay should be useful for systematic studies of actin
unfolding and refolding. Using this assay, we have investigated how the
thermal stability of actin is affected by either Ca2 + or Mg2 +
at the high affinity divalent cation binding site, by the concentration
of excess nucleotide, and by the nucleotide in different
states of phosphorylation (ATP, ADP.Pi, ADP. Vi, ADP.AlF4, ADP.BeFx,
and ADP). Actin isoforms from different species were also
compared, and the effect of profilin on the thermal stability of actin
was studied. We conclude that the thermal unfolding of G-actin is a
three-state process, in which an equilibrium exists between native
actin with bound nucleotide and an intermediate free of
nucleotide. Actins in the Mg-form were less stable than the Ca-forms,
and the stability of the different isoforms decreased in the following
order: rabbit skeletal muscle alpha-actin = bovine cytoplasmic
gamma-actin > yeast actin > cytoplasmic beta-actin. The
activation
energies for the thermal unfolding reactions were in the range 200-290
kJ.mol- 1, depending on the bound ligands. Generally, the
stability of the actin depended on the degree with which the nucleotide
contributed to the connectivity between the two domains of the
protein.
J. Mol. Biol. 1999; 294 (5): 1271-1285.
X-ray Structure Determination of Human Profilin II: A Comparative Structural Analysis of Human Profilins.
Nodelman IM, Bowman GD, Lindberg U, Schutt CE
Human profilins
are multifunctional, single-domain proteins which directly link the
actin
microfilament system to a variety of signalling pathways via two
spatially
distinct binding sites. Profilin binds to monomeric actin in a 1:1
complex,
catalyzes the exchange of the actin-bound nucleotide and regulates
actin
filament barbed end assembly. Like SH3 domains, profilin has a
surface-exposed
aromatic patch which binds to proline-rich peptides. Various
multidomain
proteins including members of the Ena/VASP and formin families localize
profilin:actin complexes through profilin:poly-L-proline interactions
to
particular cytoskeletal locations (e.g. focal adhesions, cleavage
furrows).
Humans express a basic (I) and an acidic (II) isoform of profilin which
exhibit different affinities for peptides and proteins rich in proline
residues. Here, we report the crystallization and X-ray structure
determination
of human profilin II to 2.2 Å. This structure reveals an aromatic
extension
of the previously defined poly-L-proline binding site for profilin I.
In
contrast to serine 29 of profilin I, tyrosine 29 in profilin II is
capable
of forming an additional stacking interaction and a hydrogen bond with
poly-L-proline which may account for the increased affinity of the
second
isoform for proline-rich peptides. Differential isoform specificity for
proline-rich proteins may be attributed to the differences in charged
and
hydrophobic residues in and proximal to the poly-L-proline binding
site.
The actin-binding face remains nearly identical with the exception of
five
amino acid differences. These observations are important for the
understanding
of the functional and structural differences between these two classes
of profilin isoforms.
Les Cahiers de Science & Vie 1999; 53: 78-87.
Nouveau Regard Sur le Muscle.
Kreatsoulas C, Lindberg U, Schutt CE
C'est par le mouvement coordonné de milliers de
molécules que les muscles produisent des forces. La
simulation, sur ordinateur, de ce "ballet" moléculaire
amène à formuler une nouvelle théorie de leur
contraction.
Eur. J. Biochem. 1999; 265 (1): 210-220.
Mutational Analysis of Ser14 and Asp157 in the Nucleotide-Binding Site of b-actin.
Schuler H, Korenbaum E, Schutt CE, Lindberg U, Karlsson R
This paper compares wild-type and two mutant beta-actins, one in which
Ser14 was replaced by a cysteine, and a second in which both Ser14 and
Asp157 were exchanged (Ser14-->Cys and Ser14-->Cys,
Asp157-->Ala, respectively). Both of these residues are part of
invariant sequences in the loops, which bind the ATP phosphates,
in the interdomain cleft of actin. The increased nucleotide exchange
rate, and the decreased thermal stability and affinity for DNase
I seen with the mutant actins indicated that the mutations disturbed
the interdomain coupling. Despite this, the two mutant actins
retained their ATPase activity. In fact, the mutated actins expressed a
significant ATPase activity even in the presence of Ca2+
ions, conditions under which actin normally has a very low ATPase
activity. In the presence of Mg2+ ions, the ATPase activity of
actin was decreased slightly by the mutations. The mutant actins
polymerized as the wild-type protein in the presence of Mg2+ ions, but
slower than the wild-type in a K+/Ca2+ milieu. Profilin affected the
lag phases and elongation rates during polymerization of the
mutant and wild-type actins to the same extent, whereas at
steady-state, the concentration of unpolymerized mutant actin appeared
to
be elevated. Decoration of mutant actin filaments with myosin
subfragment 1 appeared to be normal, as did their movement in the
low-load motility assay system. Our results show that Ser14 and Asp157
are key residues for interdomain communication, and that
hydroxyl and carboxyl groups in positions 14 and 157, respectively, are
not necessary for ATP hydrolysis in actin.
Acta Physiol. Scand. 1998;163 (4): 307-323.
Muscle Contraction as a Markov Process. I: Energetics of the Process.
Schutt CE, Lindberg U
Force generation during muscle contraction
can be understood in terms of cyclical length changes in segments of
actin
thin filaments moving through the three-dimensional lattice of myosin
thick
filaments. Recent anomalies discovered in connection with analysis of
myosin
step sizes in in vitro motility assays and with skinned fibres can be
rationalized
by assuming that ATP hydrolysis on actin accompanies these length
changes.
The paradoxically rapid regeneration of tension in quick release
experiments,
as well as classical energetic relationships, such as Hill's
force-velocity
curve, the Fenn effect, and the unexplained enthalpy of shortening, can
be given mutually self-consistent explanations with this model. When
muscle
is viewed as a Markov process, the vectorial process of chemomechanical
transduction can be understood in terms of lattice dependent
transitions,
wherein the phosphate release steps of the myosin and actin ATPases
depend
only on occurrence of allosteric changes in neighbouring molecules.
Tropomyosin
has a central role in coordinating the steady progression of these
cooperative
transitions along actin filaments and in gearing up the system in
response
to higher imposed loads.
J. Mol. Biol. 1998; 280 (3): 463-474.
Domain Motions in Actin.
Page R, Lindberg U, Schutt CE
Previous
crystallographic
investigations have shown that actin can undergo large conformational
changes,
even when complexed to the same actin binding protein. We have
conducted
a formal analysis of domain motions in actin, using the four available
crystal structures, to classify the mechanism as either hinge or shear
and to quantify the magnitude of these changes. We demonstrate that
actin
consists of two rigid cores, a semi-rigid domain and three
conformationally
variable extended loops. Confirming predictions about the nature of the
domain rotation in actin based on its structural similarity to
hexokinase,
we show, using an algorithm previously used only to identify protein
hinges,
that residues at the interface between the two rigid cores undergo a
shear
between alternative conformations of actin. Rotations of less than 7
degrees
in the torsion angles of five residues in the polypeptides that connect
the rigid cores enable one actin conformation to be transformed into
another.
Because these torsion angle changes are small, the interface between
the
domains is maintained. In addition, we show that actin secondary
structure
elements, including those outside the rigid cores, are conformationally
invariant among the four crystal structures, even when actin is
complexed
to different actin binding proteins. Finally, we demonstrate that the
current
F-actin models are inconsistent with the principles of actin
conformational
change identified here.
Nat. Struct. Biol. 1997; 4 (3): 169-72.
Schutt CE, Kreatsoulas C, Page R, Lindberg U
"Plugging into the 'architectonic socket' might initiate a flow of
momentum along actin filaments and through the extended structures that
contribute to cortical tension."
Structure 1997; 5 (1): 19-32.
Thorn KS, Christensen HE, Shigeta R, Huddler D, Shalaby L,
Lindberg
U, Chua NH, Schutt CE
The
determination of the Arabidopsis thaliana
profilin isoform I structure, using multiwavelength anomalous
diffraction
(MAD) to obtain structure-factor phases, is reported here. The
structure
of Arabidopsis profilin is similar to that of previously determined
profilin
structures. Conserved amino acid residues in profilins from plants,
mammals,
and lower eukaryotes are critically important in dictating the geometry
of the PLP-binding site and the overall polypeptide fold. The main
feature
distinguishing plant profilins from other profilins is a solvent-filled
pocket located in the most variable region of the fold. CONCLUSIONS:
Comparison
of the structures of SH3 domains with those of profilins from three
distinct
sources suggests that the mode of PLP binding may be similar. A
comparison
of three profilin structures from different families reveals only
partial
conservation of the actin-binding surface. The proximity of the
semi-conserved
actin-binding site and the binding pocket characteristic of plant
profilins
suggests that epitopes encompassing both features are responsible for
the
cross-reactivity of antibodies between human and plant profilins
thought
to be responsible for type I allergies.
J. Mol. Biol. 1996; 263 (4): 607-623.
The Structure of an Open State of b-actin at 2.65 Å Resolution.
Chik JK, Lindberg U, Schutt CE
The
structure
of an "open state" of crystalline profilin:beta-actin has been solved
to
2.65 Å by X-ray crystallography. The open-state crystals, in 1.8
M
potassium
phosphate, have an expanded unit cell dimension in the c direction of
185.7 Å compared with 171.9 Å in the previously solved
ammonium
sulphate-stabilized
"tight-state" structure. The unit cell change between the open and the
tight states is accompanied by large subdomain movements in actin.
Furthermore,
the nucleotide in the open state is significantly more exposed to
solvent,
and local conformational changes in the hydrophobic pocket surrounding
cysteine 374 occur during the transition to the tight state.
Significant
changes were observed at the N terminus and in the DNase-I binding
loop.
Neither the structure of profilin nor its contact with beta-actin are
affected
by the changes in the unit cell. Applying osmotic pressure to
profilin:beta-actin
crystals brings about a collapse of the unit cell comparable with that
seen in the open to tight-state transition, enabling an estimate of the
work required to cause this transformation of beta-actin in the
crystals.
The slight difference in energy between the open and collapsed states
explains
the extreme sensitivity of profilin:beta-actin crystals to changes in
chemical
and thermal environment.
A Discourse on Modeling F-Actin.
Schutt CE, Rozycki MD, Myslik JC, Lindberg U
A major challenge for structural biologists is to obtain atomic
resolution models for higher order structures. In favorable
instances, the intact particle can be crystallized and all atomic
positions and bonds within the macromolecular complex can be determined
to the resolution limit of the data. Icosahedrally symmetric
virus particles exemplify the application of crystallography to
structures of polymeric complexes at sufficient resolution to visualize
the amino-acid side chain interactions stabilizing intermolecular
surfaces (Harrison et al.,
1978). Tobacco mosaic virus is an outstanding example of the
successful phasing of a high-resolution fiber X-ray diffraction pattern
by multiple isomorphous replacement (Namba & Stubbs, 1986).
But, what can be when crystals of the intact structures cannot be
obtained or when the existing fiber patterns simply do not extend to
high enough resolution?
Structural Studies on the Ribbon-to-Helix Transition in Profilin:Actin Crystals.
Schutt CE, Rozycki MD, Chik JK, Lindberg U
Nature 1993; 365: 810-816.
The Structure of Crystalline Profilin-b-actin.
Schutt CE, Myslik JC, Rozycki MD, Goonesekere NC, Lindberg U
The three-dimensional
structure of bovine profilin-b-actin has been solved
to 2.55 Å
resolution
by X-ray crystallography. There are several significant local changes
in
the structure of b-actin
compared with a-actin as
well as an
overall
5 degrees rotation between its two major domains. Actin molecules in
the
crystal are organized into ribbons through intermolecular contacts like
those found in oligomeric protein assemblies. Profilin forms two
extensive
contacts with the actin ribbon, one of which appears to correspond to
the
solution contact in vitro.
FEBS Lett. 1993; 325 (1-2): 59-62.
A New Perspective on Muscle Contraction.
Recent experimental findings suggest that the myosin cross-bridge theory may no longer be adequate to account for certain basic facts concerning muscle contraction. A newly-proposed mechanism based on length changes in actin filaments might be the basis for a simpler explanation for how the free energy of ATP hydrolysis can be transduced into work by muscle fibers.
Proc. Nat. Acad. Sci. U.S.A. 1992; 89 (1): 319-323.
Schutt CE, Lindberg U
We
propose that the key structural
feature in the conversion of chemical free energy into mechanical work
by actomyosin is a myosin-induced change in the length of the actin
filament.
As reported earlier, there is evidence that helical actin filaments can
untwist into ribbons having an increased intersubunit repeat. Regular
patterns
of actomyosin interactions arise when ribbons are aligned with myosin
thick
filaments, because the repeat distance of the myosin lattice (429
Å) is
an integral multiple of the subunit repeat in the ribbon (35.7
Å).
This
commensurability property of the actomyosin lattice leads to a simple
mechanism
for controlling the sequence of events in chemical-mechanical
transduction.
A role for tropomyosin in transmitting the forces developed by
actomyosin
is proposed. In this paper, we describe how these transduction
principles
provide the basis for a theory of muscle contraction.
 Schutt
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