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Cite This: ACS Omega 2020, 5, 2159−2168

Tissue-Specificity of Dystrophin−Actin Interactions: Isoform-Specific

Thermodynamic Stability and Actin-Binding Function of Tandem
Calponin-Homology Domains
Vaibhav Upadhyay, Swati Bandi, Sudipta Panja, Laura Saba, and Krishna M. G. Mallela*
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz
Medical Campus, 12850 East Montview Boulevard, MS C238-V20, Aurora, Colorado 80045, United States
*
S Supporting Information

ABSTRACT: Genetic mutations in Duchenne muscular dystrophy (DMD) gene affecting

the expression of dystrophin protein lead to a number of muscle disorders collectively called
dystrophinopathies. In addition to muscle dystrophin, mutations in brain-specific dystrophin
isoforms, in particular those that are expressed in the brain cortex and Purkinje neurons,
result in cognitive impairment associated with DMD. These isoforms carry minor variations
in the flanking region of the N-terminal actin-binding domain (ABD1) of dystrophin, which
is composed of two calponin-homology (CH) domains in tandem. Determining the effect of
these sequence variations is critical for understanding the mechanisms that govern varied
symptoms of the disease. We studied the impact of differences in the N-terminal flanking
region on the structure and function of dystrophin tandem CH domain isoforms. The amino
acid changes did not affect the global structure of the protein but drastically affected the
thermodynamic stability, with the muscle isoform more stable than the brain and Purkinje
isoforms. Actin binding investigated with actin from different sources (skeletal muscle,
smooth muscle, cardiac muscle, and platelets) revealed that the muscle isoform binds to filamentous actin (F-actin) with a lower
affinity compared to the brain and Purkinje isoforms, and a similar trend was observed with actin from different sources. In
addition, all isoforms showed a higher affinity to smooth muscle actin in comparison to actin from other sources. In conclusion,
tandem CH domain isoforms might be using minor sequence variations in the N-terminal flanking regions to modulate their
thermodynamic stability and actin-binding function, thus leading to specificity in dystrophin−actin interactions in various
tissues.

■ INTRODUCTION
Duchenne muscular dystrophy (DMD) is an X-linked genetic
disorder caused by the absence or loss of function of
dystrophin protein (UniProtKB P11532).1−4 It is characterized
by progressive muscle weakness and degeneration, finally
leading to death at an early age due to cardiac or respiratory
failure.5 One of the major functions of dystrophin includes
binding to actin cytoskeleton using its N-terminal actin-
binding domain (N-ABD or ABD1).6,7 At least seven different
promoters exist on the DMD gene that lead to tissue-specific
expression of dystrophin isoforms with different lengths.2
Transcription from the first three promoters leads to the
expression of full-length (427 kDa) dystrophin isoforms:
muscle dystrophin (in skeletal and cardiac muscles), brain
dystrophin (in the hippocampus and the cortex in the brain),
and Purkinje dystrophin (in cerebellar Purkinje neurons),
named based on their major site of localization.8,9 Other
promoters lead to the expression of progressively shorter
isoforms from the N-terminus (260, 140, 116, and 71 kDa)
lacking the ABD1 (Figure 1a). Each of the three full-length
isoforms [muscle (DP427M or Dys-M), brain (DP427B or
Dys-B), and Purkinje (DP427P or Dys-P)] is encoded by a
unique first exon and a set of common exons from 2 to 79,
leading to variations in the N-terminal flanking region. In this
study, we examined how these minor sequence variations at
the N-terminus affect the structure and function of dystrophin
ABD1.
Dystrophin ABD1 is composed of two calponin-homology
(CH) domains in tandem. Similar tandem domains are present
in a number of actin-binding proteins including utrophin, α-
actinin, spectrin, and fimbrin.10−12 Previous studies have
shown that the N-terminal (CH1) and C-terminal (CH2)
domains contribute differentially to the stability and function
of dystrophin and utrophin tandem CH domains, with CH1
domains being unstable but with a higher binding affinity for
filamentous actin (F-actin).13−15 CH2 domains do not bind to
actin but provide stability to ABD1.13,15 Utrophin tandem CH
domain contains a long 23-amino-acid flanking region
compared to dystrophin tandem CH domain, and deleting
this region impacts the stability and actin-binding affinity of the
tandem CH domain, making the truncated utrophin tandem
CH domain behave similar to that of dystrophin with a shorter
flanking region.16 A similar role for the N-terminal flanking
regions has been recently proposed for other tandem CH
Received: September 6, 2019
Accepted: December 24, 2019
Published: January 10, 2020

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Figure 1. Dystrophin isoforms resulting from different promoters and sequence comparison of actin-binding tandem calponin-homology (CH)
domains of muscle, Purkinje, and brain isoforms. (A) A schematic representation of the dystrophin gene and isoforms resulting from transcription
initiating from different promoters. The position of promoters is represented on the gene with curved arrows as muscle (M), Purkinje (P) neurons,
brain (B), retina (R), brain, kidney (B3), peripheral nervous system (S), and ubiquitous (G). Transcription initiation from M, P, and B promoters
utilizes a unique first exon and produces protein products with a molecular weight of ∼427 kDa, which are represented as DP427M (Dys-M),
DP427P (Dys-P), and DP427B (Dys-B) respectively. Transcription from R, B3, S, and G promoters initiates with exon 30, 45, 56, and 63 and

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Figure 1. continued

produces protein products with molecular weights of 260 kDa (DP260), 140 kDa (DP140), 116 kDa (DP116), and 71 kDa (DP71), respectively.
(B) Sequence alignment of the tandem CH domains of Dys-M, Dys-P, and Dys-B. The amino acid sequence translated from the unique first exon is
shown in bold letters in black (Dys-M), red (Dys-P), and blue (Dys-B). The rest of the amino acid sequence is identical for the three isoforms. The
figure also shows the three actin-binding surfaces (ABS1, ABS2, and ABS3) through which dystrophin tandem CH domain interacts with actin. (C)
Multiple sequence alignment of six actin isoforms at the amino acid level. Sequence differences are shown in bold letters. Differences in the N-
terminal acidic residues are shown in green. Unique amino acid substitutions in a single isoform are represented by cyan, and substitutions in two
isoforms are represented by red. Amino acids present in the majority of isoforms are represented by black.

Figure 2. Purification and characterization of tandem CH domains of dystrophin isoforms. (A) SDS-PAGE and (B) native-PAGE of purified
dystrophin tandem CH domains. Lanes labeled Dys-M, Dys-P, and Dys-B correspond to the tandem CH domains of the skeletal muscle, Purkinje
neurons, and brain cortex isoforms of dystrophin, respectively. Lane labeled M represents the molecular weight markers (180, 130, 100, 70, 55, 40,
35, 25, 15, and 10 kDa from top to bottom). (C) Far-UV circular dichroism (CD) and (D) intrinsic tryptophan fluorescence spectra of dystrophin
tandem CH domain isoforms: Dys-M (black), Dys-P (red), and Dys-B (blue). Solid and dashed lines in panel D represent the fluorescence spectra
of native and denatured states, respectively.

domains.17−19 The ABD1 of the three dystrophin isoforms
(muscle, Purkinje, and brain) is composed of 246, 242, and
238 amino acid residues, respectively, and the only difference
lies in their N-terminal flanking regions before the first actin-
binding site (ABS1) (Figure 1b). An 11 amino acid stretch in
muscle dystrophin is replaced by 7 amino acids in Purkinje
dystrophin and 3 amino acids in brain dystrophin. We
examined how these three naturally occurring isoforms use
minor sequence variations in their flanking regions to modulate
the thermodynamic stability and actin-binding function, and
whether they lead to tissue specificity of dystrophin−actin
interactions.
Similar to dystrophin, actin also exists in multiple isoforms
with nearly identical amino acid sequences but coded by six
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different genes (Figure 1c).20 Actin isoforms have a conserved
amino acid sequence across species, and the six genes encoding
them are also evolutionary conserved. Like dystrophin, actin
isoforms show tissue-specific expression, with β- and γ-actin
ubiquitously expressed, while other actin isoforms expressed in
specific muscle tissues. This indicates the importance of
different isoforms of actin and led to the hypothesis that actin
isoforms perform nonredundant functions in respective tissues.
Animal knockout studies have shown that specific actin
isoforms perform specialized functions, which cannot be
taken over by concomitant up-regulation of other isoforms.20,21
Biochemical and biophysical studies have shown that despite a
difference of a few amino acids mainly in their N-terminal
flanking regions (Figure 1c), distinct actin isoforms have
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Figure 3. Stability comparison of the tandem CH domains of dystrophin isoforms using urea denaturation and temperature melts. (A) Change in
the CD signal at 222 nm and (B) change in the fluorescence intensity at 320 nm as a function of urea concentration. (C) Change in the CD signal
at 222 nm and (D) change in the fluorescence intensity at 355 nm plotted against temperature. Black, red, and blue colors represent the data
corresponding to the tandem CH domains of Dys-M, Dys-P, and Dys-B, respectively.

differing properties in terms of their polymerization and
depolymerization potential,22,23 Thus, similar to dystrophin,
minor variations in the amino acid sequence and tissue
specificity of actin isoforms may be an important factor in
determining actin−dystrophin interactions. In addition,
specificity might exist in actin−dystrophin interactions, i.e., a
particular dystrophin isoform may be able to distinguish
different actin isoforms in terms of actin-binding affinity, which
has not been examined before.
In this study, we examined how the tandem CH domains of
the three naturally occurring dystrophin isoforms use their
minor variations in the N-terminal flanking regions to
modulate their stability and actin-binding function. In addition
to determining the amino acid sequence of the protein (Figure
1a), transcription from different promoters can also control the
levels of transcript and protein levels, leading to tissue
specificity.8,24 Since dystrophin isoforms are expressed in
different tissues and the actin isoform expression pattern also
varies with the tissue type, we also examined whether different
isoforms of the dystrophin tandem CH domain can differ-
entiate between actin isoforms in terms of their binding
affinity.
the Materials and Methods section. Proteins were obtained
from soluble fraction and purified to homogeneity using nickel-
nitrilotriacetic acid (Ni-NTA) affinity chromatography. Figure
2a shows the sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) of the three purified isoforms.
Native-PAGE analysis (Figure 2b) shows the three isoforms as
homogenous preparations. The tandem CH domain of Dys-M
migrates slightly faster toward the positively charged electrode
compared to the other two proteins because of the presence of
two additional acidic residues in its N-terminal flanking region.
Secondary and tertiary structure characterization of the three
tandem CH domains was performed by circular dichroism
(CD) and fluorescence spectroscopy (Figure 2c,d). CD spectra
of the proteins showed a characteristic signature of α-helical
proteins with major negative bands at 222 and 208 nm.25 The
three spectra were indistinguishable suggesting similar content
of the secondary structural components in the three isoforms.
This indicates that differences in the N-terminal flanking
region did not affect the secondary structure of dystrophin
tandem CH domain isoforms, consistent with earlier studies on
other tandem CH domains.16,17 Fluorescence spectrum of the

■
tandem CH domain of Dys-M in its native state showed
emission maximum red-shifted compared to those of Dys-P
RESULTS and Dys-B. Fluorescence emission after excitation at 295 nm
Purification and Characterization of Proteins. Tandem gives selective information on the environment of tryptophan
CH domains of the three dystrophin isoforms were expressed residues.26 Fluorescence emission spectra of exposed trypto-
in high yield in Escherichia coli, using the protocols described in phan residues are red-shifted compared to buried tryptophan
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Table 1. Thermodynamic Parameters of Tandem CH Domains Obtained from Urea and Temperature Denaturation Melts
(Figure 3)a
urea denaturation (global fit of CD and fluorescence)
tandem CH domain ΔG°unf Cm (M m-value
isoform (kcal/mol) [urea]) (kcal/mol M [urea]) Tm (°C) (CD)
Dys-M 11.86 ± 0.34 5.56 ± 0.01 −2.13 ± 0.06
Dys-P 6.30 ± 0.30 5.26 ± 0.03 −1.20 ± 0.06
Dys-B 6.82 ± 0.26 5.16 ± 0.02 −1.32 ± 0.05
a
At least two data sets from two independent protein preparations were used for the data analysis.
residues. The tandem CH domain of Dys-M contains eight
tryptophan residues as opposed to six in the tandem CH
domains of Dys-P and Dys-B. The two additional tryptophans
in the tandem CH domain of Dys-M are present in its
unstructured N-terminal flanking region, which explains why
its fluorescence spectrum is relatively red-shifted compared to
those of Dys-P and Dys-B. The urea denatured state of all of
the three isoforms showed similar emission spectra with
maxima at 354 nm, suggesting similar exposure of tryptophan
residues upon denaturation.
Tandem CH Domain of Muscle Dystrophin Is More
Stable Than Those of Purkinje and Brain Isoforms. The
stability of the three isoforms was compared using urea and
thermal denaturation. The CD signal at 222 nm and
fluorescence emission at 320 nm were used as probes for the
secondary and tertiary structures of the proteins to record the
urea denaturation profiles (Figure 3a,b). For thermal
denaturation, in addition to CD and fluorescence (Figure
3c,d), optical density (OD) at 350 nm was used as a probe to
monitor protein aggregation (data is not shown). Urea and
thermal denaturation profiles were fit to a two-state unfolding
model (eqs 1, 2, and 4 in the Materials and Methods section)
to obtain the free energy of unfolding at zero denaturant
concentration (ΔG°unf), the slope of linear variation of ΔGunf
with denaturant concentration (m-value), midpoint denaturant
concentration of the melt (Cm), and midpoint temperature of
the thermal melt (Tm) (Tables 1 and S1).
Urea denaturation profiles with both fluorescence and CD
showed that the tandem CH domain of Dys-M is more stable
compared to those of Dys-P and Dys-B. ΔG°unf, Cm, and m-
values obtained for a given protein by individually fitting
fluorescence and CD denaturant melts to a two-state
equilibrium unfolding model (eqs 1 and 2 in the Materials
and Methods section) agreed well with each other (Table S1 in
the Supporting Information). Therefore, the fluorescence and
CD data were analyzed globally to fit to a two-state equilibrium
unfolding model (eqs 1 and 2) by sharing the ΔG°unf, Cm, and
m-values between the data sets. At least two data sets (from
different batches of protein purification) of each protein were
analyzed for each probe, i.e., CD and fluorescence. ΔG°unf for
Dys-M tandem CH domain was 11.86 ± 0.34 kcal/mol,
whereas that for the tandem CH domains of Dys-P and Dys-B
were 6.30 ± 0.30 and 6.82 ± 0.26 kcal/mol, respectively,
indicating that the tandem CH domain of Dys-M is stable by
∼5 kcal/mol compared to those of the other two isoforms.
Similarly, the m-value for Dys-M tandem CH domains was
−2.13 ± 0.06 kcal/(mol M) [urea] compared to −1.20 ± 0.06
and −1.32 ± 0.05 kcal/(mol M) [urea] for the tandem CH
domains of Dys-P and Dys-B, respectively. The m-value is a
measure of the change in the accessible surface area upon
denaturation and thus is also a measure of the compactness of
a protein in the native state in the case of two-state folders.27
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thermal denaturation
ΔHm, app (CD) Tm (°C) ΔHm,app (fluorescence)
(kcal/mol) (fluorescence) (kcal/mol)
60.66 ± 0.03 5.88 ± 0.13 61.55 ± 0.11 9.91 ± 1.16
58.03 ± 0.02 5.26 ± 0.05 59.96 ± 0.12 8.07 ± 0.95
58.54 ± 0.02 5.59 ± 0.06 59.63 ± 0.11 9.62 ± 1.21
However, since the dystrophin tandem CH domain contains
two domains in tandem and folds in a non-two-state manner,28
decreases in m-value might represent an increased population
of partially unfolded state(s) between native and unfolded
states.29
Temperature denaturation profiles recorded using CD at
222 nm reflect secondary structural changes with temperature
(Figure 3c), while those recorded using fluorescence emission
at 355 nm (Figure 3d) give information about changes in the
tertiary structure of the protein with temperature. Thermal
denaturation recorded using OD at 350 nm showed an
increase in light scattering, implying aggregation of these
proteins with temperature (data is not shown). Since the
thermal denaturation was irreversible, the data was analyzed
only qualitatively for Tm comparison. Like the results obtained
above for chemical denaturation, thermal denaturation also
reveals higher stability for the tandem CH domain of Dys-M
compared to those of Dys-P and Dys-B as reflected in the Tm
values measured using both CD and fluorescence as probes
(Table 1).
Isoform Specificity and Actin-Binding Function of
Dystrophin Tandem CH Domain Isoforms. Tandem CH
domains of dystrophin isoforms were tested for binding to
actin obtained from different sources (skeletal muscle, smooth
muscle, cardiac muscle, and platelets), using the co-
sedimentation assays.13,14,16,30 Like dystrophin, actin also exists
as six distinct isoforms with well-defined tissue specificity.20
Whether these differences in the tissue-specific expression of
actin or dystrophin isoforms impact actin−dystrophin
interactions has not been examined before. Isolation of pure
actin isoforms from natural sources is challenging due to small
differences in their amino acid sequences (Figure 1c), which
leads to co-purification of isoforms with one another. Thus, in
this study, actin isolated from four different natural sources was
used. Their composition in terms of actin isoforms is shown in
Table 2.
Table 2. Actin Composition from Different Tissues
actin source composition
skeletal muscle α-skeletal actin
smooth muscle γ-smooth muscle actin (80%), β-cytoplasmic actin (20%)
cardiac α-cardiac actin
non-muscle β-cytoplasmic actin (85%), γ-cytoplasmic actin (15%)
Actin co-sedimentation assays with tandem CH domains of
dystrophin isoforms were performed for skeletal muscle actin
(Figure S1 in the Supporting Information), using the protocols
described earlier.14,31 The data was fit using eq 5 with Bmax
fixed to 1, as observed previously for dystrophin tandem CH
domains.13,30,31 The calculated Kd values were 130 ± 7, 87 ± 4,
and 109 ± 3 μM for Dys-M, Dys-P, and Dys-B respectively
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(Table S2 in the Supporting Information). To determine
whether there exists a statistically significant difference in the
actin-binding affinity of different isoforms, the co-sedimenta-
tion assays were performed with a fixed concentration of F-
actin (7 μM) incubated with 60 μM of dystrophin tandem CH
domains. The bar chart in Figure 4 was obtained after
Figure 4. Actin co-sedimentation assays of tandem CH domains of
dystrophin isoforms with actin from different tissues. The molar ratio
of bound dystrophin to actin calculated from the Coomassie blue-
stained band intensities from SDS-PAGE. Actin from different tissues
is represented as skeletal muscle actin (Sk-M), smooth muscle actin
(Sm-M), cardiac muscle actin (Car-M), and non-muscle actin from
human platelets (Non-M). Data labeled as Dys-M, Dys-P, and Dys-B
correspond to the tandem CH domains of muscle, Purkinje, and brain
isoforms. The error bars represent one standard error of the mean. p-
Values ≤ 0.01 are represented as (**), p ≤ 0.001 are represented as
(***), and p ≤ 0.0001 are represented as (****).
repeating the co-sedimentation assays at least five to seven
times. The binding data reflects that the tandem CH domain of
muscle dystrophin binds weakly to all of the actin isoforms
compared to those of the Purkinje and brain isoforms. Purkinje
and brain isoforms do not show such distinction among
themselves, similar to no changes in their stabilities (Figure 3
and Table 1). Also, smooth muscle actin shows stronger
binding to all dystrophin isoforms compared to the skeletal
muscle, cardiac, and non-muscle actin.
To test the statistical significance of the results obtained
from co-sedimentation assays, data presented in Figure 4 were
analyzed using a two-way analysis of variance (ANOVA) with
Tukey’s test for comparison of the means. The two-way
ANOVA tested the impact of two independent variables,
dystrophin isoform and actin source, on the dependent
variable, which is the amount of dystrophin tandem CH
domain bound to actin (Table 3). This test also provided
information on whether or not the interaction between the two
independent variables affects the dependent variable. The
results obtained from the ANOVA are summarized in Table 4.
Table 3. Parameters Obtained from Two-Way ANOVA
Analysis of Actin-Binding Assays (Figure 4)
degrees of
freedom sum of mean
variable (n − 1) squares square f-value p-value
dystrophin 2 0.21 0.11 37.53 4.58 × 10−11
tandem CH
domain
isoforms
actin source 3 0.06 0.02 7.53 2.55 × 10−4
interaction 6 0.01 0.001 0.48 0.82
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Results show that the effect of type of dystrophin isoform on
actin−dystrophin binding was not dependent on the source of
actin, i.e., the interaction effect from the ANOVA was not
significant (p = 0.82). However, both variables, i.e., the type of
dystrophin isoform and the tissue source of actin, influenced
actin−dystrophin binding independently (p-value = 4.6 ×
10−11 and 2.6 × 10−4, respectively). A comparison of means
analysis showed that the tandem CH domain of Dys-M shows
significantly weaker binding to F-actin compared to those of
Dys-P and Dys-B (p-value <1 × 10−6 for both). Also, binding
of dystrophin isoforms to smooth muscle actin is significantly
stronger from that of skeletal muscle actin (p-value = 0.003),
cardiac actin (p-value = 5.4 × 10−4), and non-muscle actin (p-
value = 0.006) (Table 4).
■ DISCUSSION
Minor Variations in the N-Terminal Flanking Region
Determine the Thermodynamic Stability of Dystrophin
Tandem CH Domain Isoforms. According to the results
presented in this study, tandem CH domains of dystrophin
isoforms have different stabilities, with the tandem CH domain
of Dys-M being more stable by ∼5 kcal/mol than those of Dys-
P and the Dys-B. In terms of the length of the protein, a stretch
of 11 amino acid residues at the N-terminus of muscle isoform
is replaced with 7 and 3 different amino acids in Purkinje and
brain isoforms, respectively (Figure 1b). Changes in the length
of protein by introduction or deletion of amino acid residues at
the N or C-terminus can affect the protein stability.16,32−35
Also, another important determinant for protein stability, in
general, is the N and C-terminal contact.36,37 Dystrophin
tandem CH domain exists in solution in a closed conformation
with its N- and C-termini closer.38 Thus, in the closed
conformation, any residue changes at the termini of dystrophin
tandem CH domain might impact protein stability by directly
influencing the N−C contact, and our experimental results
presented here on the dystrophin tandem CH domain support
this hypothesis. Consistently, deletion of the N-terminal
flanking region in the case of utrophin tandem CH domain,
which exists in an open conformation,39 results in marginal
stability changes.16
Protein Conformation Defines Stability−Function
Relationship in Dystrophin Tandem CH Domain Iso-
forms. The variations in the N-terminal flanking regions of
dystrophin tandem CH domain isoforms are neither part of the
known actin-binding sites (ABSs) on dystrophin (Figure 1b)
nor they are part of any stable secondary structure.40−44 Still,
the results obtained in this study indicate that minor amino
acid sequence variations at the N-terminus can significantly
impact the stability and actin-binding function of tandem CH
domains of dystrophin isoforms. The variations in these
isoforms are part of the N-terminal flanking region of the CH1
domain (Figure 1b). Previous studies have shown that CH1
and CH2 domains of dystrophin contribute differentially
toward the stability and function of its tandem CH domain.13
Actin binding is primarily determined by the unstable CH1
domain, whereas stability is determined primarily by the CH2
domain, which does not bind to actin.13 This seems like a
general strategy adopted by other tandem CH domains, like
utrophin and α-actinin, to fine-tune their stability and function
by modulating inter-CH-domain interactions.13−15,45 The full-
length tandem CH domain of dystrophin is stabilized because
of the tethering of an unstable CH1 domain to a stable CH2
domain and also due to N−C-terminal contact in a closed
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Table 4. Parameters Obtained from Tukey’s Test for Comparison of Means across (a) Dystrophin Tandem CH Domain
Isoforms and (b) Actin Source at 95% Confidence Intervala
(a)
groups compared mean difference standard error of the mean q-value p-value lower control limit upper control limit
Dys-P Dys-M 0.11 0.02 9.84 <1.00 × 10−5 0.07 0.15
Dys-B Dys-M 0.12 0.02 10.73 <1.00 × 10−5 0.08 0.16
Dys-B Dys-P 0.01 0.02 0.83 0.83 −0.03 0.05
(b)
groups compared mean difference standard error of the mean q-value p-value lower control limit upper control limit
−3
Sm-M Sk-M 0.07 0.02 5.23 2.73 × 10 0.02 0.12
Car-M Sk-M −9.31 × 10−3 0.02 0.71 0.96 −0.06 0.04
Car-M Sm-M −0.08 0.02 5.95 5.38 × 10−4 −0.13 −0.03
Non-M Sk-M 4.58 × 10−3 0.02 0.35 0.99 −0.04 0.05
Non-M Sm-M −0.06 0.02 4.87 5.81 × 10−3 −0.11 −0.01
Non-M Car-M 0.01 0.02 1.07 0.87 −0.03 0.06
a
In the table, Dys-M, Dys-P, and Dys-B correspond to the data on the tandem CH domains of muscle, Purkinje, and brain isoforms, respectively.
Sk-M, Sm-M, Car-M, and Non-M correspond to actin from skeletal muscle actin, smooth muscle actin, cardiac muscle actin, and non-muscle actin
from human platelets.

conformation.38 A likely example of the importance of inter-
CH-domain interactions and N−C contact in stabilizing
tandem CH domains comes from utrophin, which, unlike
dystrophin, exists in an open conformation.39 Correspondingly,
the utrophin tandem CH domain binds to F-actin with a
higher affinity than that of dystrophin.13,30 This leads to the
hypothesis that the closed conformation provides stability to
the tandem CH domain at the expense of its actin-binding
function. Since the utrophin tandem CH domain exists in an
open conformation, deleting the N-terminal flanking region in
utrophin caused lesser stability changes (∼1.4 kcal/mol)16
compared to the dystrophin tandem CH domain (∼5 kcal/
mol) (Table 1) that exists in a closed conformation.
The results obtained in this study agree with the above
mentioned earlier studies. The Dys-M tandem CH domain is
more stable than those of Dys-P and Dys-B (Figure 3 and
Table 1) but has a lesser affinity toward binding to F-actin
(Figure 4). Lower stability of Dys-P and Dys-B tandem CH
domains could stem from decreased N−C contact leading to
more open conformation, which increases their actin-binding
affinity. Comparison of m-values in urea denaturation profiles
also showed lower m-values for tandem CH domains of Dys-P
and Dys-B (Table 1), suggesting a more open conformation
for these isoforms as compared to that of Dys-M. A similar
inverse relationship between the thermodynamic stability and
actin-binding function has been observed for tandem CH
domains of utrophin,30,46 α-actinin,47 filamin,48 and spectrin.49
Many mutations that destabilize the tandem CH domains have
increased actin-binding affinity compared to their correspond-
ing wild-type proteins.
An inverse relationship between thermodynamic stability
and actin-binding function of tandem CH domains can be
quantitatively correlated only when we can estimate the
thermodynamic stability of the CH1 domain in the tandem
CH domain, since CH1 controls the actin-binding function of
tandem CH domains.13,14 Denaturant melts of tandem CH
domains, such as those in Figure 3A,B, should in principle be
analyzed using a three-state equation with an intermediate
state where only the CH1 domain is unfolded in between the
native state (with both CH domains folded) and unfolded state
(with both CH domains unfolded).16,28 In such a three-state
analysis, the first transition corresponds to the unfolding of the
CH1 domain and the breaking of the CH1−CH2 interactions,
2165
whereas the second transition corresponds to the unfolding of
the CH2 domain. In the case of the utrophin tandem CH
domain, which exists in an open conformation with minimal
inter-CH-domain interactions,13,30,39,46 the difference in free
energy of the first transition upon N-terminal modifications
was nearly the same as the change in actin-binding free
energy.16 Such analysis cannot be done on the dystrophin
tandem CH domain, which exists in a closed conformation
with significant inter-CH-domain interactions,13,30,38 because
the first transition in the denaturant melt corresponds to the
sum of the stabilities of the CH1 domain and the CH1−CH2
interactions that differ between the three isoforms (see the
Supporting Information).
Tissue-Specificity of Tandem CH Domain−Actin
Interactions. Dystrophin tandem CH domain isoforms bind
to smooth muscle actin with a higher affinity than actin from
other tissues. Actin from smooth muscles is composed mostly
of γ-smooth muscle actin (85%) and β-actin (15%). Non-
muscle actin is composed of β-actin (85%) and γ-cytoactin
(15%). Actin from other tissues is composed of majorly α-
actin. The results presented here indicate that dystrophin
isoforms are able to distinguish γ-smooth muscle actin from
other actin isoforms. Previous studies with the utrophin
tandem CH domain have shown that it can distinguish β-actin
from α-actin and binds more efficiently to β-actin.50 Similar
studies showed a broader difference between muscle and non-
muscle actins binding to profilin,51 thymosin β4,52 ezrin,53 and
plastin.54
In summary, the results obtained in this study suggest that
naturally occurring tandem CH domains of dystrophin
isoforms utilize minor sequence variations in their N-terminal
flanking regions to modulate their stability, actin-binding
function, and tissue specificity of actin−dystrophin inter-
actions. Brain and Purkinje isoforms have lesser stability
compared to the muscle isoform. Stability and actin-binding
affinity follow an inverse correlation with less stable brain and
Purkinje isoforms binding with a higher affinity to actin.
Protein conformation, probably dictated by the N−C contact,
seems to play a major role in defining the stability and actin-
binding efficiency of tandem CH domains. Proteins in closed
conformation have higher stability and a lower affinity for
actin, while proteins in open conformation have lower stability
and a higher affinity for actin. In addition, tandem CH domains
DOI: 10.1021/acsomega.9b02911
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ACS Omega
of dystrophin isoforms were also able to distinguish smooth
muscle actin from other isoforms of actin. This differential
interaction with actin isoforms could be useful in under-
standing diverse symptoms of dystrophinopathies, which affect
tissues other than muscle and could help in designing
therapeutics better suited for such symptoms.
■
MATERIALS AND METHODS
Cloning, Expression, and Purification of Tandem CH
Domains of Dystrophin Isoforms. Genes encoding the
tandem CH domains of dystrophin isoforms (muscle, brain,
and Purkinje) were synthesized by Gene Universal and cloned
into a pET-SUMO expression vector using BamH1 and Xho1
sites. The original pET-SUMO vector leaves an additional
serine residue at the N-terminus after the expressed protein
was cleaved with the Ulp1 protease55 and hence the codon
corresponding to the serine residue was deleted using site-
directed mutagenesis. The cloned genes were transformed into
E. coli BL21 (DE3) cells. The sequence was confirmed after
isolating the plasmid from the transformed cells using a
plasmid miniprep kit from Qiagen. The bacterial constructs
encoding the three proteins were grown in lysogeny broth
(LB) media at 37 °C, and protein expression was induced with
0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at a
cell density corresponding to OD600 nm of 0.5−0.8. All of the
three proteins were expressed in soluble fraction and purified
using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column.
The N-terminal polyhistidine-SUMO tag was cleaved using
Ulp1 protease leaving no additional amino acids at the N-
terminus, and pure proteins were eluted from the Ni-NTA
column in the flow-through. Pure proteins were dialyzed in
phosphate-buffered saline (PBS) (0.1 M sodium phosphate,
0.15 M NaCl, pH 7.0). The homogeneity of the proteins was
checked using SDS-PAGE and native-PAGE.
Fluorescence and Circular Dichroism (CD) Spectros-
copy. The fluorescence spectra of tandem CH domains of
three dystrophin isoforms in PBS were recorded at a
concentration of 1 μM on a Quantamaster, PTI fluorimeter.
The samples were excited at 295 nm, and the emission was
recorded from 305 to 400 nm. CD spectra for the three
proteins were recorded at a concentration of 10 μM on an
Applied Photophysics Chirascan Plus spectrometer.
Urea Denaturation Melts. Urea denaturation melts were
performed at a protein concentration of 1 μM using both CD
and fluorescence measurements. To obtain the Gibbs free
energy of unfolding in the absence of the denaturant (ΔG°unf)
and the linear slope of the variation of Gibbs free energy ΔGunf
with denaturant concentration (m-value), changes in ellipticity
at 222 nm and fluorescence emission at 320 nm (295 nm
excitation) as a function of urea concentration were fitted to a
two-state equilibrium unfolding model,56 using the equation
0 actin-binding assays were fit to the equation
(S N + mN[D]) + (SU + mU [D]) e−(ΔGunf + m[D])/ RT
SD = 0 Bmax x
1 + e−(ΔGunf + m[D])/ RT
(1)
where SD is the measured signal as a function of denaturant
concentration [D], SN and SU are the intrinsic signals
corresponding to the native and unfolded states in the absence
of the denaturant, mN and mU are the slopes of linear
dependence of SN and SU on denaturant concentration, R is the
universal gas constant, and T is the absolute temperature in
kelvin.
2166
Article
Midpoint denaturant concentration, Cm, in the denaturant
melt was estimated by modifying the above eq 1 as
(S N + mN[D]) + (SU + mU [D]) e−(−Cmm + m[D])/ RT
SD =
1 + e−(−Cmm + m[D])/ RT
(2)
Thermal Denaturation Melts. Temperature denaturation
melts for tandem CH domains were recorded at a protein
concentration of 1 μM by monitoring the changes in ellipticity
at 222 nm with an increase in the solution temperature. The
temperature ramp rate was kept at 1 °C/min. To calculate the
midpoint melting temperature (Tm), data were fitted to a two-
state unfolding equation57,58
SD =
ΔHm 1 ΔCp
( − 1 ) − RT ((Tm − T ) + T ln( TT )))
(S N + mN[D]) + (SU + mU [D]) e−( R T Tm m
ΔHm 1 ΔCp
( − 1 ) − RT ((Tm − T ) + T ln( TT )))
1 + e−( R T Tm m
(3)
where ΔHm is the enthalpy change at Tm and ΔCp is the heat
capacity change between the native and unfolded states. ΔCp
was set to zero for the calculation of Tm from thermal melts,
which simplifies the above equation to
ΔHm 1
(S N + mN[D]) + (SU + mU [D]) e−(
1
R ( T − Tm ))
SD = ΔHm 1 1
1 + e −( R ( T − Tm ))
(4)
Since thermal melts of dystrophin tandem CH domains are
irreversible because of aggregation at higher temperatures, only
Tm values were considered for comparison between the three
isoforms.
Actin-Binding Assays. Actin from different sources
(skeletal muscle, smooth muscle, cardiac muscle, and platelets)
was purchased from Cytoskeleton Inc. For obtaining binding
curves, skeletal muscle actin was polymerized in polymer-
ization buffer (50 mM KCl, 2 mM MgCl2, 1 mM adenosine 5′-
triphosphate (ATP), and 10 mm Tris, pH 7.5), and 7 μM was
incubated with dystrophin tandem CH domains ranging in
concentration from 2 to 60 μM. The reaction mixture was
centrifuged at 100 000g for 25 min. Pellets were resuspended
in 30 μL of SDS-PAGE loading buffer, 15 μL of which was
used for gel electrophoresis. Bands of actin and dystrophin
tandem CH domains were stained with Coomassie brilliant
blue R-250. The amount of dystrophin bound to actin was
calculated by measuring the ratio of band intensities of
dystrophin to actin using the Quantity One software on Bio-
Rad Gel Doc XR. The band intensities were corrected for
differential staining of proteins with Coomassie blue, as
described before.14,59 For determining the maximal number
of binding sites (Bmax) and the dissociation constant (Kd),
y=
Kd + x (5)
where y is the molar ratio of the bound tandem CH domain to
actin and x is the unbound tandem CH domain concentration.
The data fitted well with Bmax fixed to 1, as observed previously
for dystrophin tandem CH domains. For statistical analysis, 7
μM polymerized actin from different sources was incubated at
room temperature with 60 μM of tandem CH domains in a
reaction volume of 100 μL. The reaction mixture was
DOI: 10.1021/acsomega.9b02911
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ACS Omega
processed as described above to obtain the amount of
dystrophin bound to actin. The data was presented as the
mean of at least five to seven independent experiments and was
analyzed by two-way ANOVA using the Origin Pro version 8
software to find the statistical significance of the effect of two
variables, actin tissue source and dystrophin isoform, on actin−
dystrophin binding affinity. Tukey’s test was used for
comparing the means of individual groups.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.9b02911.
List of thermodynamic parameters obtained from
individual fitting of urea denaturation melts (Table
S1), actin-binding curves with Kd values (Figure S1 and
Table S2), and a three-state analysis of urea denaturation
melts (Figure S2 and Table S3) (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: krishna.mallela@cuanschutz.edu. Phone: 1-303-724-
3576. Fax: 1-303-724-7266.
ORCID
Krishna M. G. Mallela: 0000-0001-8308-5318
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank Surinder Singh for many helpful discussions and the
Biophysics Core at the University of Colorado Anschutz
Medical Campus for access to some of the instruments used in
this study. This work was supported by a Therapeutic
Innovation Grant from The ALSAM Foundation.
■ ABBREVIATIONS
ABD, actin-binding domain; ABD1, the first or N-terminal
actin-binding domain of dystrophin; ABS, actin-binding
surface; CD, circular dichroism; Cm, midpoint denaturant
concentration; CH, calponin homology; CH1, N-terminal or
the first CH domain; CH2, C-terminal or the second CH
domain; ΔCp, heat capacity change between the native and
unfolded states; ΔG°unf, Gibbs free energy of unfolding at zero
denaturant concentration; ΔHm, enthalpy change at the
midpoint temperature of thermal denaturation; DMD,
Duchenne muscular dystrophy; Dys-M, muscle dystrophin
isoform DP427M; Dys-B, brain dystrophin isoform DP427B;
Dys-P, Purkinje dystrophin isoform DP427P; m-value, slope of
linear variation of Gibbs free energy with denaturant
concentration; Tm, midpoint temperature of thermal denatura-
tion
■
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 SUPPORTING INFORMATION
Tissue-specificity of dystrophin-actin interactions: Isoform-specific thermodynamic
stability and actin binding function of tandem calponin-homology domains
Vaibhav Upadhyay, Swati Bandi, Sudipta Panja, Laura Saba, and Krishna M.G. Mallela*
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical
Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045.

Table S1: Thermodynamic parameters of tandem CH domains obtained from individual fitting
of urea denaturation melts using CD and fluorescence spectroscopy.
Isoform Urea denaturation Urea denaturation
(CD) (Fluorescence)
ΔG0unf Cm m-value ΔG0unf Cm m-value
(kcal/mol) (M [urea]) (kcal/mol/M (kcal/mol) (M [urea]) (kcal/mol/M
[urea]) [urea])
Dys-M 11.90 ± 0.34 5.58 ± 0.01 -2.13 ± 0.06 11.88 ± 0.55 5.54 ± 0.02 -2.14 ± 0.10
Dys-P 6.30 ± 0.24 5.35 ± 0.03 -1.18 ± 0.05 6.38 ± 0.52 5.19 ± 0.02 -1.23 ± 0.10
Dys-B 6.90 ± 0.17 5.21 ± 0.02 -1.32 ± 0.03 6.83 ± 0.47 5.12 ± 0.04 -1.33 ± 0.09

Table S2: Kd values obtained for interaction of dystrophin tandem CH domain isoforms with
skeletal muscle actin.
Isoform Kd (µM)
Dys-M 130 ± 7
Dys-P 87 ± 4
Dys-B 109 ± 3

S1
Figure S1: Actin binding of tandem CH domains of dystrophin isoforms. (A-C) SDS-PAGE of
the pellets from high-speed centrifugation performed at a fixed concentration of F-actin (7 µM)
and with varying concentrations of tandem CH domains of Dys-M, Dys-P, or Dys-B. (D) Actin
binding curves obtained from the band intensities on SDS-PAGE shown in panels A-C, after
correcting for differential staining of the dye to proteins.

S2
3-state analysis of urea denaturation melts of dystrophin tandem CH domain isoforms
Since tandem CH domains unfold through an intermediate state with only CH1 domain
unfolded,1-2 denaturant melts of the three isoforms should in principle be analyzed in terms of a
3-state equilibrium,
𝑁𝑁 ⇌ 𝐼𝐼 ⇌ 𝑈𝑈
where N represents the native state with both CH1 and CH2 folded, I represents the intermediate
state with CH1 unfolded and CH2 folded, and U represents the unfolded state with both CH1 and
CH2 unfolded. In the case of such 3-state equilibrium, the equation that describes the denaturant
melt becomes,3-4

𝑆𝑆𝐷𝐷
0 0 0
∆𝐺𝐺𝑁𝑁𝑁𝑁 +𝑚𝑚𝑁𝑁𝑁𝑁 [𝐷𝐷] ∆𝐺𝐺𝑁𝑁𝑁𝑁 +𝑚𝑚𝑁𝑁𝑁𝑁 [𝐷𝐷] ∆𝐺𝐺𝐼𝐼𝐼𝐼 +𝑚𝑚𝐼𝐼𝐼𝐼 [𝐷𝐷]
(𝑆𝑆𝑁𝑁 + 𝑚𝑚𝑁𝑁 [𝐷𝐷]) + (𝑆𝑆𝐼𝐼 + 𝑚𝑚𝐼𝐼 [𝐷𝐷])𝑒𝑒 − 𝑅𝑅𝑅𝑅 + (𝑆𝑆𝑈𝑈 + 𝑚𝑚𝑈𝑈 [𝐷𝐷])𝑒𝑒 − 𝑅𝑅𝑅𝑅 𝑒𝑒 − 𝑅𝑅𝑅𝑅
= 0 +𝑚𝑚 [𝐷𝐷]
∆𝐺𝐺𝑁𝑁𝑁𝑁 𝑁𝑁𝑁𝑁
0 +𝑚𝑚 [𝐷𝐷]
∆𝐺𝐺𝑁𝑁𝑁𝑁 𝑁𝑁𝑁𝑁 ∆𝐺𝐺 0 +𝑚𝑚𝐼𝐼𝐼𝐼 [𝐷𝐷]
− − − 𝐼𝐼𝐼𝐼
1 + 𝑒𝑒 𝑅𝑅𝑅𝑅 + 𝑒𝑒 𝑅𝑅𝑅𝑅 𝑒𝑒 𝑅𝑅𝑅𝑅

(Eq. S1)
where SD is the measured signal as a function of the denaturant concentration D; SN, SI, and SU are
the intrinsic signals corresponding to the native, intermediate, and unfolded states in the absence
of denaturant; mN, mI, and mU are the slopes of the linear dependence of SN, SI, and SU on
denaturant concentration; ∆G0NI and ∆G0IU correspond to the Gibbs free energies of the first (N ⇌
I) and second (I ⇌ U) transitions in the absence of denaturant, and mNI and mIU are the slopes of
linear dependence of ∆GNI and ∆GIU with denaturant concentration.
Above Eq. S1 indicates that 3-state fit of a denaturant melt involves ten fit parameters,
specifically, SN, mN, SI, mI, SU, mU, ∆G0NI, mNI, ∆G0IU, and mIU. Unless the protein shows a clear
double sigmoidal denaturant melt with two well-separated transitions corresponding to the
unfolding of CH1 and CH2 domains or without knowing the spectral properties of the intermediate
state and their denaturant dependence, it is not possible to fit the denaturant melts shown in Figs.
3A and 3B to a unique set of parameters with much less standard errors of estimates on fit
parameters. Therefore, to minimize the number of fit parameters, we need to make reasonable
assumptions. Since the intermediate consists of unfolded CH1 domain and folded CH2 domain,
CD signal at 222 nm, which measures the α-helical secondary structure, of the intermediate and
its denaturant dependence is assumed to be half that of the native and unfolded states (SI =
(SN+SU)/2; mI = (mN+mU)/2). This assumption may not be valid in the case of denaturant melts
measured by fluorescence, because the change in fluorescence signal may not mirror the equivalent
change in the protein’s structure, since fluorescence signals depend primarily on the solvent
environment and the nearby chemical groups that quench the fluorescence.5-6 In addition, muscle
isoform contains two additional tryptophans compared to brain and Purkinje isoforms (Fig. 1B);
hence fluorescence signal of the intermediate may not be half of the corresponding native and
unfolded states. Therefore, we analyzed only the denaturant melts obtained by CD to the above 3-
state equation (Eq. S1). Since the difference between the three isoforms is only in the N-terminal
flanking region of the CH1 domain, we also assumed that the stability of CH2 domain with

S3
unfolded CH1 is the same across the three isoforms. Figure S2 shows the fit and Table S3 lists the
fit parameters.

1.0
Normalized CD @ 222 nm

0.8

0.6

0.4

0.2
Dys-M
Dys-P
Dys-B
0.0

0 2 4 6 8
[Urea] (M)
Figure S2: Fitting of CD denaturant melts to 3-state equilibrium equation S1. Solid lines represent
the fit curves with constraints listed in Table S3.

Table S3: Thermodynamic stability parameters obtained from the global analysis of CD
denaturant melts.
Fit constraints Tandem ∆G0NI = mNI = mCH1 + ∆G0IU = mIU = mCH2
CH domain ∆G0CH1 + mCH1-CH2 ∆G0CH2 (kcal/mol/M
isoform ∆G0CH1-CH2 (kcal/mol/M (kcal/mol) [urea])

(kcal/mol) [urea])

SI = (SN+SU)/2 Dys-M 11.55 ± 1.10 -2.01 ± 0.23 2.79 ± 1.44 -0.51 ± 0.34
mI = (mN+mU)/2
Dys-P 5.73 ± 1.19 -1.04 ± 0.23 2.79 ± 1.44 -0.51 ± 0.34
Dys-B 6.51 ± 0.93 -1.22 ± 0.18 2.79 ± 1.44 -0.51 ± 0.34

S4
Although the data fit well to the 3-state model with a reasonable constraint about intermediate
CD signal and its denaturant dependence, the absence of a well-separated second transition
resulted in higher standard errors of estimates on fit values of ∆G0IU and mIU.
In the above fit (Fig. S2 and Table S3), ∆G0NI corresponds to the unfolding of the CH1
domain, whereas ∆G0IU corresponds to the unfolding of the CH2 domain. For those tandem CH
domains such as that of dystrophin which exists in a closed conformation with significant
interactions between folded CH1 and CH2 domains, when CH1 unfolds, it also results in the
breaking of the inter-CH-domain interactions. Therefore, the ∆G0NI becomes the sum of intrinsic
stability of CH1 domain in the presence of CH2 (∆G0CH1) and the strength of CH1 interactions
with CH2 (∆G0CH1-CH2), as stated in Table S3. Since CH1 domain determines the actin binding
affinity of tandem CH domains and inter-CH-domain interactions determine the thermodynamic
stability of tandem CH domains rather than their actin-binding affinity,7-9 we need to know the
intrinsic stability of CH1 in the full-length dystrophin tandem CH domain (∆G0CH1) that is
distinct from that of inter-CH-domain interactions (∆G0CH1-CH2) to establish a quantitative
correlation between the thermodynamic stability and actin-binding affinity of dystrophin tandem
CH domain isoforms, which is not possible with the current data analysis. We have shown
previously that such quantitative relationship exists in the case of utrophin tandem CH domain,
which exists in an open conformation with minimal inter-CH-domain interactions (∆G0NI ≈
∆G0CH1; ∆G0CH1-CH2 ≈ 0); ∆∆G0CH1 is of the same magnitude as that of ∆∆G0 of actin binding.3

S5
REFERENCES
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tandem calponin-homology domain appears to be thermodynamically and kinetically more stable
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utrophin tandem calponin-homology domain is primarily determined by its N‑terminal domain.
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S6