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INTRODUCTION |
The two heavy chains
(MyHC)1 of a myosin molecule
dimerize through their C-terminal 1098 amino acids, forming an
-helical coiled-coil structure known as myosin rod. In all
vertebrates, sarcomeric MyHCs are represented by multiple isoforms that
are differentially expressed at various developmental stages and in different muscle fiber types (1, 2). Frequently, several MyHC isoforms
are expressed in a single muscle cell (3-5), giving rise to the
possibility of these heavy chains associating as either homo- or
heterodimeric myosin molecules.
At least five fast MyHC isoforms are alternatively expressed in
developing chicken fast skeletal muscle (6-10), and it has been shown
that at stages when either embryonic and neonatal isoforms or neonatal
and adult isoforms are being simultaneously expressed in the same
muscle fiber, chicken myosin molecules are found as dimers of identical
MyHC isoforms (11, 12). In vitro experiments, using
proteolytic rod fragments, showed that under conditions where isoform
exchange was induced by denaturation of the coiled-coil, chicken myosin
rods folded back into homodimers, indicating that the amino acid
differences in the rod domain of these isoforms were sufficient to code
for their dimerization selectivity (12).
The amino acid sequence of the rod follows the seven-amino acid repeat
(abcdefg)n characteristic of
-helical
coiled-coil proteins, in which positions a and d are mostly occupied by
hydrophobic residues and charged residues are often found at positions
e and g (13, 14). Experiments with model peptides have shown that hydrophobicity and packing effects of residues at a and d positions affect the stability and oligomerization state of the coiled-coil (15-19), while electrostatic interactions appear to control
dimerization specificity and chain orientation (parallel
versus antiparallel) (20). A large body of experimental
evidence implicates charged amino acids at g and e positions in the
regulation of dimerization specificity of Fos and Jun and other leucine
zippers (21-28). Besides interhelical ionic interactions between g and
e' residues, general unfavorable electrostatic effects in the dimer
interface have been suggested to modulate preferential
heterodimerization of coiled-coil proteins (24, 29, 30). However, it is
not clear if these interhelical electrostatic interactions control the
association of peptide sequences that preferentially form homodimeric
coiled-coils (31).
Despite the extensive work on dimerization specificity of leucine
zippers and model coiled-coils, little is known about the molecular
mechanism that controls myosin dimerization specificity. The ability of
two isoforms to form heterodimeric myosin molecules has been correlated
with sequence conservation; therefore, it has been suggested that
sequence homology controls dimerization specificity (11). However, the
amino acid sequences of chicken myosins are highly conserved (>95%
identical) (32), and yet they do not form stable heterodimeric
coiled-coils.
To investigate the sequence elements that determine the preference of
chicken myosin isoforms to associate as homodimers, we have analyzed
the dimerization of the chicken LMM domain, a rod fragment encompassing
the C-terminal 650 amino acids of the MyHC. Recombinant LMM constructs
were expressed with or without a polyhistidine tag at the N terminus,
and their ability to dimerize was analyzed by metal chelation
chromatography. Homo- and heterodimers between histidine-tagged and
untagged LMM constructs could be fractionated based on the different
affinity exhibited by LMM dimers containing zero, one or two
histidine-tagged strands for binding to the Ni-NTA resin. Our results
showed that denatured neonatal and adult LMMs could refold into
heterodimeric coiled-coils although they preferentially associated as
homodimeric molecules. The contribution of the amino acid differences
present in the LMM region of these isoforms to dimerization selectivity
was studied by construction of chimeric proteins combining neonatal and
adult sequences. Our results indicated that, although sequence homology indeed stabilized the dimer, not all amino acid differences had an
equivalent effect on heterodimer stability. Sequence identity at the N
terminus of the LMM had a greater effect on dimer stability than
sequence identity at the C terminus. In addition, our experiments indicated that dimerization selectivity of the neonatal and adult isoforms was affected differently by sequence identity at a given region of the LMM.
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MATERIALS AND METHODS |
Cloning of Recombinant LMMs--
Recombinant LMMs were expressed
using the T7 RNA polymerase-based (33) ampicillin-resistant pET
expression vectors (Novagen, Madison, WI).
Neonatal and Adult LMM Clones--
cDNA clones encoding the
rod sequences of chicken neonatal and adult MyHC isoforms have been
previously characterized (9). The cDNA clone E1 (9) was digested
with SspI, DraI, and EcoRI restriction
enzymes, and the SspI-EcoRI 2-kilobase pair
fragment coding for the neonatal LMM sequence subcloned into
pBluescript vector (digested with SmaI and EcoRI)
resulting in the LNbs clone. The 2-kilobase pair
BamHI-EcoRI fragment of LNbs clone was ligated into the pET-5c expression vector (Novagen). The resulting clone, termed pET-5cNeoLMM, encoded the 648 C-terminal amino acids of the
chicken neonatal LMM and at its N terminus contained 15 amino acids
(MASMTGGNNMGRIPH) translated from vector sequences. An analogous strategy was used to subclone the adult LMM from clone AA-4 (9) resulting in the clone pET-5cAdultLMM. These clones were used to
express neonatal (Neo) and adult (Ad) LMM constructs.
Histidine-tagged LMM Clones--
The 2-kilobase pair
BamHI-EcoRI insert of either pET-5cNeoLMM or
pET-5cAdultLMM, coding for the LMM sequence of the neonatal or adult
MyHC isoforms, respectively, was subcloned into the pET-15b vector
(Novagen). Polymerase chain reaction was used to delete one nucleotide
at the 5' end of the insert in order to correct the reading frame of
the insert. The sequence of the forward primer containing the deletion
was: 5'-GCTCGAGGATCCCCATTCACGCCAG-3'. The reverse primer within the LMM
sequence was: 5'- AGGTCCTCCACCTCATTCTG -3'. The polymerase chain
reaction-amplified region was subcloned as a
BamHI-StuI fragment into the parental vector,
replacing the corresponding wild type BamHI-StuI
fragment. The resulting clones were termed
pET-15b-NeoLMM-StuI and pET-15b-AdultLMM-StuI,
and were used to express histidine-tagged neonatal (HNeo) and
histidine-tagged adult (HAd) LMMs, respectively. DNA sequencing
confirmed the reading frame change and the absence of additional
mutations. The N-terminal 25 amino acids of the fusion proteins
(MGSSHHHHHHSSGLVPRGSHMLGDPH) expressed from these constructs were
encoded by vector sequences and included a hexahistidine tag flanked by
a thrombin proteolytic site.
Chimeric LMM Clones--
Chimeric LMM proteins A386, A278, and
A183 were constructed by substituting 386, 278, or 183 amino acids at
the N terminus of the neonatal LMM with the corresponding adult
sequences. Plasmids pET5c-NeoLMM and pET5c-AdultLMM were digested with
BamHI and either NcoI, SstI, or
StuI restriction enzymes. BamH-NcoI
(1158 base pairs), BamHI-SstI (834 base pairs),
and BamHI-StuI (549 base pairs) fragments of the
adult LMM sequence were ligated to the double-digested pET-5c-NeoLMM
plasmid to substitute the corresponding neonatal
BamH-NcoI, BamHI-SstI, or
BamHI-StuI fragments. An analogous strategy was
used to produce construct N183. N386 was constructed by replacing the
StuI-EcoRI fragment at the 3' end of the neonatal sequence in pET5c-NeoLMM with the corresponding adult sequence. The
correct clones were identified by their restriction digestion patterns.
Expression and Purification of Recombinant
LMMs--
BL21(DE3)pLysS E. coli cells (33) transformed
with recombinant pET plasmids were cultured according to
manufacturer's directions (Novagen). The bacterial cultures were
harvested 3 h after induction, centrifuged at 5000 × g for 10 min in a GSA rotor, and the pellet stored at
80 °C. The frozen bacterial pellets were thawed on ice,
resuspended in low salt buffer (20 mM KCl, 2 mM
KH2PO4, 1 mM EGTA, pH 6.8), 10 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride in
1/40 of the volume of the bacterial culture, and sonicated on ice to
decrease viscosity. The LMM proteins were purified according to the
method previously described to purify myosin from chicken muscle (34),
and further purified by gel permeation chromatography under denaturing
conditions. The LMM preparations were denatured by dialysis against 4 M (or 6 M) GdmCl, 40 mM
NaPPi, pH 7.5, 10 mM DTT, centrifuged
(14,000 × g, 15 min., 4 °C), and the supernatant loaded onto a Bio-Gel A-15m (Bio-Rad) column (2 × 50 cm)
equilibrated with the same buffer. The column was eluted with the
equilibration buffer at 10 ml/h at room temperature. The fractions with
the highest degree of homogeneity were pooled, renatured, and stored at
20 °C in 80 mM NaPPi, pH 7.5, 10 mM DTT, 50% glycerol.
LMM constructs containing a polyhistidine tag at the N terminus were
purified by Ni-NTA chromatography under denaturing conditions. The
procedure was similar to the one described below for Ni-NTA column
except that 4 M GdmCl was added to all the buffers and that
after washing with 10 mM imidazole buffer, the column was eluted with 100 mM imidazole. The eluted fractions were
pooled, concentrated, and further purified by gel permeation
chromatography under denaturing conditions.
Determination of Protein Concentration--
Concentration of
protein preparations was estimated by scanning densitometry (Pharmacia
LKB, Bromma, Sweden) of Coomassie Blue-stained SDS-PAGE gels.
Increasing amounts of the protein preparations were loaded, and the
slope obtained for each protein preparation was compared with the slope
of a standard LMM preparation of know concentration. The concentration
of the standard had been estimated by lyophilization of the protein and
resuspension to a known concentration.
Circular Dichroism--
Circular dichroism measurements were
carried out on a Jasco-700 spectropolarimeter (Jasco Inc., Easton, MD)
at 25 °C, under nitrogen flush, in a 0.1-cm cell, with protein
concentration of 0.1-0.4 mg/ml. The instrument was calibrated with
ammonium d(+)-10-camphor sulfonate at 290.5 nm. CD spectra
were the average of five scans obtained by collecting data at 10 nm/min, with a time constant of 2 min. The mean residue molar
ellipticity ([
], degrees cm2 dmol
1) was
calculated as described by Zhou et al. (35). Denaturation curves of the recombinant LMMs were obtained at 25 °C by following the decrease in ellipticity at 222 nm in the presence of increasing concentrations of GdmCl as described by Pace et al. (36).
The molarity of the GdmCl solutions was determined by refractive index measurements as described by Nozaki (37). Parameters of the unfolding
reaction were calculated as described by Pace et al. (36).
Polyacrylamide Gel Electrophoresis--
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as
described by Laemmli (38). Native gel electrophoresis was carried out
on 1.5-mm slab gels according to the method of Waller and Lowey
(39).
LMM Denaturation and Renaturation--
Equal amounts of two
proteins in 40 mM NaPPi buffer (1-1.5 mg of
total protein), only one of which contained a polyhistidine tag, were
mixed and denatured by addition of denaturation buffer (8 M
GdmCl, 40 mM NaPPi, pH 7.5, 10 mM
DTT) to a final 5 M GdmCl concentration. Protein
concentration was adjusted to 1 mg/ml and, after incubation at room
temperature for 2 h, the preparation was renatured by dialysis in
40 mM NaPPi, pH 7.5, 10 mM DTT for 4 h at 4 °C. The renatured samples were dialyzed overnight
against Ni-NTA loading buffer (10 mM imidazole, 0.5 M NaCl, 0.4 M GdmCl, pH 7.8, 5 mM
-mercaptoethanol) at 4 °C. After centrifugation (10,000 × g for 30 min. at 4 °C), the sample was loaded onto a Ni-NTA column.
Ni-NTA Column Chromatography--
The renatured sample
equilibrated in the Ni-NTA loading buffer was loaded onto 0.8 ml of
packed Ni-NTA resin. All exchange experiments were carried out with
Ni-NTA resin (Quiagen, lot nos. R9419 and RA97008). The column was run
in a continuous flow mode at 10 ml/h. The loading buffer was 10 mM imidazole, 0.5 M NaCl, 0.4 M
GdmCl, 5 mM
-mercaptoethanol, pH 7.8. The column was
washed with 12 column volumes of loading buffer and eluted with 18 column volumes of 50 mM imidazole buffer (50 mM
imidazole, 0.5 M NaCl, 0.4 M GdmCl, 5 mM
-mercaptoethanol, pH 7.8), followed by 12 volumes of
1 M imidazole buffer (1 M imidazole, 0.5 M NaCl, 0.4 M GdmCl, 5 mM
-mercaptoethanol, pH 7.8). Protein containing fractions were
detected by SDS-PAGE. Aliquots corresponding to 1/10 of the volume of
each of the three protein peaks eluted from the resin were
trichloroacetic acid-precipitated (40). 20 µl of a
-galactosidase solution (0.32 mg/ml in 0.1 M sodium phosphate buffer, pH
7.5) was added to each of the three fractions (10 mM, 50 mM, and 1 M imidazole) prior to precipitation.
Increasing amounts of each precipitated fraction were loaded onto
SDS-PAGE gel and the
-galactosidase and LMM protein bands analyzed
by densitometry. The slope of the plot of the LMM absorbance
versus loaded volume for each one of the three fractions (10 mM, 50 mM, 1 M imidazole) was
standardized to the slope obtained for the
-galactosidase of the
corresponding fraction. The amount of heterodimers formed in each
exchange experiments was estimated either as the percentage of
recovered protein eluting in the 50 mM imidazole fraction
or, alternatively, as the percentage of loaded protein eluting in the
50 mM imidazole fraction. In those exchange experiments in
which the two methods were used, the values obtained were very similar
(±6%).
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RESULTS |
Expression, Purification, and Structural Characterization of LMM
Constructs--
Recombinant LMM sequences encompassing the C-terminal
648 amino acids of the chicken fast neonatal and adult isoforms were expressed either with or without a hexahistidine tag at the N terminus
(Fig. 1), and purified as described under
"Materials and Methods." Fig.
2A shows samples of these
preparations analyzed by SDS-PAGE. The proteins were estimated to be
>95% homogeneous by scanning densitometry.

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Fig. 1.
Diagram of LMM constructs. LMM
constructs encompassed the 648 C-terminal amino acids of the neonatal
and adult MyHCs. The amino acids at the N terminus of the constructs
derived from vector sequences are indicated, and the hexahistidine tag
underlined. The N terminus of the chimeric LMM constructs,
contained 183, 278, and 386 residues of the adult (constructs A183,
A278, A386) or neonatal (N183, N386) sequence; in each case, the
remaining sequence corresponded to the reciprocal isoform.
Black circles on the diagram of the myosin rod
represent the position of the 18 amino acid differences between
neonatal and adult LMMs at heptad positions a, d, e, and g, which can
participate in interhelical interactions and contribute to dimer
stability. The repeat number corresponds to the 28-amino acid
periodicity present in the myosin rod (14).
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Fig. 2.
Electrophoretic mobility of LMM
constructs. A, SDS-PAGE of purified recombinant LMMs.
Lane 1, protein Mr
standards; lane 2, adult LMM obtained by
chymotryptic digestion of myosin purified from adult chicken pectoralis
muscle; lane 3, recombinant neonatal LMM; lane
4, recombinant adult LMM; lane 5, HNeo
LMM; lane 6, HAd LMM; lane
7, chimera A183N; lane 8, chimera
A278N; lane 9, chimera A386N. B, NDE.
Lane 1, myosin purified from adult chicken
pectoral muscle; lane 2, proteolytic rod
fragment; lane 3, chymotryptic LMM fragment;
lane 4, recombinant neonatal LMM expressed in
bacteria; lane 5, recombinant adult LMM;
lane 6, HNeo LMM; lane 7,
HAd LMM; lane 8, chimera A183N; lane
9, chimera A278N; lane 10, chimera
A386N.
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Non-denaturing electrophoresis and Circular Dichroism analysis of the
recombinant LMM preparations were consistent with these constructs
being stabilized in a dimeric coiled-coil conformation. The mobility of
the recombinant LMMs on NDE gels was intermediate between that of the
rod and the LMM fragments obtained by chymotryptic digestion of native
myosin molecules (Fig. 2B). This was expected for dimers of
these constructs since their Mr was higher than that of LMM fragments obtained by proteolysis (Fig. 2A). The
CD spectra of the recombinant LMM preparations presented a double minima at 222 and 208 nm as well as a maximum close to 190 nm. The high
molar ellipticity value at 222 nm indicated that the protein was
predominantly
-helical (Fig.
3A). The molar ellipticity ratio was greater than 1 ([
]222/[
]208 = 1.044), which has been described to be characteristic of
-helical
coiled-coils (18).

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Fig. 3.
Characterization of recombinant LMMs.
A, CD profile of Neo-LMM construct in 40 mM
NaPPi buffer (pH 7.5) in the absence (thick
line) or presence (thin lines) of
increasing guanidinium chloride concentration. B,
denaturation curve of recombinant neonatal LMM.
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In the dimerization experiments described in this report, strand
exchange between LMM constructs was promoted by denaturant-mediated unfolding of the coiled-coil followed by refolding upon removal of the
denaturing agent. As shown in Fig. 3B, GdmCl concentrations below 1 M had no effect on the folding state of the LMM,
whereas the unfolding was practically complete above 2 M
GdmCl. The transition midpoint was at about 1.5 M GdmCl
([GdmCl]1/2) (Table
I). The conformational stability of the
LMM constructs in the absence of denaturant (Table I) was estimated
assuming a two-state transition, as described by Pace et al.
(36). No significant differences were found in the denaturation of the
Neo, Ad, and HNeo LMM constructs.
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Table I
Summary of denaturation experiments of recombinant LMMs
[GdmHCl]1/2 is the midpoint of the unfolding transition,
i.e. guanidinium concentration at which 50% of the LMM
molecules are unfolded. Since the free energy of unfolding,
Gu, varies linearly with GdmCl concentration in
the limited region where Gu can be measured, the
conformational stability of the protein in the absence of GdmC
( GH2O) was estimated by
extrapolation of the regression line defined by the G
measured at the various [GdmC].
GH2O, and the m values
represent the y axis intercept and the slope of that
regression line, respectively (36).
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Analysis of Dimerization between Histidine-tagged and Untagged LMM
Constructs by Ni-NTA Chromatography--
The hexahistidine tag confers
upon the LMM constructs the ability to selectively bind to the Ni-NTA
resin. In the presence of 10 mM imidazole, untagged LMMs do
not bind to the column and elute in the wash fraction. In contrast,
histidine-tagged LMMs bind to the column at 10 mM imidazole
and elute at 80-100 mM imidazole. This different affinity
is shown in Fig. 4A, where a
mixture of HNeo and Ad LMMs was fractionated by the metal chelation
column into two protein components.

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Fig. 4.
Fractionation of homo- and heterodimeric
coiled-coils by Ni-NTA chromatography. A, Ad and HNeo
LMMs were denatured and renatured independently, mixed after
renaturation and loaded on the column at 10 mM imidazole.
The column was eluted stepwise with 50 mM and 1 M imidazole buffers. The protein concentration of every
fourth fraction was analyzed by densitometry of SDS-PAGE gels. The
elution profile (on the left) shows that two protein peaks
are recovered at 10 mM and 1 M imidazole,
respectively. Analysis of these fractions by Western blot (shown on the
right) with mAb 2E9 (neonatal isoform-specific) and mAb
EB165 (adult isoform-specific) indicates that the protein eluting at 10 mM imidazole corresponds to Ad LMM, whereas the protein
found in the 1 M imidazole fraction corresponds to HNeo
LMM. No protein elutes in the 50 mM imidazole fraction.
B, an equimolar mixture of Ad LMM and HNeo LMM were mixed,
denatured, and refolded prior to loading onto the column. In contrast
to the results shown in A, three protein peaks were
recovered. The new protein fraction eluting at 50 mM
imidazole reacted with both the neonatal and adult specific mAb, as
indicated in the Western blot (on the right of the figure).
C, an equimolar mixture of HAd LMM and Neo LMM was
codenatured and refolded prior to loading the column. As in
B, three protein peaks are recovered. SDS-PAGE analysis of
the eluted fractions (shown on the left of the figure) shows
that these two proteins can be resolved by SDS-PAGE, and indicates that
the two constructs are present in the 50 mM imidazole
fraction in 1:1 ratio, whereas only NeoLMM or HAd LMM are recovered in
the 10 mM and 1 M imidazole fractions,
respectively. AU, arbitrary units of protein concentration;
, relative protein concentration; , imidazole concentration in
the buffer.
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When the histidine-tagged and untagged constructs were denatured
together and then renatured prior to loading, three protein peaks were
recovered from the column, indicating the presence of a new protein
species of intermediate affinity (Fig. 4, B and C). The fact that this new species, eluting in the 50 mM imidazole fraction, was recovered only when strand
exchange between tagged and untagged LMMs had been promoted by
denaturing and refolding of the coiled-coil structure indicated that it
probably corresponded to heterodimers between tagged and untagged
constructs containing a single histidine tag per dimer. The Western
blot and SDS-PAGE analysis of Ni-NTA column fractions shown in Fig. 4
(B and C) indicated that both tagged and untagged
LMM constructs were present in the 50 mM imidazole fraction
and that the two proteins were in equimolar amounts. The presence of
untagged LMMs in a fraction bound to the Ni-NTA column would require
association with a histidine-tagged strand. Furthermore, the
observation that this fraction was eluted from the resin at lower
imidazole concentration than histidine-tagged dimers is consistent with
the formation of hybrid molecules, containing one tagged and one
untagged strand, during renaturation.
Chicken Neonatal and Adult LMMs Associate Preferentially as
Homodimeric Molecules--
In order to analyze dimerization between
neonatal and adult LMMs, equimolar amounts of a tagged and an untagged
LMM construct were mixed, denatured in guanidinium, and renatured by
dialysis. The renatured sample was loaded onto a Ni-NTA column, and the 50 mM imidazole heterodimer fraction was quantitated.
In exchange experiments between HNeo and Neo LMMs, the 50 mM fraction represented about 47.4 ± 2.6% of the
total protein in the three fractions, close to the 50% value expected
for random exchange. In exchange experiments between HNeo and Ad LMMs,
the 50 mM fraction represented about 25.4 ± 2.1% of
the total protein (Fig. 5). Similarly, in
exchange experiments between HAd and Ad LMMs, around 48.3 ± 3.9%
of the protein was recovered in the heterodimer fraction, whereas in
exchange experiments between HAd and Neo LMMs, the heterodimer
represented 28.5 ± 4.1% of the total protein (Fig. 5).
Therefore, the heterodimers formed during exchange experiments between
different isoforms was about half that of the 50 mM
imidazole fraction in exchange experiments between identical LMM
sequences.

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Fig. 5.
Neonatal and adult LMM fragments do form
heterodimers. Pairwise exchange experiments between neonatal or
adult LMMs with either HisNeo or HisAdult LMMs were carried out as
described under "Materials and Methods." The three protein
fractions recovered from the Ni-NTA column at 10 mM, 50 mM, and 1 M imidazole were precipitated and
quantitated. The percentage of recovered protein present in the 50 mM imidazole heterodimer fraction is presented for each
pair. The bars represent the average values of at least
three determinations, and the error bars indicate
standard deviation.
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Exchange Experiments between Chimeric LMMs and Either Neonatal or
Adult His-tagged LMMs--
The LMM sequences of the neonatal and adult
isoforms differ in 35 amino acids, only 18 of which are at heptad
positions a, d, g, and e, which participate in interhelical contacts
and thus can have the largest contribution to dimer stability and
specificity (41, 42). In order to investigate which of these amino acid differences were relevant to the preferential formation of homo- versus heterodimeric LMMs, three chimeric LMM proteins were
constructed that combined neonatal and adult sequences (Fig. 1). The
N-terminal 183, 278, and 386 amino acids of the neonatal LMM were
substituted with the corresponding adult sequences, resulting in the
chimeric LMMs termed A183, A278, and A386, respectively. The three
chimeric LMMs were virtually identical to the neonatal and adult
recombinant LMMs, as judged by NDE mobility (Fig. 2B). Their
reactivity with isoform-specific mAb reflected that their sequence
contained both neonatal and adult epitopes (9).
In exchange experiments between HNeo and the chimeric LMMs, the
proportion of eluted protein present in the 50 mM imidazole fraction was 28.9 (± 2.7), 37.1 (± 2.8), 38.4 (± 2.3)%, for
chimeric LMMs A386, A278, and A183, respectively (Fig.
6). The amino acid sequences of these
three chimeric LMMs contained 22, 15, and 11 differences (10, 6, and 4 at a, d, g, and e positions) with respect to the neonatal sequence.

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Fig. 6.
Amount of heterodimers formed in exchange
experiments with chimeric LMMs. The bars represent the
percentage of the total protein present in the 50 mM
imidazole fraction of exchange experiments between three chimeric LMMs
and either HNeo (A) or HAd (B) LMM constructs.
The values obtained in exchange experiments with the original neonatal
and adult LMM constructs (Fig. 5) are presented for comparison. The
average values of at least three determinations and the standard
deviations are represented. The numbers inside each
bar indicate the number of amino acid differences present in
each pair of constructs (i.e. for the A278
bar in A, the numbers 15 and 6 indicate that the sequences of HNeo and A278 LMMs
differ at 15 positions, 6 of which are found at heptad positions a, d,
g, and e).
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In exchange experiments with HAd LMM, the proportion of heterodimers
formed was 47.3 (± 2.6), 49.0 (± 3.2), and 49.6 (± 4.1)%, for
chimeric constructs A183, A278 and A386, respectively. The amino acid
sequences of these three chimeric LMMs contained 24, 20, and 13 differences (14, 12, and 8 at a, d, g, and e positions) with respect to
the adult sequence. The differences in amount of heterodimers formed in
exchange experiments between HAd and either one of these chimeric LMMs
or the Ad LMM were not statistically significant at the 5% level.
The fact that replacement of the 183 N-terminal amino acids of the
neonatal LMM with the corresponding adult sequence was sufficient to
obtain virtually random exchange between chimera A183 and HAd LMM
suggested a predominant role of these residues in the determination of
the dimerization selectivity. To test if absence of substitutions in
the N-terminal 183 amino acids was sufficient to stabilize LMM dimers,
we built construct N183 (N-terminal 183 residues corresponded to the
neonatal sequence and the remaining 465 C-terminal amino acids to the
adult sequence) and tested its ability to form dimers with the HNeo and
HAd LMMs.
Chimera N183 formed 36.1% heterodimers with HNeo LMM. Although this
amount was considerable higher than that obtained in exchange experiments between Ad and HNeo LMMs, it did not represent random dimerization. In exchange experiments with HAd LMM, N183 formed about
28.0% heterodimers, virtually the same values obtained in exchange
experiments between HAd and Neo LMMs.
Further incrementing the extent of neonatal sequence at the N terminus
(chimera N386) resulted in an additional increase in the amount of
heterodimers formed between the chimera and HNeo (41.5% heterodimers),
without reaching values expected for non-selective dimerization.
 |
DISCUSSION |
The present work addressed the problem of which sequence elements
control dimerization specificity in the coiled-coil of the chicken
myosin rod. We have developed a method using Ni-NTA chromatography that
is capable of fractionating coiled-coil dimers according to the number
of histidine-tagged strands they contain. Subsequently, we have used
this method to study dimerization specificity of chicken neonatal and
adult myosin LMM fragments by monitoring dimerization between
histidine-tagged and untagged LMM constructs.
We showed that, during in vitro refolding of denatured
neonatal and adult LMMs, dimerization between sequences of identical isoforms was favored. The 35 amino acid differences between the neonatal and adult LMMs reduced by 50% the extent of strand exchange between histidine-tagged and untagged LMM constructs. It is not likely
that the preferential homodimer formation between neonatal and adult
LMMs is caused by trapping of folding intermediates at low temperature,
as described for
and
tropomyosin isoforms (43), since
increasing the temperature of refolding did not favor heterodimer
formation (data not shown). In addition, in the case of
and
tropomyosin the relative instability of the 
homodimers favored
the formation of the 
heterodimer (44). In our case, no
differences in the free energy of dissociation of neonatal and adult
LMMs were observed (Table I).
In studies on the dimerization specificity of full-length chicken
neonatal and adult myosin rods, heterodimers represented less than 5%
of the refolded dimers (12). Our observation that neonatal and adult
myosin LMMs could form up to 28% heterodimers could be explained if
amino acid differences outside the LMM region contribute to further
destabilization of the heterodimeric myosin rod. However, we cannot
rule out the possibility that the sensitivity of the two methods used
to quantify heterodimers is different. Nevertheless, the neonatal and
adult LMMs still exhibit preferential dimerization with the homologous sequence.
Exchange experiments conducted between HNeo and chimeric LMMs A386,
A278, or A183 indicated that the lower the number of amino acid
differences between the two polypeptides, the higher the degree of
heterodimerization. These results were consistent with the proposal
that sequence homology plays a key role in myosin dimerization
specificity (11). However, any of these chimeric LMMs was able to
dimerize with HAd to virtually the same extent as the adult LMM (Ad),
despite all the amino acid differences present. The extent of
dimerization between A183 and HAd was especially remarkable, since the
sequences of the two constructs differed in 24 positions out of the 35 amino acid differences present between neonatal and adult LMMs. The
observation that removing 11 amino acid differences in the N terminus
of the molecule remarkably increased the amount of heterodimers formed
(compare HAd × A183 versus HAd × Neo), whereas
removing 13 amino acid differences from the C terminus had little
effect on dimerization (compare HNeo × A386 versus
HNeo × Ad), indicated that not all substitutions had the same
contribution to destabilization of heterodimers and suggested that the
N terminus of the LMM played a crucial role in dimerization
selectivity. However, chimeric construct N183, where the N-terminal 183 amino acids corresponded to the neonatal sequence, did not dimerize
randomly with HNeo. HNeo formed about the same amount of heterodimers
with N183 as with A278. Thus, a stretch of 183 amino acids of identity
at the N terminus of the molecule stabilized the heterodimer to the
same extent as a stretch of 372 amino acids at the C terminus. These
results were also consistent with the idea of sequence identity at the N terminus of he LMM being more relevant to dimer stabilization than
sequence identity at the C terminus.
The amount of heterodimers formed for each pair of proteins is a
function of the difference between the free energy of dissociation of
the heterodimer and the half sum of the free energy of dissociation of
the two homodimeric species. Since the untagged homodimer species in
exchange experiments (HAd × A183) and (HNeo × A183) is the same (Fig. 7), if we assume similar free
energy of dissociation for HNeo and HAd dimers (as would be expected
based on the data in Table I), then the higher amount of heterodimers
formed between HAd and A183 indicated that the HAd-A183 heterodimer is
more stable than HNeo-A183 heterodimer, despite the fact that the
former pair of constructs differ at 24 positions and the latter pair of
constructs only at 11 positions. Similarly, the higher amount of
heterodimers obtained in exchange experiments (HNeo × N183)
versus (HAd × N183) indicated that the HNeo-N183 dimer
is more stable than HAd-N183 dimer, despite the higher number of amino
acid differences. Thus, in either case heterodimers between pairs of
constructs with no amino acid differences at the N-terminal 183 residues are more stable than heterodimers between constructs with no
amino acid differences at the C-terminal 465 residues.

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Fig. 7.
Schematic representation of the coiled-coil
species formed in exchange experiments between histidine-tagged LMMs
and either N183 or A183 LMM constructs. For each pair of
constructs, the percentage of total protein recovered in the
heterodimeric fraction is indicated in parentheses.
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