From the Rosenstiel Basic Medical Sciences Research Center,
Brandeis University, Waltham, Massachusetts 02254-9110
The regulatory domain (RD), or neck region of the
myosin head, consists of two classes of light chains that stabilize an
-helical segment of the heavy chain. RD from chicken skeletal muscle
myosin was prepared in Escherichia coli by coexpression of
a 9-kDa heavy chain fragment with the essential light chain.
Recombinant regulatory light chain (RLC), wild type or mutant, was
added separately to reconstitute the complex. The affinity of RD for
divalent cations was determined by measuring the change in fluorescence
of a pair of heavy chain tryptophans upon addition of calcium or
magnesium. The complex bound divalent cations with high affinity,
similar to the association constants determined for native myosin. The intrinsic fluorescence of the tryptophans could be used as a donor to
measure the fluorescence resonance energy transfer distance to a single
labeled cysteine engineered at position 2 on RLC. Dansylated
Cys2 could also serve as a donor by preparing RLC with a
second cysteine at position 79 which was labeled with an acceptor
probe. These fluorescence resonance energy transfer distances (24-30
Å), together with a previous measurement between Cys2 and
Cys155 (Wolff-Long, V. L., Tao, T., and Lowey, S. (1995) J. Biol. Chem. 270, 31111-31118) suggest a
location for the NH2 terminus of RLC that appears to
preclude a direct interaction between the phosphorylatable serine and
specific residues in the COOH-terminal domain.
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INTRODUCTION |
The nucleotide- and actin-binding sites are located in the
globular portion of the myosin head
(S1),1 whereas the light
chains are wrapped around a long (~10 nm)
-helical region that
includes about 70 residues of the COOH terminus of the S1 heavy chain
(1) (see Fig. 1). The light chain-binding region is also known as the "regulatory domain" (RD), because regulation of the actin-activated ATPase activity is achieved either by
calcium binding to the essential light chain (ELC) of molluscan myosins
(2), or by phosphorylation of the regulatory light chain (RLC) of
smooth and non-muscle myosins (3). The RLC of vertebrate striated
muscle can also be phosphorylated at a homologous serine residue, but
here phosphorylation has only a modulatory effect on activity (4). How
these signals are transmitted from RD to the active sites remains a
major unsolved problem.

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Fig. 1.
Ribbon diagram of the RD domain of
myosin. ELC (blue) and RLC (green,
COOH-terminal lobe; and yellow, NH2-terminal
lobe) are wrapped around an -helical HC peptide (gray)
that serves to connect the motor domain to the rod. The NH2
terminus of RLC (red) starts at Phe20 since
there is no electron density for the first 19 residues in the chicken
S1 structure (1). The phosphoserine would be at residue 14 in chicken
skeletal RLC. The position of Cys and Trp residues (red) and
the Mg2+-binding site (blue) in RLC, and the Trp
pair (red) at the sharp bend of HC are indicated. This
structure is of scallop RD (2) which is very similar to chicken RD in
its overall fold. Numbering is for the chicken sequence.
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A stable fragment containing RLC and ELC bound to a 9-kDa heavy chain
peptide was first isolated by proteolytic digestion of scallop myosin
(5). This fragment bound calcium with high affinity in the presence of
magnesium, mimicking the behavior of intact scallop myosin.
Crystallization of RD led to a high resolution structure that revealed
the detailed interactions of the three chains in the complex, and their
contribution to a calcium-specific binding site in ELC (2). The overall
fold of the LCs in scallop RD is similar to that in the neck region of
chicken skeletal S1 (1), although there are differences that are
probably related to the unique regulatory mechanisms in the two myosins
(6).
Our initial goal was to see if a correctly folded RD complex,
consisting of two light chains and a heavy chain peptide, could be
prepared in an expression system. Not only would this approach eliminate microheterogeneity introduced by proteolysis, but the opportunity would arise to study light chain-heavy chain interactions by site-directed mutagenesis. To date it has not been possible to
express sufficient quantities of a striated muscle myosin for biophysical studies. We show here that homogeneous RD, retaining the
high affinity nonspecific calcium/magnesium site found in all muscle
myosins, can be prepared in good yield in a bacterial expression
system. Moreover, by labeling a single engineered cysteine (Cys2) with 1,5-IAEDANS at the NH2 terminus of
RLC, it was possible to determine the fluorescence resonance energy
transfer distance between Cys2 and a cluster of two
tryptophans located at the sharp bend of the heavy chain
-helix,
which served as a donor to IAEDANS. Dansylated Cys2 could
also be used as a donor by engineering a second cysteine at position
79, which was subsequently labeled with an acceptor molecule. These two
FRET distances, together with a previously determined distance between
Cys2 and Cys155 (7), were used to define an
approximate position for the NH2 terminus of RLC. The first
19 residues of RLC are not visible in the crystal structure of chicken
S1, probably due to their flexibility (1). The structure of scallop RD
is without 11 NH2-terminal RLC residues, since these are
removed by proteolysis during the preparation of RD (2). The location
of the missing sequence by fluorescence resonance energy transfer,
despite the low resolution of this technique, provides a structural
framework for considering how the actomyosin interaction can be
enhanced by phosphorylation of a single serine on a light chain far
removed from the active site.
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MATERIALS AND METHODS |
Expression of Heavy Chain Peptide and ELC--
Embryonic chicken
skeletal muscle myosin heavy chain cDNA, provided by J. Robbins
(8), was used as a template to obtain cDNA encoding residues
Leu774 to Leu844 by the polymerase chain
reaction method. Restriction sites EcoRI and
BamHI were created at the 5'- and 3'-ends, respectively, for cloning into the pT7-7 expression vector. This construct produces 4 extra residues (MARI) at the NH2 terminus of the expressed
protein.
The cDNA obtained for chicken skeletal muscle ELC (LC1 isoform) was
missing 45 bases from the 5'-end that encodes the first 15 residues of
LC1 (9). An oligonucleotide containing the additional bases was ligated
to the cDNA, and the completed sequence was cloned into an
expression vector designated pWY. This vector was engineered from two
plasmids, pT7-7 and pGP1-2 (10), for the purpose of having a different
origin of replication and antibiotic resistance (kanamycin) from pT7-7
(ampicillin). The light chain expressed from the pWY/LC1 construct has
an NH2-terminal tag of 4 residues (MARI) from the vector
followed by Pro2 as the first residue of LC1 instead of
Ala. Sequencing of the cDNA revealed a six-base deletion (encoding
Pro-Ala) in the 5'-coding region, which results in six pairs of Pro-Ala
instead of the seven pairs found in tandem in the native protein.
Comparison of LC1 sequences from different species indicates a high
degree of variability in the NH2-terminal regions (11), and
the deletion was considered unimportant for the experiments presented
here.
A construct with both LC1 and wild-type RLC in the same pWY vector was
also designed. The pWY/LC1 clone was digested with PstI/BamHI and the 700-base pair
PstI/BglII fragment from pT7-7/LC2 encoding RLC
(12) was ligated into this clone. Thus, the cDNA for each light
chain was positioned in tandem with the promoter and stop codon. This
construct was used to express both light chains together with the pT7-7
vector containing the 9-kDa heavy chain fragment. Protein expression
was in BL21(DE3), and bacterial cultures were grown at 37 °C for
16-18 h in the presence of ampicillin and/or kanamycin as described in
Wolff-Long et al. (13).
Preparation of RLC--
The expression and purification of
wild-type and mutant chicken skeletal RLC in the vector pT7-7 has been
described (14). Wild-type RLC contains two endogenous Cys residues at
positions 126 and 155. In some RLC mutants, e.g.
Cys126/Cys155 and Cys2, the
endogenous tryptophan at position 138 was changed to phenylalanine in
order to eliminate the contribution of Trp fluorescence. In other
mutants the endogenous Cys residues were selectively removed by
mutagenesis to alanine, as in Cys126, and new Cys residues
were introduced at positions 2 and 79 as in Cys2 and
Cys2/Cys79. Table I gives a description of all
the RLCs, and the location of specific residues is shown in the ribbon
diagram of the regulatory domain in Fig. 1.
Purification of the Regulatory Domain--
When the 9-kDa heavy
chain fragment and ELC are coexpressed, they are found in inclusion
bodies, whereas simultaneous expression of heavy chain fragment
together with RLC and ELC results in a soluble complex in the
cytoplasm. An advantage of the first method is that the soluble
bacterial proteins can be readily separated from the inclusion bodies
by centrifugation, followed by washing of the pellet with a mild
detergent (14). The relatively pure protein in the inclusion bodies can
then be used to prepare complexes with a variety of unlabeled or
labeled mutant RLCs. Although simultaneous expression of the three
chains is in some ways a simpler procedure, a major drawback is the
presence of a much higher concentration of contaminating bacterial
proteins that are difficult to remove. For this reason, and since we
usually wished to incorporate a labeled RLC into the regulatory
complex, the first preparative procedure was adopted.
The cell pellet from a 1-liter culture of Escherichia coli
BL21(DE3) coexpressing 9-kDa HC and LC1 was lysed, and the inclusion bodies isolated as described (14). The washed pellet was resuspended in
8 M guanidine, 10 mM DTT in PBS (10 mM sodium phosphate, pH 7.2, 150 mM NaCl, 3 mM NaN3), to which RLC (20-25 mg
unchromatographed, or purified and labeled) was added with stirring at
room temperature for 1 h. Insoluble cell debris was removed by
centrifugation (100,000 × g for 30 min), and the
proteins were renatured by dialysis against PBS, 1 mM DTT,
1 mM MgCl2 at 4 °C. Denatured insoluble
proteins were removed by centrifugation, and the supernatant was
dialyzed against 10 mM sodium phosphate, pH 7.5, 1 mM MgCl2, 1 mM DTT, 3 mM NaN3 for 4 h, clarified, and applied to
a hydroxylapatite column (1.5 × 10 cm) equilibrated with the same
buffer. The proteins were eluted with a linear gradient (10-250
mM sodium phosphate in a total volume of 200 ml), and the
fractions containing the regulatory complex were pooled and further
purified on a DEAE-Sephacel (Pharmacia) column (1 × 10 cm)
equilibrated in 10 mM imidazole, pH 7.0, 20 mM
NaCl, 2 mM MgCl2, 1 mM DTT, 1 mM NaN3. After eluting the proteins with a
linear gradient to 0.5 M NaCl (total volume 100 ml), the
fractions were analyzed by 15% SDS-PAGE and pooled. Trace amounts of
remaining impurities were removed by gel filtration chromatography on a
Superose 12 column (1 × 30 cm, FPLC, Pharmacia). The yield was
10-15 mg of complex.
Labeling of RLC--
Purified RLC (lyophilized with sucrose) was
dissolved in 8 M guanidine, PBS, 10-20 mM DTT,
and stirred at room temperature for 1 h in order to completely
reduce the cysteines before dialysis against PBS, 50 µM
DTT, with at least one change of buffer. The single Cys mutant,
Cys2 (Table I), was reacted with 2.5-5-fold molar excess
of 1,5-IAEDANS (Molecular Probes) over total thiols for 2 h on ice
in the dark before stopping the reaction by adding DTT to a final
concentration of 10 mM. The double Cys mutant, C2/C79, at a
concentration of 5-7 mg/ml, was reacted with a 3-fold molar excess of
1,5-IAEDANS for 2 min on ice. The reaction was stopped with 50 mM DTT, and the sample dialyzed against 50 mM
sodium phosphate, pH 7.0, 0.2 mM EDTA, 1 mM
DTT, and 3 mM NaN3. The protein was
chromatographed on a DEAE-Sephacel (FPLC, Pharmacia) column (1.5 × 10 cm) equilibrated in the above buffer and eluted with a linear
gradient to 300 mM sodium phosphate (total volume 150 ml).
Fractions containing unreacted, singly labeled (C2DAN/C79),
and doubly labeled RLC were identified by urea-PAGE.
C2DAN/C79 (in PBS, 50 µM DTT) was
subsequently reacted with a 4-fold molar excess of DDPM (Aldrich) at
room temperature for 4 h to obtain
C2DAN/C79DDP. Iodoacetamide was used to block
any unreacted cysteines in control experiments. The light chains were
dialyzed exhaustively against PBS to remove excess reagent before being
used to prepare the RD complex.
Spectrophotometric analysis of the labeled light chains was used to
determine the extent of labeling. Protein concentration was determined
by the method of Bradford (15) using wild-type RLC as a standard
(absorbance of 0.5 at 280 nm, 1 mg/ml). A molar extinction coefficient
of 6200 M
1 cm
1 at 340 nm was
used for IAEDANS-labeled proteins. The ratio of DANS to light chain was
~0.9. The molar extinction coefficient for DDPM-labeled RLC was taken
as 2930 M
1 cm
1 at 442 nm (7).
The ratio of DDP to RLC was approximately 1.
Titration with Divalent Cations--
RLC and the regulatory
domain (with either wild-type or mutant RLCs) were dialyzed against 0.1 M Hepes, pH 7.2, 0.1 M NaCl, 1 mM
NaN3 for 10-14 h and clarified at 100,000 × g for 30 min. EGTA was added to a final concentration of 1 mM before titration with calcium ions. Similarly, 1 mM EDTA was used in titrations with magnesium ions.
Proteins were diluted to an absorbance of less than 0.05 to reduce the
effect of light scattering (and the Raman scattering was removed by
subtraction of a buffer blank). Small aliquots (2-20 µl) of the
stock CaCl2 or MgCl2 solution were delivered by
a microsyringe (Hamilton Co.) into 2 ml of the protein solution with
constant stirring. A SLM 48000 spectrofluorometer was used to measure
tryptophan fluorescence (emission maximum at 340 nm) upon excitation at
295 nm. The emission spectra were recorded from 300 to 400 nm at 1-nm
intervals with 2-s integration at each wavelength. The sample
compartment temperature was maintained at 20 °C. Percent decrease in
tryptophan fluorescence (after volume correction) was plotted against
-log free metal ions (pM) to obtain the concentration
(equivalent to 1/Ka, the association constant) at
which the half-maximal change in fluorescence occurs. The program used
to calculate free metal ion concentrations was based on the methodology
of Storer and Cornish-Bowden (16).
Energy Transfer Measurements--
Steady-state fluorescence
energy transfer measurements were performed on an ISS PC1 photon
counting spectrofluorometer equipped with a rhodamine B (4 mg/ml in
ethanol with a RG 630 filter) quantum counter. Spectra were recorded in
ratio mode to correct for lamp intensity fluctuations. The spectral
bandwidths for excitation and emission were 8 nm. Emission spectra were
recorded at 1-nm intervals with 2-s integration at each wavelength, and
were later corrected with software supplied by ISS. A 305 nm cut-off
filter was used in the emission path for tryptophan spectra. When
IAEDANS was used as a donor, 320 and 470 nm cut-off filters were placed in the excitation and emission paths, respectively.
The cluster of two tryptophans in the heavy chain peptide of RD could
be used as an intrinsic energy donor. The acceptor was a single
cysteine on RLC labeled with 1,5-IAEDANS. For preparation of RD, RLC
mutants were used in which tryptophan residue 138 was replaced with
phenylalanine. Two samples of RD had to be independently prepared, one
containing Cys2 blocked with iodoacetamide, and the other
containing IAEDANS-labeled Cys2. Proteins were dialyzed
against Hepes buffer containing 1 mM magnesium chloride and
clarified as described above. The tryptophan emission spectra were
recorded under identical optical conditions for the unlabeled and
labeled complexes diluted into the dialysate or 7 M
guanidine hydrochloride. Contributions to the spectra due to Raman
scattering were removed by subtraction of the buffer blank. In
guanidine, where the complex is fully dissociated and energy transfer
is presumably zero, the tryptophan intensities of the two complexes are
directly proportional to their concentrations. Therefore, the ratio of
the tryptophan fluorescence intensities for the labeled and unlabeled
RD complexes in the native state should be the same as in the denatured
state, if no energy transfer occurs. After normalizing the
concentrations of the two species, any observed decrease in
fluorescence of the acceptor-labeled RD relative to the unlabeled
sample can be attributed to energy transfer between donor and acceptor.
The same procedure was applied to RD specimens in which RLC labeled
with IAEDANS was used as the donor and a second cysteine introduced
into RLC was labeled with the acceptor DDPM.
The efficiency of energy transfer (E) was calculated
according to the following equation,
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(Eq. 1)
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where Fda and Fd
are the measured fluorescence intensities in the presence and absence
of the acceptor, respectively. The degree of energy transfer is
dependent upon the distance (R) between donor and acceptor
and is given by Equation 2,
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(Eq. 2)
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where R0, the Forster critical distance
at which the energy transfer is 50%, was calculated by experimentally
determining the parameters in Equation 3,
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(Eq. 3)
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where Qd is the quantum yield of the donor,
n is the refractive index of the medium, taken here as 1.4, J is the overlap integral expressed in mol
1
cm3, and
2 is the orientation factor. The
quantum yield, which depends upon the chemical environment of the
donor, was calculated for the free light chain and RD by the procedure
described by Chen (17) using quinine sulfate (in 0.05 M
sulfuric acid) as a standard. The absorbance of proteins at 295 nm and
the quantum yield of 0.70 for quinine sulfate (18) were used in the
following equation,
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(Eq. 4)
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In order to determine the overlap integral, J, the
emission spectrum of the donor (tryptophan excited at 295 nm) and the absorption spectrum of the acceptor (IAEDANS) were recorded at 1-nm
intervals from 320 to 380 nm. The value of J was calculated according to the expression,
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(Eq. 5)
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where
a is the extinction coefficient
(mol
1 cm
1) of the acceptor and F
is the fluorescence intensity of the donor at wavelength
(cm).
Steady-state polarization measurements were performed on RLC and RD in
order to estimate the upper and lower limits of the orientation factor
2 (19) essentially as described in Ref. 20. The limiting
polarization anisotropies for donor and acceptor were determined using
L-format with the emission monochromator set at a fixed wavelength (340 and 480 nm for donor and acceptor, respectively). Sucrose was added to
the samples to provide a range of viscosities at 20 °C for
extrapolation to infinite viscosity in Perrin plots (21).
Other Procedures--
SDS-PAGE was performed as described by
Laemmli (22). The extent of labeling was monitored by urea-PAGE
analysis (23). The extinction coefficients of the recombinant RDs were
determined by measuring the absorbance at 280 nm and determining the
concentration by the method of Bradford (15). Sedimentation velocity
runs were made in a Beckman Model E analytical ultracentrifuge. Myosin light chains were phosphorylated as described previously (24).
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RESULTS |
Purification and Characterization of Recombinant RD--
The 9-kDa
heavy chain fragment (HC) could not be obtained in any significant
amount when expressed without the light chains, as determined by
SDS-PAGE analysis of the crude extracts from overnight cultures (data
not shown). Therefore, it was necessary to coexpress HC with both light
chains, ELC (LC1 isoform) and RLC (LC2) (Fig.
2A, lane 1) or with ELC alone
(lane 2). In the latter method, HC and ELC were segregated
into inclusion bodies, which could be readily separated from the
bacterial proteins (lane 3). After resuspension in 8 M guanidine, RLC was added to solubilize the complex upon
subsequent dialysis into a benign solvent (lane 4). RD was
purified by ion-exchange chromatography on hydroxylapatite (lane
5) followed by DEAE-Sephacel (lane 6) to remove any
last trace of impurities. The recombinant RLC added to the HC·ELC
complex was one of several mutants described in Table
I. The purified RD remained stable at all
concentrations (up to 20 mg/ml) when stored at 4 °C.

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Fig. 2.
Purification of RD. A, HC peptide
(9 kDa) was coexpressed with ELC (LC1) and RLC (LC2) (lane
1) or with ELC alone (lane 2). The latter method
segregated HC and ELC into inclusion bodies, which were separated from
the bacterial proteins (lane 3), and resuspended in 8 M guanidine. Addition of RLC solubilized the complex
(lane 4), which was purified on hydroxylapatite (lane
5) followed by DEAE-Sephacel (lane 6). The added RLC,
labeled or unlabeled, was one of several mutants described in Table I.
B, sedimentation velocity pattern for purified RD showed a
single, homogeneous peak.
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Various methods were used to establish the composition of RD. The
co-elution of the light and heavy chains from the ion-exchange columns
during purification suggested that they form a complex. Upon gel
filtration chromatography, RD eluted between the chymotryptic S1
(~100 kDa) and the free light chains (~20 kDa) consistent with a
molecular mass of ~50 kDa. The sedimentation velocity pattern for
purified RD showed a single, homogeneous peak with a sedimentation coefficient of 3.3 S (Fig. 2B). The extinction coefficients
of RDs made with wild-type and Cys126/Cys155
were 0.5 and 0.4, respectively, which agrees well with the theoretical values based on the number of tryptophans and tyrosines in a 1:1:1 complex (25). Although the ratio of the two LCs and HC as determined by
densitometry of the Coomassie Blue-stained SDS gels was 1:1:2, it is
probable that the large number of charged residues in the heavy chain
peptide caused a disproportionate absorption of dye.
Intrinsic Fluorescence of Heavy Chain Tryptophans--
There are
three tryptophans in wild-type RD, two in the heavy chain peptide
(Trp829-Pro830-Trp831) and one in
wild-type RLC (Trp138) (Fig. 1). A mutant RLC was prepared
in which Trp138 was replaced by Phe so that Trp
fluorescence from the heavy chain could be used as a single reporter
group or donor site in FRET experiments. Tryptophan emission spectra
for RD containing wild-type or mutant RLC were compared with free
wild-type RLC (Fig. 3). RD(mut)
containing two tryptophans from the heavy chain showed far less
fluorescence than wild-type RLC which has only a single tryptophan. The
diminished intensity of fluorescence suggests intramolecular quenching
by the heavy chain tryptophans due to their close proximity in the
structure (WPW). In contrast, the single Trp138 in
wild-type RLC appears to increase its fluorescence in the more
hydrophobic environment of the heavy chain complex, as judged by the
enhanced fluorescence of RD(wt) compared with the sum of the
fluorescence intensity of wild-type RLC and RD(mut). In a denaturing
solvent such as guanidine hydrochloride, the fluorescence intensity is
more proportional to the tryptophan content of the protein:
i.e. the fluorescence of RD(wt) is equal to the sum of the
fluorescence of RD(mut) and wild-type RLC (Fig. 3). The emission maxima
in guanidine hydrochloride showed a red shift (353 nm) compared with
the emission maxima at 343 and 340 nm for WT and RDs, respectively, in
a benign solvent. This can be explained by the increased exposure of
the Trps to solvent in an unfolded, denatured state.

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Fig. 3.
The corrected tryptophan fluorescence
emission spectra of RD and RLC. The excitation wavelength is 295 nm. Molar protein concentration is constant for all species. RD(wt)
(solid line) contains two heavy chain Trp(WPW) and a
wild-type RLC that has a single Trp, see Table I. RD(mut) (dotted
line) contains the two heavy chain Trp, and the mutant RLC
(C126/C155) that has no Trp. The low fluorescence intensity of RD(mut)
relative to wild-type RLC(WT) (broken line) in a benign
solvent (PBS) suggests intramolecular quenching of the heavy chain
Trps. In a denaturing solvent, such as guanidine hydrochloride, the
fluorescence intensity more closely approximates the tryptophan content
of the protein: i.e. RD(wt) > RD(mut) > WT.
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Binding of Divalent Metal Ions to RLC and RD--
Titrations with
divalent cations were carried out to determine if expressed RD would
bind calcium and magnesium with affinities similar to those found for
native myosin. First, the intrinsic fluorescence of Trp138
in RLC was measured as a function of calcium (or magnesium) to determine the binding of these ions to free RLC (data shown only for
Ca2+ binding) (Fig. 4,
A and B). The total decrease in fluorescence intensity was ~18% for Ca2+ and ~12% for
Mg2+ ions. The association constants of 105
M
1 for Ca2+ and 103
M
1 for Mg2+ obtained here agree
well with those reported by Alexis and Gratzer (26) for rabbit skeletal
RLC. Despite the considerable distance between the cation binding site
in the NH2-terminal domain and the single Trp in the COOH
terminus of RLC, the conformational change associated with metal
binding must be transmitted to the Trp, as evidenced by a change in
signal.

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Fig. 4.
Binding of calcium ions to RLC and RD.
A and B, the decrease in tryptophan fluorescence
intensity (emission maximum at 340 nm) of RLC as a function of calcium
ions was used to measure the binding of Ca2+ to free RLC.
Emission spectra at the beginning (EGTA, solid line) and at
the end of the titration (calcium, dotted line) are shown.
Similarly, the data for RD(wt) and RD(mut) are shown in C
and D. The greater percentage fluorescence change with
RD(mut) indicates that the heavy chain tryptophans are the major source
of the fluorescence change upon metal binding. Excitation was at 295 nm.
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Similarly, the decrease in fluorescence for RD(wt) or RD(mut) as a
function of calcium (or magnesium) was used to determine the affinity
of RLC for metal ions in the heavy chain bound state. Association
constants of 107-108
M
1 for Ca2+, and 106
for Mg2+ were obtained (Fig. 4, C and
D). The smaller fluorescence change with RD(wt) compared
with RD(mut) can be attributed to the high level of background
fluorescence contributed by Trp138 in wild-type RLC. The
large fluorescence change in RD(mut) (~32%), despite the low
absolute intensity of the signal, suggests that the heavy chain
tryptophans are perturbed to a greater degree by metal binding than the
single Trp in RLC. This may be related to the hydrophobic interactions
of residues in the NH2-terminal domain of RLC with the
tryptophans in the COOH terminus of the heavy chain (2). The binding
constants obtained here are in good agreement with earlier values
(3 × 107 M
1 for
Ca2+) determined by more direct binding methods (27).
Fluorescence Energy Transfer between Heavy Chain Tryptophans and
IAEDANS-labeled RLC--
The intrinsic fluorescence of the two heavy
chain tryptophans can also be used as a donor in energy transfer
experiments. By introducing a single IAEDANS acceptor probe into the
Cys2 residue of RLC, we could measure the energy transfer
distance between the cluster of Trp at the bend of the heavy chain
helix and the NH2 terminus of RLC. The tryptophan emission
spectra of RD (excitation at 295 nm) prepared either with or without
the IAEDANS-labeled RLC are shown in Fig.
5A. The tryptophan emission is
reduced by about 9% when Cys2 is labeled with the IAEDANS
acceptor (RD(C2DAN)). Although donor quenching is small,
the large enhancement in IAEDANS fluorescence at 500 nm compared with
the free labeled light chain (C2DAN) is strong qualitative
evidence for energy transfer from the heavy chain tryptophans to the
dansyl probe on RLC.

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Fig. 5.
Fluorescence energy transfer between heavy
chain tryptophans and IAEDANS-labeled RLC. A, corrected
tryptophan emission spectra (excitation at 295 nm) of RD in the absence
(RD[Cys2], solid line) and presence
(RD[Cys-2DAN], dotted line) of acceptor.
Although donor quenching is small (~10%), the large enhancement in
acceptor fluorescence at 500 nm compared with the free labeled light
chain (Cys-2DAN, broken line) is strong
qualitative evidence for energy transfer from the heavy chain
tryptophans to the dansylated Cys2. B,
excitation spectra (emission monitored at 500 nm) of dansylated RD
(dotted line) shows a tryptophan component between 270 and
300 nm which is evidence for energy transfer. C2DAN
(broken line) has no tryptophan and therefore shows little
or no fluorescence when excited in this region. C126DAN
(solid line) shows strong fluorescence when the single Trp
at position 138 is excited due to its close proximity to the dansyl
probe. The molar protein concentration was the same for all
species.
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We also examined the excitation spectra for several constructs that
contained either no tryptophan or had tryptophans located at different
distances from the acceptor probe, IAEDANS (Fig. 5B). The
appearance of a tryptophan component between 280 and 300 nm in the
excitation spectrum of IAEDANS (emission measured at 500 nm) is strong
evidence that energy transfer has occurred. C2DAN has no
tryptophan and therefore shows little fluorescence when excited in the
region of 290 nm. C126DAN shows strong fluorescence when
the single tryptophan at position 138 is excited due to the close
proximity of the labeled Cys126. Similarly, the tryptophan
cluster (WPW) on the heavy chain shows energy transfer, although much
weaker, to the dansylated Cys2 in
RD(C2DAN).
The efficiency of energy transfer (E) between the heavy
chain Trp and the IAEDANS probe on RLC, as determined from emission spectra, is given in Table II. In order
to convert E into a distance, the quantum yield
(Qd) and the overlap integral (J) had to
be determined for RD(mut) (see "Materials and Methods"). The low
value of 0.02 obtained for Qd is not surprising
since the tryptophan emission is unusually low for this complex (see Fig. 3). The overlap integral for the Trp/IAEDANS pair was calculated to be 6.1 × 10
15 M
1
cm3. The orientation factor,
2, used in the
calculations was the isotropically averaged value of 2/3. Measurements
of the limiting anisotropy of the Trp donor and IAEDANS acceptor in RD
gave values of 0.14 to 0.16, indicating considerable randomization of
the probe orientation (21). A value of 2/3 for the orientation
factor has generally been used in distance calculations, with no major
errors in distance resulting from this assumption. Based on the above
parameters, R0, the distance at which
E is 50%, was 16 Å which yields a distance of 23-24 Å between -WPW- and Cys2 in RD. This distance did not change
significantly upon phosphorylation of RLC, or in the presence or
absence of divalent cations.
As a control, the distance between Trp138 and
Cys126 was determined in the RLC mutant, Cys126
(see Table I) using IAEDANS as the acceptor. Between 80 and 85%
quenching was observed in the presence or absence of metal ions. A
Qd of 0.14 was obtained for the single Trp in RLC,
which is close to the value reported for free tryptophan (17). The
value for the overlap integral was assumed to be the same as for the
above, particularly, since a similar J value was reported
for a Trp cluster and single IAEDANS-labeled Cys mutant in a membrane
protein (20). The range of anisotropy values measured for
Trp138/IAEDANS in RLC was also similar to RD, and a value
of 2/3 was taken for the orientation factor. These parameters led to a
value of 22 Å for R0, from which a distance of 16-17 Å was calculated (Table II). This FRET distance agrees well with the
distance of 18 Å for the corresponding residues in the crystal
structure of scallop RD (2), and with an earlier distance determination by fluorescence spectroscopy (28).
Fluorescence Energy Transfer between Donor/Acceptor Probes on RLC
Bound to the Heavy Chain--
To fix the position of Cys2
in RD, a minimum of three distances needs to be determined. In an
earlier study, the FRET distance between Cys2 and
Cys155 in the heavy chain-bound RLC was measured (7). For a
third distance, Cys2 and Cys79 in RLC were
labeled with IAEDANS and DDPM as donor and acceptor probes,
respectively, and reconstituted with heavy chain peptide in RD. The
greater reactivity of Cys2 compared with Cys79
again enabled us to prepare the RLC with donor and acceptor probes in a
1:1 stoichiometry (7). The emission spectra of RD containing mutant RLC
labeled with donor, IAEDANS, at position 2 (C2DAN/C79) and
RD containing RLC with donor and acceptor
(C2DAN/C79DDP) is shown in Fig.
6. The fluorescence of IAEDANS is
strongly quenched by the non-fluorescent acceptor, DDPM, see Table II. In the presence of guanidine hydrochloride no energy transfer occurs,
and the fluorescence intensity of the light chains with or without
acceptor was the same. A distance of 29-30 Å was calculated between
Cys2 and Cys79, using parameters from the
literature for these probes (7). As with the other sites,
phosphorylation had no effect on the distance.

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Fig. 6.
Fluorescence energy transfer between donor
and acceptor probes on RLC bound to the heavy chain. Emission
spectrum (excitation at 340 nm) of RD containing mutant RLC labeled
with donor (RD[C2DAN/C79], solid line) shows
strong quenching when the non-fluorescent acceptor DDP is attached to
position 79 (RD[C2DAN/C79DDP],
broken line). In guanidine hydrochloride no energy transfer
occurs, and the fluorescence intensity of the light chains is the same
in the presence or absence of acceptor (dotted and
broken lines superimpose).
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DISCUSSION |
The structure and function of myosin have been studied
historically by a reductionist approach: first, myosin was cleaved by
proteolytic enzymes into heavy meromyosin and light meromyosin, which
allowed a separation of the enzymatic from the assembly properties of
this macromolecule. Next, the active globular head region,
subfragment-1 (S1), was cleaved from the long
-helical rod (29).
Although proteolytic procedures could be used to reduce S1 even further
(5, 30), it became far simpler to prepare the "motor domain" by
molecular biological methods (31). Here we describe the first
preparation of the "regulatory domain" or neck region of chicken
skeletal myosin S1 by recombinant methods.
The regulatory domain appropriately derived its name from the structure
of the invertebrate, scallop myosin, a molecule that is regulated by a
Ca2+-specific site in the neck region of the myosin head
(32). The RD consists of an essential (ELC) and a regulatory (RLC)
light chain, which together stabilize the S1 heavy chain in an
-helical conformation as it joins the
-helical coiled-coil rod
(1). The crystal structure of scallop RD (2) first showed that this multisubunit interaction creates a Ca2+-binding site on the
NH2 terminus of the ELC, a result which could not be
predicted from the sequence, since it fails to correspond to a typical
EF-hand (2). The only other myosins regulated by Ca2+ are
the "unconventional" myosins which contain primarily calmodulin in
the neck region of the head (33).
Unlike the invertebrate and unconventional myosins, the vertebrate
myosin IIs are regulated to varying degrees by a phosphorylated serine
in the NH2 terminus of the RLC (34). Smooth and nonmuscle myosins are completely inactive in the dephosphorylated state, whereas
the activity of skeletal myosin is only modulated by the state of
phosphorylation (4). This raises the intriguing question of which
properties of myosin dictate whether a single phosphorylation site in
the regulatory domain can accelerate product release in the motor
domain? In a recent study, it was suggested that the RLC must possess
certain arginine residues in its COOH-terminal domain which can bond to
the phosphoserine in the NH2 terminus (35). Only thick
filament-regulated myosins have conserved arginine residues in the E to
H-helices (nomenclature refers to calmodulin) of RLC that can serve as
possible candidates for such an interaction. This might explain why
skeletal RLC, which lacks these conserved Arg residues, cannot replace
smooth RLC in the regulation of smooth muscle myosin (36).
Location of the NH2 Terminus of RLC--
The two FRET
distances determined here, together with an earlier determination of
the distance between Cys2 and Cys155 in RLC
exchanged into myosin (7), define a locus for Cys2, which
marks the beginning of the sequence for RLC (Fig.
7). The 19 residues in RLC, that are not
visible in the crystal structure of chicken S1, appear to lie at an
angle to the first A-helix of the EF-hand that forms the
Mg2+-binding site. The region of closest approach between
the phosphoserine and the remainder of the RLC molecule would be in the
vicinity of the D/E linker segment that joins the NH2- and
COOH-terminal lobes. Although, theoretically, Cys2 could
lie 180° away from this location, this orientation is preferred insofar as it maximizes side chain interactions between the
NH2-terminal peptide and the linker region. Interestingly,
the NH2 terminus of troponin C is located in approximately
the same position relative to the A-helix and the central linker helix
(37). An Arg residue in the NH2 terminus of TnC has been
shown to form a H-bond with a Glu residue in the D-helix of the
NH2-terminal domain (37). Assuming that skeletal and smooth
RLC share a similar structure, the localization studies described here
preclude a direct interaction between the phosphoserine and specific
arginine residues in the COOH-terminal domain. However, before
discussing alternative mechanisms for the activation of myosin's
activity, one should question: 1) how faithfully the recombinant
regulatory domain mimics the neck region of the native myosin molecule,
and 2) how reliable are resonance energy transfer measurements in
determining distances?

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Fig. 7.
Stereo ribbon diagram of RD showing only the
binding site of RLC. Based on the distance measured between
Cys2 and WPW (24 Å) and the distance between
Cys2 and Cys79 (30 Å), see Fig. 1 and Table
II, together with the FRET distance determined in myosin between
Cys2 and Cys155 (30 Å) (7), a location for the
NH2 terminus of RLC (Cys2) is defined.
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The structural determination of the regulatory domain of scallop myosin
was carried out on a complex produced by a series of proteolytic
digestions of the native protein (2, 5). Despite the absence of the
motor domain, the structure of scallop RD was remarkably similar in its
overall features to that of chicken S1 (1), implying that RD is a
stable folding unit, and that the structure of RD in all myosin IIs is
highly conserved. The remarkable stability of RD is evident from its
ability to fold spontaneously upon replacement of a denaturing solvent
with a benign solvent (5). The recombinant chicken RD used in this study was also prepared by reconstitution out of a denaturant, although
the same complex could be prepared by simultaneous expression of the
two LCs and the heavy chain peptide, which avoids the use of
denaturants. The only functional property that can readily be measured
is the binding affinity of RD for divalent cations. The good agreement
between the binding constants obtained here and those reported for
myosin, provides strong evidence that RD retains its native properties
and is a good model system. An important advantage in using RD for
structural studies compared with S1 is that the endogenous tryptophans
on the heavy chain can be used as a donor group without introducing
potentially disruptive extrinsic probes. Thr79 was chosen
for mutagenesis to Cys, because its position on the D-helix is
solvent-exposed, and a probe at this site would be expected to have a
minimal effect on RLC/heavy chain interactions.
The accuracy of distance measurements by resonance energy transfer is
thought to be limited by assumptions about the orientation factor,
2 (see "Discussion" in Wolff-Long et al.
(7) and references therein). Even with this uncertainty, usually less
than 20%, FRET distance measurements are in a range that is
sufficiently narrow to determine whether an interaction is likely to
occur between the NH2 and COOH termini of RLC. An
unexpected experimental problem encountered here was the low quantum
yield of the two neighboring tryptophans in the heavy chain peptide.
This led to a low value for R0 (16 Å), and
therefore very little quenching of the Trp fluorescence by the acceptor
probe (<10%). To perform these measurements, it was necessary to
prepare RD with a high degree of purity since any contamination by
fluorescent impurities would seriously compromise the data. The
reproducibility of the results, and the demonstration of acceptor
excitation, enhanced our confidence in the validity of these distance
determinations.
Regulation by Phosphorylation--
Protein phosphorylation
stabilizes different conformational states of regulated molecules in
order to enhance or repress biological activity. A classic example of
such a mechanism is glycogen phosphorylase, whose crystal structure has
been solved in both the active and inactive form (38). It was shown
that phosphorylation of the NH2-terminal peptide results in
a structural change from a disordered state to a distorted
310 helix, which is stabilized at the subunit interface of
the glycogen phosphorylase dimer by a series of hydrogen bonds and van
der Waals interactions. These hydrophobic interactions disrupt COOH-
and NH2-terminal regions and new contacts are made which
promote an active state.
It is difficult to envision how this type of allosteric mechanism can
be readily applied to the myosin molecule. The two subunits of myosin
are relatively independent entities in the head or S1 regions, and do
not become joined until the junction with the
-helical coiled-coil
rod. Computer modeling of the head/rod junction has suggested that the
principal contact between the S1s occurs between the RLCs of
neighboring heads (39). Experimentally, it was shown that mutant RLCs
containing cysteines in the NH2-terminal domain formed
intermolecular disulfide bonds between the heads (13). It is quite
conceivable, therefore, that phosphorylation of the NH2
terminus might induce a structural change that could disrupt the RLC
interface interactions and free the S1 heads from their constrained
configuration. As interhead interactions are weakened, the intrasubunit
interactions between the RLC and the heavy chain may be strengthened,
thereby propagating a structural change to the active site that
enhances the release of the products of ATP hydrolysis.
What is the evidence in support of such a mechanism? It is well
established that single-headed smooth muscle myosin is active independent of phosphorylation (40, 41). This observation is consistent
with the hypothesis that the "off" state at a minimum involves
interactions between the RLCs of the two heads, but it does not tell us
about the affinities of these interactions. It has been shown that
mutations in the COOH-terminal region of smooth RLC can markedly reduce
the affinity of RLC for the heavy chain, in parallel with a loss of
myosin's motor activity (34, 42). We have found that a single point
mutation in skeletal RLC, which lowers its affinity to the heavy chain,
also causes a reduction in the in vitro motility of skeletal
myosin, both in the single- and double-headed
state.2 The general principal
that emerges from these studies is that strong binding of RLC to the
heavy chain is a prerequisite for the "on" state. This conclusion
can also be applied to single-headed myosin I, where removal of
calmodulin results in a loss of motor activity (43). Even recent
physiological experiments in skeletal muscle fibers have shown that
phosphorylation increases Ca2+ binding to RLC, an effect
that would be expected to increase the RLC/heavy chain interaction, and
may therefore be responsible for the increase in force production at
submaximal levels of Ca2+ (44).
This brings us back to the question of why mutating six residues in the
COOH-terminal domain of skeletal RLC to those present in smooth RLC (of
which four were Arg) had the effect of restoring regulation to a smooth
myosin heavy chain, which was locked permanently in the off state with
wild-type skeletal RLC (36). Instead of proposing a direct coordination
between the phosphoserine and Arg residues (35), we suggest that the
mutated skeletal RLC interacts more strongly with smooth heavy chain
than wild-type skeletal RLC, with the consequence that phosphorylation
can now activate this chimeric myosin. This alternative explanation
pictures myosin regulation to be based on a complex series of molecular interactions between the RLC and the heavy chain that can be perturbed by phosphorylation, and transmitted to the active site through the
regulatory domain.
We thank Dr. Steven H. Grossman for help in
fluorescence measurements at initial stages of this work. We also
acknowledge the contribution of Dr. Weiyi Yang in the construction of
plasmid pWY. We thank Dr. Terence Tao for life-time measurements and
helpful discussions, Dr. Anne Houdusse for providing the stereo images of the RD, and Dr. Kathleen Trybus for suggestions in the preparation of this manuscript.