From the
Department of Biochemistry,
Eötvös Loránd University, Budapest 1117, Hungary and
Department of Pharmacology, Gunma University
School of Medicine, Maebashi, Gunma 371, Japan
Received for publication, April 22, 2003
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ABSTRACT |
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INTRODUCTION |
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Enzymatic and motor activity in the myosin superfamily can be controlled by a variety of mechanisms and regulatory systems associated with either the actin filament or the myosin molecule. Regulation through the RD can be achieved by reversible phosphorylation or direct Ca2+ binding. Vertebrate smooth muscle and non-muscle myosin IIs are activated by phosphorylation of the RLC by a Ca2+/CaM-dependent myosin light chain kinase (3). In the animal kingdom the Mollusca conventional myosins are unique in that they are activated by direct Ca2+ binding to the ELC (4). In the lower eukaryote Physarum polycephalum, in contrast to any other known conventional myosins, increase in Ca2+ concentration inhibits the activity of myosin (5, 6). The diverse classes of unconventional myosins are also regulated in part by the interaction of Ca2+ with the CaM subunits; the consequences of the Ca2+ binding can be more complex and are not as well understood as in the myosin II class. Elevated Ca2+ concentration has, in general, an inhibitory effect on the motility of vertebrate myosin I and myosin V as well as the plant-specific myosins (79).
Structural studies of the isolated RD (a three-chain complex of ELC, RLC,
and a 10-kDa fragment of the heavy chain) of scallop myosin II revealed
that the triggering Ca2+-binding site is localized in
the first EF-hand of ELC (10,
11). The conformation of the
N-terminal domain of ELC containing the bound Ca2+ is
closed rather than open, which is found in the conventional
Ca2+-saturated EF-hands. RLC of scallop and all the
known higher eukaryotic myosin II contains a divalent metal-binding site in
the first EF-hand that preferentially binds Mg2+ and has
only a structural role (10,
11).
Cytoplasmic streaming in plasmodia of the myxomycete P. polycephalum is exceedingly vigorous. The cytoplasm streams at a rate about 1.3 mm/s and changes direction every 23 min. This oscillatory process is regulated by Ca2+ and is thought to be driven by a conventional myosin (12). Ca2+ regulates the contractile system of Physarum plasmodia by direct binding to the myosin, and inhibiting its ATPase activity and in vitro motility (5, 6). It was proposed that the ELC is the Ca2+-binding subunit of Physarum myosin (1315). Other mechanisms could also be involved in the regulation or modulation of the activity of the myosin. Phosphorylation of the heavy chain and RLC by heavy chain and light chain kinases, respectively, increases the affinity of myosin to actin; activity of the kinases is suppressed by Ca2+; however, at high actin concentrations existing in vivo in the plasmodium, phosphorylation has a negligible effect on regulation of myosin (6). A caldesmon-like actin-binding protein can stimulate the ATPase activity of myosin. This effect is inhibited by Ca2+-CaM (16, 17).
To compare the structural basis of the two, oppositely regulated Ca2+-binding conventional myosins, we initiated studies to localize and characterize the regulatory calcium-binding site of Physarum myosin using recombinant LCs and RD. The results reveal that there is one Ca2+-binding site per head in the myosin. By mutating all of the EF-hands, which were predicted to be competent metal-binding sites based on sequence analysis, we demonstrate that the first EF-hand of the ELC is responsible for Ca2+ binding. Moreover, our results suggest that the N-terminal domain of ELC remains in a closed conformation even in the presence of Ca2+.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification of Recombinant ProteinsRecombinant proteins were expressed in Escherichia coli strain BL21(DE3)pLysS as described previously (19). The light chains were purified under denaturating conditions. Cells were suspended in 6 M guanidine hydrochloride, 0.5 M NaCl, 20 mM sodium phosphate, pH 7.8, 0.5 mM phenylmethylsulfonyl fluoride, 20 µg/ml pepstatin A and homogenized with sonication. The homogenate was centrifuged at 50,000 x g, and the supernatant was loaded onto a Ni2+ chelate column (ProBond, Invitrogen); the column was washed with a buffer containing 8 M urea, 0.5 M NaCl, 20 mM sodium phosphate at pH 6.0 and 5.3, and the recombinant protein was eluted at pH 4.0. The RD was purified under non-denaturing conditions; cells were suspended in 0.5 M NaCl, 20 mM sodium phosphate, pH 7.8, 3 mM NaN3, 0.5 mM phenylmethylsulfonyl fluoride on ice. After sonication and centrifugation of the homogenate, the supernatant was loaded on a ProBond column and washed with the same buffer at pH 6.0 containing 50 mM imidazole, and the RD was eluted by increasing the concentration of imidazole to 0.5 M. The eluted light chains and RD were dialyzed against 20 mM NaCl, 10 mM Tris/HCl, pH 7.6, 3 mM NaN3, 0.1 mM EDTA, 0.1 mM DTT and loaded onto a Mono Q ion exchange column equilibrated with the same buffer. The absorbed proteins were eluted by a linear gradient of NaCl to 0.5 M.
Purification of Physarum MyosinPlasmodia of Physarum were grown on Quaker oatmeal according to Camp (20) with some modifications (21). Myosin was isolated from the collected plasmodia by the high salt extraction method as described earlier (5).
Calcium Binding ExperimentsThe extent of
Ca2+ binding was measured by the flow dialysis method
according to Nakashima et al.
(22,
23) with
45CaCl2 (PerkinElmer Life Sciences) in 0.1 M
NaCl, 20 mM MOPS/NaOH, pH 7.0, at 25 °C in the presence or
absence 2 mM MgCl2. The protein concentrations were
25100 µM. The loss of radioactive ligand during the
experiments and the nonspecific Ca2+ binding to the flow
dialysis cell were corrected. In some experiments we measured
Ca2+ binding by equilibrium dialysis as described
previously (24) at 22 °C
in a buffer containing 50 mM NaCl, 20 mM MOPS/NaOH, pH
7.0, 3 mM NaN3, 0.11
mM45CaCl2, and various concentrations of EGTA
to obtain the desired free Ca2+ concentration. The
resulting Ca2+-binding data were analyzed by fitting to
the Adair-Klotz equation (25)
for single binding site,
![]() | (Eq. 1) |
![]() | (Eq. 2) |
ELC Labeling with 1,5-IAEDANSWild-type and mutant ELCs containing a single cysteine (Cys-10) were labeled with 3-fold molar excess of 1,5-IAEDANS (Sigma-Aldrich) in 6 M guanidine hydrochloride, 10 mM Tris/HCl, pH 7.5, for 30 min at room temperature. Labeling was terminated with 1 mM DTT, and then the samples were dialyzed overnight against a buffer containing 50 mM NaCl, 50 mM HEPES, pH 7.5, 0.1 mM DTT.
Transient Kinetic AssaysTransient kinetic assays were
performed using an Applied Photophysics SX18MV stopped flow apparatus with a
150 watt Xe lamp at 22 °C. The dead time of the system was 1.5 ms, as
determined earlier (26). The
assay buffer contained 50 mM NaCl, 50 mM HEPES, pH 7.5,
and 0.1 mM DTT. The IAEDANS-labeled ELCs were excited at 336 nm,
and the emission was monitored through a GG455 filter.
Ca2+- and Mg2+-saturated
IAEDANS-labeled ELCs were rapidly mixed with excess EDTA, respectively.
Ca2+ dissociation from RD were investigated by mixing
the Ca2+-saturated RD with excess of the calcium
indicator Quin 2 (Sigma-Aldrich), which rapidly binds
Ca2+, reducing the free calcium concentration to
nanomolar range. The samples were excited at a wavelength of 366 nm, and the
fluorescence was measured through the GG455 filter. During the determination
of Mg2+ dissociation rate from RD by displacement of
Mg2+ with Ca2+, the decrease in
Ca2+ concentration was detected by using the low
affinity calcium indicator Calcium Green 5N (Molecular Probes). The excitation
wavelength was 506 nm, and fluorescence was detected through a 530-nm cut-off
filter. All data were collected on a logarithmic time base and analyzed by
fitting to exponential functions using Origin, version 5.0 (Microcal
Software). Second order rate constants (kon) for
Ca2+ and Mg2+ binding to ELC and
RD were also calculated using the equation,
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Steady-state Fluorescence StudiesSteady-state fluorescence of labeled ELCs was measured at 22 °C in an assay buffer containing 50 mM NaCl, 20 mM MOPS/NaOH, pH 7.2, 0.1 mM DTT using a Spex Fluoromax spectrofluorometer. The samples were excited at 336 nm, and the fluorescence spectra were collected between 450 and 520 nm. Intrinsic Trp fluorescence of Physarum RD was measured in the same assay buffer, samples were excited at 297 nm, and fluorescence spectra were collected between 310 and 370 nm. The desired free Ca2+ (Mg2+) concentrations were adjusted by adding 0.1 mM EGTA (EDTA) and the appropriate amount of CaCl2 (MgCl2), calculated by the program ALEX (a gift of Dr. M. Vivaudou).
Limited Proteolysis of RDRD (0.5 mg/ml) was incubated at 20 °C with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated bovine trypsin (Sigma-Aldrich) at a ratio of 1:100 (w/w) in a buffer containing 0.1 M NaCl, 20 mM MOPS/NaOH, pH 7.0, 1 mM MgCl2, and 0.1 mM EGTA. The samples were digested in the presence and absence of 0.2 mM CaCl2 for 030 min. At predetermined times aliquots were removed, and 1 mM phenylmethylsulfonyl fluoride was added. The fractions were immediately boiled in SDS sample buffer and separated by 15% SDS-PAGE.
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RESULTS |
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Expression and Purification of Recombinant Light Chains and Regulatory DomainThe wild-type and mutant ELCs, the C- and N-terminal domains of ELC, RLC, and Dictyostelium ELC, were expressed in E. coli to investigate their Ca2+-binding properties. Recombinant Physarum myosin RD was produced by co-expressing ELC, RLC, and the HC fragment (residues 771841) in E. coli (see "Experimental Procedures"). The RLC and ELC (mutant or wild type) co-purified with the HC fragment containing a His6 affinity tag (see Fig. 6, 0 min), suggesting that the three polypeptide chains can assemble into RD in the bacterial cell and that mutations of ELC do not affect binding of the light chains to the heavy chain fragment. The average yields of RDs and LCs were 1520 and 3545 mg of protein/liter of culture media, respectively.
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Localization and Characterization of the Ca2+-binding Site by Direct Binding AssayCa2+ binding measurements are summarized in Figs. 1, 2, 3, and macroscopic dissociation constants, calculated by the Adair-Klotz equation (see "Experimental Procedures"), are summarized in Table II. The wild-type ELC and RD bind one Ca2+ (mol/mol). The RD showed considerably higher Ca2+ affinity than those of ELC; however, the divalent cation-binding site is not highly specific to Ca2+ in either molecule (Fig. 1). Examination of recombinant Dictyostelium ELC by flow dialysis also indicated the presence of one divalent cation-binding site, whereas Physarum RLC did not show any metal binding (Fig. 2). Binding studies of the two separate lobes of Physarum ELC revealed that the Ca2+-binding site is located in the N-terminal domain (Fig. 2). In accordance with these results, the mutants E97A ELC and S124A/D126A ELC (EF-hand III and IV mutants, respectively) show similar calcium-binding capacity to the wild type ELC. Only partial loss of Ca2+ binding was observed in the mutant E26A ELC (EF-hand I mutant), although the glutamic acid in the z position of a canonical metal binding loop normally provides bidentate coordination for Ca2+. Introduction of two additional mutations in the first and third position of loop I (D15A/D17A/E26A ELC) completely abolished Ca2+ binding to both ELC and RD, indicating that EF-hand I of ELC is the only binding site for divalent cations in Physarum myosin (Fig. 3). Ca2+-binding studies of myosin by equilibrium dialysis showed that Physarum myosin binds 2 mol of Ca2+/mol of protein non-cooperatively with a macroscopic dissociation constant of 4.4 µM in the presence of 2 mM MgCl2 (Fig. 1, inset).
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Transient Kinetics of Divalent Cation DissociationBecause
the metal-binding site in the ELC of Physarum myosin has moderate
selectivity for Ca2+ (see
Fig. 1 and
Table II) it could be occupied
by Mg2+ under physiological conditions. To assess
whether bound Mg2+ could be exchanged at a
physiologically relevant time scale upon increase in
Ca2+ concentration, dissociation rates of the two ions
from ELC and RD were measured by stopped flow fluorimetry. The results are
summarized in Table III and
Fig. 4. The divalent
cation-sensitive fluorescence of IAEDANS-labeled ELC was used to determine the
Ca2+ and Mg2+ dissociation rates
of ELC. Wild-type and mutant ELCs containing a single cysteine residue
(Cys-10) in the A helix of EF-hand I were stoichiometrically labeled with
1,5-IAEDANS. Ca2+- or
Mg2+-saturated IAEDANS-labeled light chains were mixed
with an excess of EDTA. The association of divalent cations with EDTA is fast
enough (kon of EDTA 4.5 x 106
M1s1 for
Ca2+
(31)) that the rate-limiting
steps in these experiments were the dissociation of
Ca2+/Mg2+ from ELC with a
koff value of 671.1 s1 for
Ca2+ and with a nearly 40-fold lower value for
Mg2+ (koff = 18.1
s1). Ca2+ off-rate of RD
was determined by mixing the Ca2+-saturated RD with
excess of the calcium indicator Quin 2. The association of
Ca2+ with Quin 2 is a fast, diffusion controlled process
with a kon
109
M1s1
(32); therefore, the
rate-limiting step was Ca2+ dissociation from the RD.
The observed koff value of 2.03
s1 for Ca2+ dissociation
from the RD is similar to the Ca2+ off-rates of a
(Ca2+)4·CaM·target peptide
complex (33).
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The koff value for Mg2+ was obtained by measuring the rate of chasing of Mg2+ with Ca2+ from the RD. The change in Ca2+ concentration was followed with the low affinity calcium indicator, Calcium Green 5N, suitable for fast kinetic studies (koff = 104 s1 for Ca2+ (31)). Mg2+ dissociates from the Mg2+·RD complex with a value of 1.08 s1, which is nearly 20-fold faster than koff of Mg2+ from the divalent metal-binding site of RLC in skeletal muscle myosin S1 (34). Second order rate constants (kon) for Ca2+ and Mg2+ binding to ELC and RD were also calculated from the dissociation rate constants and macroscopic dissociation constants, determined by flow dialysis method (see "Experimental Procedures"). Mg2+ binding to both ELC and RD is 1020-fold slower process compared with the rate of association of Ca2+ with ELC and RD, respectively (Table III).
Steady-state Fluorescence StudiesTo examine the conformational changes induced by divalent cation binding, steady-state fluorescence studies were performed on both ELC and RD. The microenvironment of the 1,5-IAEDANS-labeled cysteine in wild-type ELC is markedly influenced by metal binding, monitored by changes of AEDANS fluorescence (Fig. 5). The fluorescence titration curve has similar shape as the plot of direct Ca2+ binding. The addition of Ca2+ or Mg2+ to the labeled D15A/D17A/E26A ELC did not affect the fluorescence. EF-hand III and IV mutant ELCs (E97A ELC and S124A/D126A ELC) had a similar fluorescence profile to the wild type upon increasing the Ca2+ level (not shown). Ca2+-induced conformational change of RD was also detected by intrinsic Trp fluorescence measurements. The four Trp residues of Physarum RD are located in the heavy chain fragment; three of them are clustered at the RLC binding region, whereas the fourth Trp is in the first IQ motif, which binds ELC. The observed 5.4% change in fluorescence is likely due to the latter. Mg2+ binding induces a smaller fluorescence change in ELC (or RD) (Fig. 5.); however, this cation does not have an inhibitory effect on the activity of Physarum myosin (5).
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Limited ProteolysisLimited proteolysis of RD with trypsin was performed in the presence and absence of Ca2+ (Fig. 6). As the time course of the digestions shows, the first event was the cleavage of the HC fragment of RD, presumably just after the His6 tag, where a thrombin cleavage site is found, followed by the further fragmentation of the HC. Subsequently, the cleavage of ELC and then that of RLC was observed. The cleavage of ELC, similar to the fragmentation of HC, was slower in the presence of Ca2+, demonstrating the diminished accessibility of trypsin to the internal cleavage sites as a consequence of Ca2+ binding.
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DISCUSSION |
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Physarum ELC and RD bind Ca2+ with a
macroscopic dissociation constant of 20 and 7.3 µM, respectively
(Table II).
Ca2+ has three times higher affinity to RD than to the
isolated ELC, which could be explained by stabilizing interactions of the
binding EF-hand with the other two chains of the RD complex. Interestingly,
the Mg2+ affinity of RD is approximately the same as
that of the isolated ELC. In the case of scallop myosin,
Ca2+ binds with high affinity and selectivity to the
EF-hand I of ELC only in the three-chain complex of RD
(38). Physarum myosin
shows an 50-fold higher Ca2+ affinity over RD in
the presence of 2 mM MgCl2 (the apparent dissociation
constant of Ca2+ is 4.4 µM; see
Table II and
Fig. 1, inset),
indicating that the divalent cation-binding site has considerably higher
Ca2+ discrimination in the full-size myosin molecule
(Kd of Mg2+ is greater than
1 mM in myosin). Thus, not only the interchain interactions within
the RD but also the presence of interhead and/or motor domain-ELC interactions
could increase the Ca2+ selectivity of the binding site.
The Ca2+ affinity of Physarum myosin is in the
same range as the average Ca2+ affinity to the EF-hands
of CaM (39). The
interplasmodial concentration of Ca2+ and
Mg2+ is in the micromolar range and
1
mM, as determined by injecting aequorin into plasmodia
(40) and by an NMR technique
(41), respectively. Therefore,
it seems likely that in vivo the binding site of myosin is at least
partially saturated by Mg2+. It is worth noting that CaM
does not have an absolute selectivity for Ca2+ either.
Mg2+ was shown to compete with
Ca2+ at or near physiological conditions; however,
Mg2+ binds to the EF-hands of CaM without inducing the
conformational changes responsible for its biological functions
(4244).
Regarding the physiological relevance of the competitive binding of the two
divalent cations to Physarum myosin, it was important to investigate
the exchange rate between Ca2+ and
Mg2+. As determined by transient kinetic measurements,
the calcium off-rate of ELC and RD is 671.1 and 2.03
s1, respectively. Similar dissociation rates were
observed in the case of CaM and CaM-target peptide complexes
(33). The estimated
Ca2+ off-rate of scallop RD (25
s1) is faster compared with that of
Physarum RD (45).
However, the difference is only 10-fold, and it should be taken into account
that the cytoplasmic streaming driven by Physarum myosin is a much
slower process than contraction of scallop adductor muscle. The replacement of
Mg2+ by Ca2+ at the potential
regulatory site of ELC on the increase of intracellular
Ca2+ concentration is limited by the rate of
Mg2+ dissociation, a value of 1.08
s1. Apparently, this
Mg2+-Ca2+ exchange rate is
considerable faster than that observed at the nonspecific divalent
cation-binding sites of scallop and chicken skeletal muscle RLC (0.05 and
0.057 s1, respectively
(34)) and also faster than the
change in direction of cytoplasmic streaming (several seconds
(12)); therefore, we conclude
that the Ca2+ binding to Physarum ELC could act
as a regulatory signal despite its limited selectivity.
The fact that direct Ca2+ binding to Physarum myosin inhibits its activity is unique among conventional myosins, but a similar type of regulation exists in many unconventional myosins where usually CaM is the light chain subunit. By increasing the Ca2+ concentration, CaMs may dissociate from some unconventional myosins, but others are inhibited without any loss of CaM (4649). In case of myosin IC, the C-terminal lobe of CaM was found to be responsible for inhibition of motor activity (8). The molecular mechanism of Ca2+ regulation must be different in the Physarum conventional myosin, where the inhibitory binding site is located in the N-terminal lobe of ELC. This is the first known example where a canonical EF-hand in the N-terminal domain of a light chain or CaM is involved in the inhibitory regulation of myosin activity.
Models of apo-CaM-myosin IQ complexes show that the N-terminal domain of CaM is in a closed conformation (50, 51). Based on the above structural models and the high sequence similarity of the Physarum myosin LCs to CaM we assume that the N-terminal domain of Physarum ELC is also in a closed conformation in the absence of Ca2+. Moreover, our results suggest that the site remains in the closed state upon Ca2+ binding since the 12th residue of the binding loop (z position), in contrast to the standard EF-hands, does not take part in metal coordination; a replacement of the z position Glu to Ala did not abolish Ca2+ binding, i.e. the characteristic closed-to-open transition of canonical EF-hands probably does not occur in the N-terminal lobe of Physarum ELC. Structural models of scallop RD show that the N-terminal domain of ELC is also in a closed state both in the Ca2+-bound and the Ca2+-free form (10, 11).2 Despite the opposite effect of Ca2+ binding on the motor activity of scallop and Physarum conventional myosins (activation versus inhibition) and the relatively low sequence similarities of the two Ca2+-binding ELCs, the two myosin motors have a common structural basis for Ca2+ regulation; that is, a closed N-terminal lobe of ELC both in the absence and presence of Ca2+. Nevertheless, the mechanism underlying Ca2+ regulation in the two conventional myosins is strikingly different; the scallop myosin RD acts as a bona fide on/off Ca2+ switch, whereas the Ca2+ binding to Physarum RD has only a modulatory effect (partial inhibition (5)) on the motor activity.
Steady-state fluorescence and limited proteolysis studies indicate that Ca2+ binding affects both the microenvironment of the binding site (as shown by fluorescence studies) and the overall structure of the RD (as shown by the decreased accessibility of tryptic sites of RD in the presence of Ca2+). The latter results could be interpreted as a decrease in the internal mobility of the RD and/or changes in the interactions between and/or within the three chains of the regulatory complex. Preliminary results using recombinant myosin fragments indicate that Ca2+ inhibition of in vitro motility is achieved only by a double-headed heavy meromyosin construct, whereas the single-headed subfragment-1 is not regulated,3 suggesting that head-head and/or head-rod interactions could contribute to the inhibitory state. In case of the Ca2+- and phosphorylation-activated conventional myosins (scallop and smooth muscle myosins, respectively), head-head and head-rod interactions stabilize the motor protein in the low activity off state, which is an asymmetric structure where the two heads interact and immobilize each other (52, 53).
This work provides evidence that the inhibitory Ca2+-binding site of Physarum myosin is located in the first EF-hand of ELC. Our results indicate that Ca2+ and Mg2+ compete for the binding site. However, the exchange of divalent cations is fast enough compared with the time scale required to regulate the cytoplasmic streaming; consequently, we conclude that the inhibitory system could operate in the plasmodia of Physarum myosin. We have recently obtained crystals of Physarum myosin RD, and its high resolution structure is currently being refined.4 The structure confirms the localization of the Ca2+-binding site in the first EF-hand motif of ELC and also verifies the surprising finding of this work that the Ca2+-saturated N-terminal lobe of ELC is in a closed conformation. Detailed analysis of the Physarum myosin RD structure could shed more light of this unique Ca2+ inhibitory mechanism, and its comparison with the scallop regulatory switch could deepen our general understanding of the Ca2+ regulation of conventional myosins.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Biochemistry, Eötvös Loránd University, Budapest, PázmányP.sétány 1/C, H-1117. Tel.: 361-381-2171; Fax: 361-381-2172; E-mail: nyitray{at}cerberus.elte.hu.
1 The abbreviations used are: RLC, regulatory light chain; ELC, essential
light chain; CaM, calmodulin; RD, regulatory domain; HC, heavy chain; E26A
ELC, Physarum ELC containing point mutation E26A; D15A/D17A/E26A ELC,
Physarum ELC containing point mutations D15A, D17A, and E26A; E97A
ELC, Physarum ELC containing point mutation E97A; S124A/D126A ELC,
Physarum ELC containing point mutations S124A/D126A; 1,5-IAEDANS,
5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid;
koff, dissociation rates constant;
kon, second order rate constant for divalent cation
binding; MOPS, 3-morpholinopropanesulfonic acid; DTT, dithiothreitol.
2 C. Cohen, personal communication.
3 H. Kawamichi, A. Nakamura, L. Nyitray, and K. Kohama, unpublished
results.
4 J. Debreczeni, L. Farkas, K. Kohama, and L. Nyitray, unpublished
results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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