 |
INTRODUCTION |
Descriptions of the molecular mechanism of muscle function tend to
focus on the dynamic properties of the myosin motor or the steric shift
of the tropomyosin/troponin
(Tm1·Tn) complex on actin.
Actin appears to take the role of a static, rigid lattice upon which
the other muscle proteins shift in order to produce and regulate muscle
movement. However, with numerous studies showing the dynamic nature of
filamentous actin (F-actin), this may not, in fact, be the case.
F-actin, depending on the ligands bound and/or the stage in the
cross-bridge cycle, becomes more or less flexible (2, 3), twists on its
axis (4, 5), and goes through marked structural changes at the C
terminus (6-8), and in subdomain 2 (3, 9, 10). Molecular modeling and
biochemical studies of F-actin indicate that the C terminus and
subdomain 2 are part of the intermolecular interface (11-20). There is
also evidence, both modeling (21) and biochemical (22, 23), indicating
direct or indirect interactions between subdomain 2 and the myosin head
(S1). Modifications of the C-terminal Cys374 are also known
to cause reagent-dependent changes in the actomyosin ATPase
activity and the in vitro motility of actin filaments (7). Thus, there are certainly strong indications that the dynamic properties of actin in general, and of subdomain 2 in particular, may
play important roles in actomyosin interactions.
The position of Tm on F-actin has been modeled to a high degree of
resolution (13, 24-27). Residues likely involved in Tm-F-actin interactions, as suggested by these models, are all located in subdomains 3 and 4. While subdomain 2 does not seem to be involved in
the Tm-F-actin interaction, an interesting biochemical study by
Bonafé et al. (28) implicates (either through direct
or indirect effects) the DNase I loop of subdomain 2 in such an
interaction. In this study the presence of Tm and the Tm·Tn complex
interfered with the cross-linking of Lys50 of actin to the
central 48 kDa of the S1 heavy chain. This inhibition of cross-linking
indicates Tm- and Tm/Tn-induced changes in either the reactivities of,
and/or the distances between, the cross-linking sites on actin and S1.
A more explicit suggestion of subdomain 2 movement during actin
regulation was advanced by Squire and Morris (1) in their review on
muscle thin filament regulation. According to their hypothesis, the
structural changes seen in electron microscopy and x-ray diffraction
studies of regulated thin filaments may be due not only to shifts of
the Tm·Tn complex, but also to subdomain movements within each actin
protomer. This hypothesis, together with the results of Bonafé
et al. (28), prompted us to examine the role of subdomain 2 movements in the regulatory process.
To study the role of subdomain 2 in the Tm/Tn regulation of actomyosin,
we attached fluorescent probes (1,5-IAEDANS or pyrene maleimide) to
three fully functional mutant yeast actins, Q41C/C374S, D51C/C374S, and
Q41C at positions 41 or 51, and in the case of Q41C, also at position
374 (Fig. 1). The first two mutants have the reactive
Cys374 replaced by Ser and new reactive cysteine residues
replacing Gln41 and Asp51, respectively. The
third mutant retains the reactive Cys374 while adding an
additional reactive cysteine in place of Gln41. Using these
labeled mutants, we monitored their fluorescence by means of direct
excitation, energy transfer, and, in the case of the doubly labeled
Q41C, excimer fluorescence during the various regulatory
steps. We also used Gln41/ANP (azidonitrophenyl
putrescine)-labeled
-skeletal actin (both cross-linked and
uncross-linked) in conjunction with rhodamine phalloidin, and in this
way, measured the quenching of rhodamine fluorescence by ANP during
regulation. Also, using the cross-linked species, we were able to
investigate the regulatory properties of F-actin with an immobilized
subdomain 2.
Our results show that there are no significant changes in the probe
environment, nor in the relative position of subdomain 2, in the course
of Ca2+ regulation by Tm/Tn. In addition, we found that
despite intrastrand cross-linking, the ANP-labeled
-skeletal actin
remained fully regulated, indicating that a mobile subdomain 2 is not a
necessary component in actomyosin regulation.
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MATERIALS AND METHODS |
Reagents--
ATP, ADP, dextrose, DTT, phalloidin, and
phenylmethylsulfonyl fluoride were purchased from Sigma.
5-(2((Iodoacetyl) amino)ethyl)aminonaphthalene-1-sulfonate (1,5-IAEDANS) and N-(1-pyrene)maleimide were purchased from
Molecular Probes, Inc. (Eugene, OR). The
QuikChangeTM site-directed mutagenesis kit, DNA restriction
enzymes, and plasmid purification kit were purchased from Stratagene
(La Jolla, CA), New England Biolabs (Beverly, MA), and Qiagen
(Valencia, CA), respectively. Yeast extract and tryptone were purchased
from Difco (Detroit, MI). DNase I was purchased from Worthington
Biochemical Co. (Lakewood, NJ).
N-(4-Azido-2-nitrophenyl)-putrescine (ANP) was a generous
gift from Dr. G. Hegyi.
Oligonucleotide-directed Mutagenesis--
The mutagenesis was
performed according to the Stratagene (La Jolla, CA)
QuikChangeTM procedure using oligonucleotides purchased
from Genset Corp. (San Diego, CA). As a template for the mutagenesis we
used double stranded yeast shuttle plasmid pTD24 (29), which carries a
copy of the ACT1 gene. Two oligonucleotide primers,
5'-GGGTCAAAAAtgtTCCTACGTTGG-3' and
5'-CCAACGTAGGAacaTTTTTGACCC-3', were used to introduce the D51C actin mutation. Two primers,
5'-CAGAGATTAGAAgcttTTGTGGTGAAC-3' and
5'-GTTCACCACAAaagcTTCTAATCTCTG-3', were used to generate
the C374S actin mutation. The mutated sequences are shown in lowercase and the selective restriction enzyme markers are underlined (in D51C
the PleI site is lost while in C374S the HindIII
is added). Actin genes from screened plasmid clones were
sequenced (GeneMed, San Francisco, CA) to confirm the absence
of random errors. The construction of the Q41C and Q41C/C374S mutants
was reported in a previous publication (20).
Yeast Transformation--
Mutant plasmids carrying the HIS3
marker were transformed into the diploid yeast strain TDS101 (29) that
has a single ACT1 gene copy on the plasmid with a URA3
marker and genomic copies of the ACT1 gene deleted. The
original plasmid carrying the ACT1 gene was sorted out by
plasmid shuffling (30). The cells were screened for HIS plus and URA
minus phenotype, which resulted in yeast cells carrying a homozygous
copy of the mutated actin gene.
Proteins--
Skeletal myosin and actin were prepared from
rabbit back muscle according to Godfrey and Harrington (31) and Spudich
and Watt (32), respectively. S1 and heavy meromyosin were prepared from
myosin using the protocols of Weeds and Pope (33) and Kron et
al. (34), respectively. The cardiac troponin and tropomyosin were
generous gifts from Dr. L. Tobacman. Ca2+-independent
bacterial transglutaminase was a generous gift of Dr. K. Seguro,
Ajimoto Co., Inc. Japan. Yeast actins were purified over a DNase I
affinity column (35). To avoid possible contamination of yeast actin
with cofilin, the DNase I column was washed with 1.0 M NaCl
in G-actin buffer (5 mM TES, 0.2 mM
CaCl2, 0.2 mM ATP, 1.0 mM DTT, pH
7.5) (36) prior to the elution of yeast actin. After elution, the
purified actins were stored on ice in G-actin buffer.
The labeling of both Q41C/C374S and D51C/C374S actin mutants with
1,5-IAEDANS was done in the following manner. After elution from DNase
I column, actin was exchanged into a DTT-free G-actin buffer using
Sephadex G-50 spin columns. A 1.3-fold molar excess of 1,5-IAEDANS
dissolved in N,N-dimethylformamide was added to the actin and the airspace was then purged with N2. The
reaction, which was allowed to proceed overnight on ice, was stopped
with an excess of DTT. The labeled actin was further purified via a polymerization-depolymerization cycle. An extinction coefficient of
5700 M
1 cm
1 was used to
determine the degree of labeling, which was generally in excess of
90%. The labeling of Q41C, which has two reactive cysteines,
Cys41 and Cys374, with pyrene maleimide was
performed according to a previously described protocol (37). The extent
of labeling was ~1.8 pyrene maleimide/actin.
The ANP-labeled and uncross-linked skeletal actin was prepared using a
procedure based on that of Hegyi et al. (18). 1.0 mg/ml
G-actin in 5.0 mM Tris-HCl, 0.2 mM
CaCl2, and 0.4 mM ATP, pH 8.0, was incubated
with 0.1 mM ANP and 0.4 unit/ml bacterial transglutaminase
at room temperature, in the dark, for 2 h. This solution was
placed under a stream of N2 for 7 min to remove the dissolved O2. To quench the attached ANP and prevent a
cross-linking reaction, 2.0 mM DTT was added to actin prior
to its UV irradiation with a Model C-62 UV Illuminator (Ultra-Violet
Products Inc., San Gabriel, CA) under a constant N2 stream
(for 7 min). The labeled sample was polymerized by the addition of 2.0 mM MgCl2. The resulting F-actin was pelleted by
centrifugation at 40,000 rpm in a Beckman Ti 70 rotor, incubated on ice
for 2 h, and homogenized in 5 mM Tris-HCl, pH 8.0, 2 mM MgCl2, and 0.2 mM ATP. The
degree of actin labeling was 98 ± 5%. The preparation of
ANP-labeled and cross-linked F-actin proceeded similarly. G-actin was
labeled by incubating with ANP and bacterial transglutaminase and was
immediately polymerized by 2.0 mM MgCl2. The
labeled F-actin was pelleted and homogenized in 5.0 mM
Tris-HCl, pH 8.0, 2 mM MgCl2, and 0.2 mM ATP. After homogenization it was placed under
N2 stream and then irradiated by UV light as described
above, except for the absence of DTT. The level of cross-linking
achieved was ~85%.
Regulated Actin-activated ATPase--
The rates of S1 Mg-ATPase
activated by regulated actin ± Ca2+ were obtained as
described previously (38) using light scattering to monitor the
clearing time of regulated F-acto-S1 solutions. Thin
filaments were reconstituted using bovine cardiac troponin, and bovine
cardiac tropomyosin and each of the following actins: wild type,
Q41C/C374S, Q41CAEDANS/C374S, D51C/C374S,
D51CAEDANS/C374S, Q41C, Q41Cpyrene, and
skeletal
-actin ANP. The concentrations of actin, Tm, Tn, and S1
were 4.0, 2.0, 1.0, and 1.0 µM, respectively. Experiments
were carried out at 23 °C using a Mg-ATP concentration of 0.1 mM and either 1.0 mM EGTA or 0.2 mM
CaCl2. The course of Mg-ATP hydrolysis was monitored by
measuring the light scattering at 350 nm from the above solutions (38)
in a Spex Fluorolog (Spex Industries Inc., Edison, NJ).
Actin Polymerization--
Polymerization of the G-actins (4.0 µM) by MgCl2 (3.0 mM) was
monitored by measuring light scattering from actin solutions in a Spex
Fluorolog set at 325 nm.
Fluorescence Measurements--
Fluorescence emissions spectra
for the labeled actins were recorded at 23 °C in Spex Fluorolog
using excitation wavelengths of 338 nm for direct excitation of the
AEDANS probe and 295 nm for measurements of energy transfer from
tryptophan residues to the probes. For monitoring the pyrene excimer
formed in Q41Cpyrene F-actin (37), the excitation
wavelength was set at 344 nm. For measurements of rhodamine quenching
in ANP-labeled actin, an excitation wavelength of 554 nm was used.
Acrylamide quenching measurements of AEDANS-labeled actins were carried
out using an excitation wavelength of 338 nm and an emission wavelength
of 492 nm. Acrylamide was added in 10 mM increments and the
data were fitted to the Stern-Volmer equation (39) in order to
determine the quenching constant KSV. The
concentrations of actin, Tm, Tn, and S1 used in fluorescence
experiments were 4.0, 2.0, 1.0, and 1.0 µM, respectively, unless stated otherwise. Yeast actin was stabilized by equimolar amounts of phalloidin.
In Vitro Motility Assays--
The in vitro motility
assays were performed according to a previously described protocol
(29). Movement was initiated by applying an assay buffer (25 mM KCl, 1.0 mM EGTA, 5.0 mM
MgCl2, 10 mM DTT, 0.2% methylcellulose, 10 mM imidazole, pH 7.4) containing 1.0 mM ATP and
an oxygen scavenging system (40). An ExpertVision System (Motion
Analysis, Santa Rosa, CA) was used to quantify the sliding speeds of
individual filaments. Individual filaments were judged to be moving
smoothly and were used for statistical analysis if the standard
deviation of their sliding speeds was less than one-third of their
average velocity (41).
 |
RESULTS |
The Actins and Their Function--
The flexibility of subdomain 2 in actin has been demonstrated by a number of studies (3, 9, 10) and
there is evidence indicating that Tm has a sufficient influence on
either this subunit or on the S1 bound to actin to abrogate their
cross-linking (28). To test for possible changes in the conformational
states of subdomain 2 due to regulatory proteins and the regulation of
actin, we employed three fluorescently labeled actin
mutants of subdomain 2 and
Gln41-labeled skeletal
-actin (Table
I, Fig.
1). All the polymerized actins used in
this study, whether labeled or unlabeled, had fully regulated acto-S1
ATPase activities when complexed with Tm/Tn. Ca2+-induced
activation of acto-S1 ATPase, as measured by the ratio of MgATPase
activities in the presence of 0.2 mM Ca2+ and
1.0 mM EGTA, ranged between 10- and 16-fold for these
actins (Table I), except for the double labeled Q41C actin, which
showed 5-fold activation (Table I). The courses of
Mg2+-induced polymerization of the actins, both labeled and
unlabeled, were similar, as were the speeds of the yeast actin
filaments in the in vitro motility assays (3.0 ± 0.3 µm/s). Previous work showed that ANP labeling of
-actin at
Gln41 had no significant effect on its in vitro
motility, while the intermolecular cross-linking by this reagent
between residues Gln41 and Cys374 strongly
impaired in vitro motility (42).

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Fig. 1.
A, two protomers from the
F-actin model of Holmes et al. (48) showing the locations of
residues 41 (red), 51 (blue), and 374 (black), all of which were mutation sites in the yeast
actins. The turquoise residues represent, from
left to right, Trp340 and
Trp356, while the violet residues represent
Trp86 and Trp79 (left to
right). B, enlarged diagram showing
the spatial relationship between tryptophan residues 340 and 356 (turquoise) and 86 and 79 (violet), and the
locations on yeast actin where AEDANS probes were attached (residues 41 (red) and 51 (blue)). The distances (Å) of the closest
tryptophans (79 and 86 on the same protomer and 340 and 356 on the
adjacent protomer) to residue 41 are shown as examples.
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Emission Spectra of Unregulated Q41CAEDANS/C374S and
D51CAEDANS/C374S--
We monitored the fluorescence of the
two AEDANS-labeled yeast actin mutants with an excitation wavelength of
338 nm (Figs. 2, A and
B). Polymerization of Q41CAEDANS/C374S
Ca2+-G-actin was accompanied by a marked increase in the
fluorescence and a blue shift of ~9 nm (from a
max of
495 nm for G- to a
max of 486 nm for
F-Q41CAEDANS/C374S) (Fig. 2A), indicating the
withdrawal of the probe from a more aqueous to a more hydrophobic
environment. These changes are qualitatively similar to the changes
observed for dansylethylenediamine attached to Gln41 in
-actin (43, 44).

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Fig. 2.
Emission spectra of AEDANS-labeled
Q41C/C374S and D51C/C374S mutant yeast actins. The spectra (in
arbitrary units) were recorded at 23 °C using an excitation
wavelength of 338 nm. The concentrations of actin, Tm, and Tn were 4.0, 2.0, and 1.0 µM, respectively. Ca2+ and EGTA
(for the Ca2+ state) concentrations were 0.2 and 1.0 mM, respectively. A, the emission spectrum of
Q41CAEDANS/C374S Ca2+-G-actin (solid
line) has max of 495 nm. Emission spectra of
F-actin alone and F-actin with Tm or Tm-Tn ± Ca2+
were identical, with a max of 486 nm, and are
represented by a single line (dashed).
B, emission spectra of D51CAEDANS/C374S
Ca2+-G-actin (solid line), F-actin alone and
with Tm (dashed line), and F-actin with Tm-Tn ± Ca2+ (dash-dotted line). The
max for all three plots is 494 nm.
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The polymerization of D51CAEDANS/C374S
Ca2+-G-actin decreased the AEDANS fluorescence
significantly (solid and dashed line spectra in
Fig. 2B) with no discernible shift in
max
(494 nm). This could indicate an extension of the probe into a more
aqueous environment. To test the possibility of shifts to different
environments for probes at Cys41 and Cys51, we
performed fluorescence titration experiments using acrylamide as the
quenching agent. The Stern-Volmer constants for the two Ca2+-G-actins were very similar; 8.4 M
1 for Q41CAEDANS/C374S and 8.5 M
1 for D51CAEDANS/C374S, showing
equal accessibility of the probes at Cys41 and
Cys51 to acrylamide. For the F-actins the
Stern-Volmer constants were different, 4.7 M
1
for Q41CAEDANS/C374S versus 5.9 M
1 for D51CAEDANS/C374S. This
suggests that at both locations AEDANS is buried upon actin
polymerization, albeit to a smaller degree at Cys41 than at
Cys51.
Emission Scans of Regulated
Q41CAEDANS/C374S--
To test for possible subdomain 2 changes that would account for the effect of Tm on S1 cross-linking to
Lys50 on actin (28), we examined the fluorescence of the
probe at Cys41 under regulatory conditions corresponding to
the blocked and closed states of the McKillop and
Geeves (46) three-state model. The sequential additions
of Tm, Tm/Tn + Ca2+ (both resulting in the
predominantly closed state (13, 46)), and Tm/Tn
Ca2+ (blocked state) had virtually
no effect on the fluorescence spectra of F-actin, justifying
their visually simplified representation by a single emission spectrum
in Fig. 2A (dashed line).
Fluorescence Resonance Energy Transfer (FRET) in Unregulated and
Regulated Q41CAEDANS/C374S--
While the shift from the
blocked to closed state may not significantly
impact probe fluorescence, this still does not preclude a shift of
subdomain 2 of actin, relative to the rest of the molecule, during
regulation. We therefore monitored the energy transfer from tryptophan
residues to the AEDANS acceptor on Cys41 (Fig. 1). Based on
the tryptophan substitution study of Doyle et al. (45) that
revealed only small contributions of Trp79 (~1%) and
Trp86 (~11%) to total actin fluorescence, and the
relative proximity to the probe of Trp340 and
Trp356 in same-strand actin protomers adjacent to (above)
the labeled actins (Fig. 1), these last two tryptophan residues appear
to be major energy donors to AEDANS on Cys41 (see
"Discussion"). As shown in Fig.
3A, the presence of AEDANS on
Cys41 led to a decrease in tryptophan fluorescence for both
G-actin and F-actin. Since the decrease in tryptophan emission was much larger in F-actin than in G-actin, polymerization increases energy transfer from the tryptophans to the AEDANS probe. This increase in
energy transfer can be accounted for by the contribution of Trp340 and Trp356 on the adjacent actin
protomer in F-actin.

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Fig. 3.
Emission spectra of AEDANS-labeled and
unlabeled Q41C/C374S actin mutant using an excitation wavelength of 295 nm. The plots show tryptophan fluorescence and, in the case of the
labeled actins, reveal energy transfer from tryptophan residues to the
AEDANS probes positioned in subdomain 2. The concentrations of protein,
Ca2+, and EGTA were the same as those listed in the legend
to Fig. 2. A, spectra of Q41C/C374S and
Q41CAEDANS/C374S Ca2+-G-actin (thin
and thick solid line, respectively) and F-actin alone
(thin and thick dashed line, respectively).
B, spectra of Q41CAEDANS/C374S
F-actin alone and F-actin with Tm (dashed line) and F-actin
with Tm-Tn ± Ca2+ (solid line).
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The addition of Tm had no effect on Q41CAEDANS/C374S
F-actin emission (Fig. 3B). The formation of the
F-actin·Tm·Tn complex increased tryptophan fluorescence (due to the
tryptophan residues on Tn) but had no effect on AEDANS emission, both
in the presence (closed state) and absence
(blocked state) of Ca2+ (Fig. 3B).
This evidence implies that not only does the creation of the
closed state (through the addition of Tm) not change the environment of the AEDANS probe, but the regulatory shift from closed to blocked states is also not accompanied
by probe movements relative to the tryptophan residues in the adjacent actin.
Excimer Fluorescence Measurements in Unregulated and Regulated
Q41CPYRENE--
As a means of further investigating the
possibility of dynamic transitions at the subdomain 2/1 interface in
F-actin, we used the yeast actin mutant Q41C (that has two reactive
cysteine residues at positions 41 and 374) labeled with pyrene (Fig.
1). Upon polymerization, the pyrene label at Cys41 forms an
excimer with the label at Cys374 of the next monomer in the
strand (37). Movements of subdomain 2 relative to the C terminus of the
following monomer would abolish or change the excimer and thus signal
changes in subdomain 2 position in the closed
versus blocked regulatory states. As expected
(37), the spectrum of pyrene-labeled Q41C G-actin did not show an
excimer band (Fig. 4). The F-actin
spectrum, however, had a decreased pyrene peak and an excimer band at
max = 476 nm (Fig. 4). The addition of regulatory
proteins had no effect on the excimer peak, indicating no changes in
the position of the stacked pyrene probes (Fig. 4). This is further
evidence for the seemingly static nature of actin subdomain 2 in the
transition from the blocked to closed regulatory
states.

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Fig. 4.
Pyrene emission spectra of the labeled
yeast actin mutant Q41C. Spectra for Ca2+-G-actin
(solid line), and for F-actin alone and F-actin with Tm or
with Tm-Tn ± Ca2+ (dashed line) were
recorded using an excitation wavelength of 344 nm. All concentrations
were the same as those listed in the legend to Fig. 2.
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Emission Scans of Regulated D51CAEDANS/C374S--
The
second subdomain 2 mutant used in this work, D51C/C374S, gave us an
additional point of reference for observing possible subdomain 2 movements during actin regulation. Furthermore, it allowed us to
monitor such movements in the vicinity of a residue (Lys50)
involved in a cross-linking to S1 that is inhibited by Tm (28). The
addition of Tm (i.e. creation of the closed
state) had no effect on the AEDANS fluorescence of F-actin (Fig.
2B, dashed line represents both F-actin and
F-actin + Tm). Interestingly, the addition of Tn (leading to the
formation of the F-actin·Tm·Tn complex) increased fluorescence to a
point between G- and F-actin where it remained, unaffected by any shift
from the closed to the blocked state
(i.e. the presence or absence of Ca2+) (Fig.
2B, dashed dotted line corresponds to both states). Thus, even at a location immediately adjacent to Lys50, we did
not detect any regulation-induced changes in the probe's environment.
Fluorescence Energy Transfer in Unregulated and Regulated
D51CAEDANS/C374S--
As with
Q41CAEDANS/C374S, we measured FRET from tryptophan residues
to the AEDANS probe, which in this case was attached at residue
Cys51 (Fig. 1). Here too, the decrease in tryptophan
emission in the presence of the AEDANS acceptor was larger in F-actin
than in G-actin (5A). This polymerization related increase in energy
transfer from the tryptophans to the AEDANS probe is most likely due to the Trp340 and Trp356 donors on the adjacent
protomers in F-actin.
As was the case when the AEDANS probe was directly excited, the
addition of Tm had no effect on the stimulated fluorescence, while the
formation of the F-actin·Tm·Tn complex increased both tryptophan
and AEDANS fluorescence without any state dependent (Ca2+-related) changes in the spectra (Fig.
5B). Thus, we did not detect any "closed state"-induced changes in energy transfer
efficiency, nor any movements due to shifts between the
closed and blocked states of actin.

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Fig. 5.
Emission spectra of AEDANS-labeled and
unlabeled D51C/C374S actin mutant using an excitation wavelength of 295 nm. The plots show tryptophan fluorescence and, in the case of the
labeled actins, reveal energy transfer from tryptophan residues to the
AEDANS probes positioned in subdomain 2. All concentrations were the
same as those listed in the legend to Fig. 2. A, spectra of
D51C/C374S and D51CAEDANS/C374S G-actin (thin
and thick solid line, respectively), and D51C/C374S and
D51CAEDANS/C374S F-actin alone (thin and
thick dashed line, respectively). B, spectra of
D51CAEDANS/C374S F-actin alone and F-actin with Tm
(dashed line) or with Tm-Tn ± Ca2+
(solid line).
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Quenching of Rhodamine Fluorescence in Unregulated and Regulated
ANP-labeled Skeletal
-actin--
Finally, we examined one more
system that provided an additional point of reference regarding
possible subdomain 2 movements, and also enabled us to evaluate all
three regulatory states: blocked, closed, and open (the tryptophans of S1, which is
required for the shift to the open state, would have
interfered with the probes used in the previous experiments). We
labeled Gln41 on skeletal
-actin with ANP, which
is an excellent acceptor of energy from rhodamine (Fig.
6). The quenching of rhodamine phalloidin
fluorescence by ANP allowed us to monitor the possible distance changes
between this probe and ANP on Gln41.

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Fig. 6.
Fluorescence spectra
( Excitation = 554 nm) of
rhodamine phalloidin (2.5 µM)
complexed with: 4.0 µM skeletal
F-actin (solid line), ANP-labeled skeletal F-actin
(dashed line), and cross-linked ANP-labeled skeletal
F-actin (dash-dotted line). Inset,
spectral overlap between rhodamine emission spectrum of complex of
skeletal F-actin (4.0 µM) and rhodamine phalloidin (2.5 µM) (solid line) and the absorbance spectrum
of 4.0 µM ANP-labeled skeletal F-actin (dashed
line). Optical density (O.D.) ordinate for ANP
absorbance is on the right side of the graph.
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|
Fig. 6 shows the fluorescence of rhodamine phalloidin bound to
unlabeled actin, labeled/uncross-linked actin, and labeled/cross-linked actin. We collected emission spectra for both uncross-linked and cross-linked ANP-labeled F-actin alone, in the predominantly
closed (+Tm, +Tm/Tn (+Ca2+)) and
blocked (+Tm/Tn (
Ca2+)) regulatory states, and
for the uncross-linked species in the open (+Tm/Tn and S1)
state (Table II). Our results indicate
that there were no significant changes in the quenching of rhodamine fluorescence by ANP in any regulatory state, including that
created in the presence of S1 (the open state). Thus, even
the shift to the open state did not induce sufficient
subdomain 2 movement to change the degree of rhodamine quenching by
ANP.
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Table II
Fluorescence intensities of rhodamine phalloidin-ANP F-actin complexes
Fluorescence intensities of rhodamine phalloidin bound to ANP-labeled
(cross-linked and uncross-linked) F-actin were recorded with the
excitation and emission wavelengths set at 554 and 570 nm,
respectively. Fluorescence data from two separate ANP-labeled actin
preparations are given in arbitrary units (A.U.). Protein
concentrations for actin, Tm, Tn, and S1 were, 4.0, 2.0, 1.0, and 4.0 µM, respectively. Rhodamine phalloidin concentration was
2.5 µM, and the concentrations for Ca2+ and EGTA
(for the Ca2+ condition) were 0.2 and 1.0 mM,
respectively.
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|
Upon exposure to UV radiation the ANP moiety cross-links to
Cys374 on the next actin protomer in the strand,
immobilizing the DNase I loop (18). We found that the ANP cross-linked
-actin remained fully regulated. Heretofore, we have shown that
regulation of actin is not accompanied by discernible subdomain 2 movements. The fact that the cross-linked actin remains fully regulated
shows that such movements, even if present, are neither a necessary nor
an important part of actin regulation.
 |
DISCUSSION |
The purpose of this study was to test the hypothesis that
subdomain 2 movements are an important component of actin regulation by
the Tm·Tn complex. Squire and Morris (1) raised this possibility, suggesting that changes in x-ray diffraction patterns of regulated actin could, in part, be attributed to subdomain 2 movements and not
solely to the shifting of the regulatory proteins. To explore this
possibility, we used labeled skeletal
-actin and yeast actin mutants
whose reactive cysteines on the DNase I loop (Cys41 or
Cys51) were labeled with fluorescent probes. We anticipated
that changes in the environment of these probes and in their distance
from native actin tryptophan residues would be reflected in their
fluorescence and in the fluorescence energy transferred to them. While
such FRET measurements between tryptophans and the AEDANS probes in actin mutants were suitable for monitoring regulatory shifts between the blocked (+Tm-Tn;
Ca2+) and the
closed (+Tm-Tn; +Ca2+) states, they could not be used
to investigate the open state because of the strong
tryptophan fluorescence of S1. By using ANP-labeled skeletal
-actin,
we were able to look for changes in the quenching of rhodamine
phalloidin fluorescence by ANP possibly stemming from shifts between
the closed and open states. In its cross-linked
state, ANP-actin also allowed us to investigate whether subdomain 2 movement is required for the regulation of actin by Tm-Tn.
Functional assays of labeled
-actin and yeast actin mutants
confirmed their suitability for testing regulation-linked changes in
actin structure. All actins polymerized well and were fully regulated
in acto-S1 ATPase experiments (Table I). In vitro motilities were similar for wild type and both labeled and unlabeled mutant actins. A previous study showed that uncross-linked ANP-labeled skeletal
-actin has sliding speeds virtually the same as those of
unlabeled
-actin, while the sliding of cross-linked ANP-actin is
impaired (42).
Emission Spectra of Labeled Yeast Actin Mutants--
As revealed
by our results, adding regulatory proteins to
Q41CAEDANS/C374S produced no significant changes in probe
fluorescence, indicating that whatever effect Tm/Tn have on this
subdomain, it is not discernible in any environmental changes of the
probe. In the case of D51CAEDANS/C374S, addition of Tn to
the F-actin·Tm complex increased the probe fluorescence by ~10%.
Yet, since Tn is added to actin-Tm in the presence of Ca2+,
there should be no shift in Tm position, as both Tm alone and the
Tm·Tn complex (+Ca2+) lie in the closed state
(13, 25). As the presence or absence of Tm does not change the probe
environment, the small effect of Tn on AEDANS fluorescence in this
actin mutant appears unrelated to the state of regulated actin. This
conclusion is confirmed by the lack of fluorescence differences between
the blocked (
Ca2+) and closed
(+Ca2+) regulatory states of this actin, indicating an
absence of regulation-dependent changes in the probes
environment. At this stage, the nature of the Tn-specific and
Ca2+ insensitive perturbation at the Cys51 site
on actin is unclear.
The availability of the actin mutant Q41C with its two reactive
cysteines, Cys41 and Cys374, allowed us to
examine the subdomain 2/1 interface in regulated F-actin. Upon
polymerization this double pyrene-labeled mutant exhibits an excimer,
indicating the stacking of pyrene labels attached to Cys41
and Cys374 on adjacent protomers within the same filament
strand (37). As reported under "Results," neither the addition of
regulatory proteins nor the switch from the blocked to the
closed state had any effect on the excimer fluorescence.
This suggests that there is little, if any, change in the overlap of
pyrene probes, and/or regulation-induced movement at the subdomain 2/1
interface in F-actin.
Fluorescence Energy Transfer Experiments--
We took advantage of
the inherent sensitivity of FRET measurements to distance changes (in
the donor-acceptor pair), and of the now available information on the
relative fluorescence levels of the four actin tryptophans, to monitor
possible subdomain 2 movements due to actin regulation.
According to the Doyle et al. (45) analysis of tryptophan
yeast actin mutants, the contributions of Trp79,
Trp86, Trp340, and Trp356 to actin
fluorescence are 1, 11, 37, and 51%, respectively, while the
R0 value for the tryptophan-AEDANS
donor-acceptor pair is 22 Å (47). These contributions permit the
calculation of FRET efficiency in F-actin using the distances from the
tryptophans to Gln41 and Asp51 derived from the
Holmes et al. (48) (Fig. 1) and Lorenz et al.
(49) models of F-actin. Obviously, such calculations of intra- and
inter-protomer FRET in F-actin are model-dependent and
their predictive value is further reduced by the unknown orientation of
AEDEANS at the Cys41 and Cys51 sites. Despite
these limitations and regardless of the exact partitioning of energy
transfer between the different tryptophan residues, these calculations
provide strong evidence for a major contribution to FRET by the
inter-protomer (on adjacent actins within the same strand) transfer of
energy from Trp340 and Trp356 to AEDANS on
either Cys41 on Cys51. This conclusion is
consistent with the much greater efficiency of FRET in F- than in
G-actin (Figs. 3A and 5A). Thus, changes in
AEDANS fluorescence during excitation at 295 nm (i.e. at the tryptophan excitation band) should monitor, to a large extent, distance
changes between the probe and Trp340 and Trp356
on the adjacent protomer.
Our main results show that the stimulated fluorescence of AEDANS
attached to either Cys41 or Cys51 does not
change upon the binding of Tm to F-actin or upon the switch of the
actin·Tm·Tn complex from the blocked to the
closed state (EGTA/Ca2+ switch). The fact that
acceptor probes located at two different sites in subdomain 2 yield the
same results regarding distance changes during Ca2+
regulation of these well regulated actins suggests that subdomain 2 remains static during this process. Our strategy of probing (by FRET)
distance changes from tryptophan donors to two acceptor sites makes it
unlikely that a movement of subdomain 2 escapes our detection due to
compensating changes in energy transfer. Moreover, the absence of any
perturbation of the pyrene excimer band in the course of regulatory
transitions in Q41C yeast actin adds credence to the static picture of
subdomain 2.
ANP-labeled Skeletal
-Actin--
Our experiments involving the
yeast actin mutants were limited to evaluating two states of the
"three-state model" of actin regulation (46); the
blocked and closed states. The transition to the
open state requires the presence of S1, which would
introduce additional energy donors to the environment of the AEDANS
probes on actin. We were able to surmount this complication by using ANP-labeled (at Gln41)
-actin and monitoring the
quenching of rhodamine phalloidin fluorescence by ANP. The ANP-labeled
actin allowed us to look for subdomain 2 movements during the shift to
the open state since the presence of S1 has no effect on
rhodamine fluorescence. Not only did this particular experiment allow
us to "fill-in the gap" left by our yeast actin mutant experiments,
it provided yet another donor/acceptor configuration (with rhodamine
phalloidin as a donor) for following actin regulatory transitions. Our
results show no significant differences in energy transfer between
rhodamine and ANP in any of the actin regulatory states, including the
open state (Table II). These findings confirm our results
obtained with AEDANS-labeled actin mutants, namely, that there seem to be no significant movements of subdomain 2 during actin regulation.
The labeling of the skeletal actin with ANP had the additional
advantage of allowing us to arrest subdomain 2 movement through the
cross-linking of Gln41 to Cys374 on adjacent
protomers (18). We found that despite this modification, which
significantly impairs the motility of F-actin, the thin filaments
remained regulated. This outcome alone does not preclude subdomain 2 movement during regulation; it simply demonstrates that such movement
is not a requirement for regulation, and it serves to reinforce our
results from the previous experiments that indicate no discernible
regulation-based subdomain 2 movements.
Care must always be exercised in interpreting data generated through
the use of probes. Probes may affect the characteristics of their host
proteins and alter the dynamic properties of mobile sites. In the case
of labeled actins, probes may stabilize or destabilize DNase I loop/C
terminus interactions, or even produce a particular conformation that
may not reflect normal function. However, the fact that the probes did
not introduce marked changes in the in vitro motility of the
actins, and especially in the regulation of their acto-S1 ATPase
activities, indicates that they do not introduce noteworthy changes in
the actins. Moreover, our multiple experiments, each yielding similar
results, suggest that subdomain 2 movements seem neither to occur
during, nor play an important role in, the regulation of actin by
tropomyosin and troponin.