©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cross-linking and Fluorescence Study of the COOH- and NH-terminal Domains of Intact Caldesmon Bound to Actin (*)

(Received for publication, May 18, 1995; and in revised form, August 23, 1995)

Philip Graceffa (§)

From the Muscle Research Group, Boston Biomedical Research Institute, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The NH(2)- and COOH-terminal domains of muscle caldesmon are separated by a long alpha-helical stretch. Cys-580, in the COOH-terminal domain, can be rapidly and efficiently disulfide-cross-linked to Cys-374 of actin by incubation with actin modified with 5,5`-dithiobis(2-nitrobenzoic acid) (Graceffa, P., and Jancso, A.(1991) J. Biol. Chem. 266, 20305-20310). Upon further incubation (±tropomyosin), a second cross-link was slowly formed between Cys-153 in the NH(2)-terminal domain and Cys-374 of another actin monomer. The yield of the second cross-link was relatively insensitive to increasing ionic strength, whereas the caldesmon-actin binding strength decreased considerably, suggesting that the NH(2)-terminal domain is largely dissociated from actin and becomes slowly cross-linked to it during collisions with the actin filament. In support of these conclusions, the yield of photocross-linking actin to caldesmon specifically labeled with benzophenonemaleimide at Cys-580 was high, but close to zero for caldesmon labeled at Cys-153, and the fluorescence intensity and polarization of tetramethylrhodamine iodoacetamide attached to Cys-580 showed large changes, while there were no changes for the probe at Cys-153 upon binding caldesmon to actin (±tropomyosin). These findings are consistent with the knowledge that COOH-terminal fragments of caldesmon bind to actin, whereas NH(2)-terminal fragments do not. Since the NH(2)-terminal domain of caldesmon binds to myosin, a dissociated NH(2)-terminal domain may account for caldesmon's ability to link myosin and actin filaments.


INTRODUCTION

Smooth muscle contraction is primarily activated by myosin phosphorylation by a Ca/calmodulin-dependent kinase and deactivated by dephosphorylation by a phosphatase(2) . However, physiological evidence indicates that this mechanism cannot fully explain regulation(3, 4) . For example, tension can be inhibited although myosin is phosphorylated(5) , and full tension can be maintained at low levels of myosin phosphorylation and ATP hydrolysis (6) . This latter, so-called ``latch state'' is thought to be responsible for smooth muscle tone, i.e. tension maintenance with low energy expenditure.

The search for an additional regulatory system has focused on the thin filament protein caldesmon. Chicken gizzard caldesmon is a single 756-residue polypeptide chain forming an elongated molecule with three domains: a COOH-terminal domain, which binds to actin and inhibits actomyosin ATPase activity; an NH(2)-terminal domain, which contains a binding site for the ``neck region'' of myosin; and a central domain consisting of a long alpha-helical ``spacer rod'' (7, 8) separating the other two domains (for review, see (9, 10, 11) ). The molecule contains two cysteine residues at position 153 in the NH(2)-terminal domain and position 580 in the COOH-terminal domain (see (8) for a schematic domain map). Since the inhibition of actomyosin ATPase activity by caldesmon can be reversed by Ca/calmodulin binding to caldesmon, it has been assumed that this reversible inhibition plays a role in regulation. Physiological evidence lends support to this assumption (12, 13, 14) . Since the NH(2)- and COOH-terminal domains of caldesmon bind to myosin and actin, respectively, it has also been proposed that a simultaneous binding of caldesmon to myosin in the thick filament and actin in the thin filament could link the filaments and possibly 1) account for the passive tension of the latch state(15, 16, 17, 18) , 2) enhance a ``productive'' interaction between myosin and actin(19) , or 3) promote the assembly of myosin filaments in the vicinity of actin filaments(20) . In fact, there is evidence that thick and thin filaments are linked into parallel arrays or bundles only in the presence of caldesmon (20, 21) and that caldesmon can form a link between myosin and actin thin filaments(19, 22) .

It is well established that COOH-terminal fragments of caldesmon bind to actin, whereas NH(2)-terminal fragments bind very weakly or not at all(23, 24, 25, 26, 27) . However, the interaction of the NH(2)-terminal domain of intact caldesmon with actin cannot be excluded from fragment studies since proteolytic digestion of caldesmon might directly or indirectly interfere with actin binding. Furthermore, in the intact molecule, the actin binding of the COOH-terminal domain might allow for significant interaction between the NH(2)-terminal domain and actin by increasing the effective concentration of the NH(2)-terminal domain (see discussion by Goody and Holmes(28) ). If, however, the NH(2)-terminal domain of actin-bound caldesmon is dissociated, then it might be able to interact with the neck region of myosin and provide the molecular basis for the caldesmon link between thick and thin filaments. Therefore, it is the object of this study to investigate the interaction of the NH(2)-terminal domain of intact caldesmon with actin by comparing the cross-linking and fluorescence of the COOH- and NH(2)-terminal domains of caldesmon bound to actin.

The basis of this work was our initial observation that Cys-580 in caldesmon's COOH-terminal domain can be easily disulfide-cross-linked to Cys-374 in actin's COOH terminus(29, 30) . Once this cross-link was formed, I could then follow the disulfide cross-linking between Cys-153 in caldesmon's NH(2)-terminal domain and another actin monomer in the actin filament. I also compared the photocross-linking and fluorescence of probes specifically attached to either Cys-580 or Cys-153 of caldesmon. Specific labeling was made possible by our ability to label Cys-153 of caldesmon whose Cys-580 had been disulfide-cross-linked to actin (29) and by using porcine stomach caldesmon, which contains a single cysteine at or close to residue 580 (30) .


EXPERIMENTAL PROCEDURES

Protein Preparations

Preparation of rabbit skeletal muscle actin and modification of actin at Cys-374 with 5,5`-dithiobis(2-nitrobenzoic acid) (Nbs(2)) (^1)were carried out as detailed earlier (29) except that the reaction was carried out at an Nbs(2)/actin molar ratio of 2.6 for 4 h at 0 °C. The Nbs(2) stock solution was freshly prepared by dissolving solid Nbs(2) to 5 mM in 0.1 M Mops, 0.4 mM EDTA, pH 8.0. Smooth muscle tropomyosin was prepared from chicken gizzard as described(31) , and smooth muscle actin from chicken gizzard was prepared by the ``low-salt/EDTA'' method of Strzelecka-Golaszewska et al.(32) . The preparation of smooth muscle caldesmon, using a heat treatment step (33) , from chicken gizzard or from porcine stomach has been described (29) . Protein concentrations were determined as reported(8, 29) .

SDS-PAGE

SDS-PAGE was run according to Laemmli(34) , and densitometry of the resulting gels was carried out as described(29) . Electrophoretic bands were cut out and rerun on SDS-PAGE in the presence of DTT as detailed earlier(29) .

Native Caldesmon

Native caldesmon, i.e. prepared from chicken gizzard without a heat step, was obtained as follows. Fresh gizzards were cleaned, cut into small pieces, and then homogenized in a blender for 3 times 30 s. The mince was washed (35) by homogenization, as described above, once in 4 volumes of buffer A (40 mM NaCl, 1 mM MgCl(2), 1 mM EGTA, 20 mM Tris, pH 7.5, 1 mM DTT plus protease inhibitor mixture (0.25 mM phenylmethylsulfonyl fluoride, 0.3 mM benzamidine, 1 µg/ml leupeptin) plus 0.2 µg/ml pepstatin A) plus 0.05% Triton X-100 and twice in buffer A. Each wash was sedimented at low speed in a centrifuge, and the supernatant was discarded. Caldesmon was extracted from the final pellet by resuspension in 4 volumes of extraction buffer (0.3 M NaCl, 50 mM Mops, pH 7.0, 1 mM EGTA, 0.5 mM MgCl(2), 10 mM mercaptoethanol plus the above inhibitors), homogenized, and centrifuged as described above, and the supernatant was saved. The 30-50% saturated (NH(4))(2)SO(4) fraction of the supernatant was taken up in a small volume of 30 mM NaCl in DEAE buffer (0.2 mM EDTA, 10 mM Mops, pH 7.0, 0.1 mM DTT plus the above inhibitors) and dialyzed overnight against the same buffer. The dialyzed sample was clarified by high speed centrifugation and by filtration through a 0.45-µ Millipore filter and loaded onto a TSK DEAE-650S column (Supelco Inc.). Protein was eluted by fast protein liquid chromatography with a 30-600 mM NaCl gradient in DEAE buffer. Caldesmon fractions, as determined by SDS-PAGE, were pooled and further purified by phosphocellulose P-11 (Whatman) chromatography (36) and calmodulin affinity chromatography(37) , with the additional presence of the above protease inhibitors throughout. In some cases, it was necessary to then carry out a second chromatography on phosphocellulose to obtain the desired purity. The yield of caldesmon by this procedure was 20-25% of that achieved by including a heat treatment step. If pepstatin A, which has been used previously to reduce proteolysis of caldesmon(38) , was omitted from the preparation, the yield was considerably lower still.

Thrombin Digestion

Thrombin digestion of caldesmon was done according to Mornet et al.(39) .

Actin Binding

Actin binding of caldesmon was determined by sedimenting actin filaments for 30 min in either a Beckman Airfuge at 20 p.s.i. (room temperature) or a Beckman TL-100 ultracentrifuge at 80,000 rpm (at 0 or 40 °C). Caldesmon in the supernatant were determined by densitometry following SDS-PAGE.

Disulfide Cross-linking of Caldesmon Cys-153 to Actin Cys-374

This was performed as follows. First, a high yield of the disulfide cross-link between Cys-580 of caldesmon and Cys-374 of actin was formed by incubating caldesmon with Nbs(2)-modified actin (±tropomyosin) for 2 min at room temperature in buffer B (40 mM NaCl, 5 mM Mops, 2 mM MgCl(2), pH 7.5, plus protease inhibitor mixture) (see Fig. 4of (29) or Fig. 2A of this work). A cross-link between caldesmon Cys-153 and another actin Cys-374 then formed slowly by further incubation for various times under these conditions or at other NaCl concentrations between 40 and 500 mM and at 0 or 40 °C. The NaCl concentration was changed by adding a small volume of 5 M NaCl. Disulfide cross-linking was stopped by adding excess N-ethylmaleimide, at the appropriate time, in order to block free sulfhydryl groups. These experiments were performed at molar ratios of actin to caldesmon of 14-20 and of actin to tropomyosin of 7 and at an actin concentration in the range of 15-35 µM.


Figure 4: Photocross-linking of BPM-labeled caldesmon to actin (±tropomyosin) for 0 or 30 min. Shown is 6.7% SDS-PAGE of the gel region showing caldesmon (CaD) and its cross-linked product with actin (CaD times A). A, cross-linking to skeletal muscle actin of BPM-labeled heat-treated chicken gizzard caldesmon (lane 1), BPM/Cys-153-labeled heat-treated chicken gizzard caldesmon (lanes 2-5), and BPM/Cys-580-labeled heat-treated porcine stomach caldesmon (lanes 6-9); B, cross-linking to gizzard muscle actin of BPM/Cys-153-labeled heat-treated chicken gizzard caldesmon (lanes 1-4), BPM/Cys-580-labeled heat-treated porcine stomach caldesmon (lanes 5 and 6), and BPM/Cys-153-labeled native chicken gizzard caldesmon (lanes 7 and 8). In B, the bands between CaD and CaD times A are small impurities in the actin. Chicken caldesmon migrates faster than porcine caldesmon (30) .




Figure 2: A, time course of the reaction between chicken gizzard caldesmon and Nbs(2)-modified actin (±tropomyosin (Tm)) by 6.7% SDS-PAGE. The reaction was carried out at room temperature in 40 mM NaCl, 5 mM Mops, 2 mM MgCl(2), pH 7.5. M indicates the internally disulfide-cross-linked caldesmon monomer, and D indicates the disulfide-cross-linked caldesmon dimer(43) , which are present in caldesmon at time 0 before addition to Nbs(2)-modified actin. X indicates the disulfide-cross-linked product between Cys-580 of caldesmon and Cys-374 of actin(29) . B, the X and X1 bands rerun in the presence of DTT on 10% SDS-PAGE.



Nonspecific Labeling of Gizzard Caldesmon

Nonspecific labeling of gizzard caldesmon with the photocross-linker benzophenonemaleimide (BPM) (Molecular Probes, Inc.) was performed by incubating caldesmon in buffer C (40 mM NaCl, 5 mM Mops, 0.2 mM EDTA, pH 7.5, plus protease inhibitor mixture) with a 10-fold molar excess of BPM for 1.5 h at room temperature and for 1 h on ice. BPM was added from a freshly prepared solution in N,N-dimethylformamide. The reaction was stopped with 1 mM DTT and dialyzed versus buffer C. All operations with BPM were done in a room lighted by indirect incandescent light, or the sample was covered with aluminum foil to prevent unwanted photocross-linking.

Specific Labeling of Cys-580

Specific labeling of the Cys-580 region of caldesmon with BPM or the fluorescent probe tetramethylrhodamine 5-iodoacetamide (TRIA) (Molecular Probes, Inc.) was accomplished with the use of caldesmon from porcine stomach, which contains a single cysteine residue close to or at position 580(30) . The labeling was carried out as performed for nonspecific labeling of gizzard caldesmon (see above) except that a 5-fold molar excess of label was used.

Specific Labeling of Cys-153

Specific labeling of Cys-153 of gizzard caldesmon with BPM or TRIA was performed by modifications of an earlier procedure(29) . Caldesmon was incubated at room temperature for 2-2.5 min with a 15-20-fold molar excess of Nbs(2)-modified actin in buffer B in order to form a disulfide cross-link between Cys-580 of caldesmon and Cys-374 of actin (see Fig. 4of (29) or Fig. 2A of this work, which show the rapid formation of this product, referred to as the X band). BPM or TRIA, at a probe/caldesmon molar ratio of 10-12, was then added in order to react with the free Cys-153 of caldesmon. The reaction was allowed to proceed for 1.5 h at room temperature and then followed by 0.5-1.5 h on ice with periodic mixing. 5 M NaCl was then added to a final concentration of 0.5 M in order to dissociate caldesmon not cross-linked to actin. The labeling reaction was continued for another 1-2 h on ice since the cross-linked caldesmon is tethered to, but otherwise dissociated from, actin under these high salt conditions and might therefore react more completely with BPM or TRIA. The reaction mixture was then centrifuged at 4 °C in a Beckman TL-100 ultracentrifuge for 30 min at 80,000 rpm in order to sediment actin with its cross-linked Cys-153-labeled caldesmon, leaving uncross-linked caldesmon in the supernatant. The tightly packed pellet was stirred at 4 °C for 1-2 h in a solution containing freshly prepared 0.5 M NaI (to dissolve the pellet by depolymerizing actin(40) ), 10 mM DTT (to reduce the disulfide bond between actin and caldesmon), 2 mM MgCl(2), 0.2 mM CaCl(2), 0.2 mM ATP, 2 mM Mops, pH 7.5, such that the final actin concentration was <3 mg/ml. The solution was dialyzed against 0.5 M NaCl (to repolymerize actin but keep the labeled caldesmon dissociated from actin), 2 mM MgCl(2), 0.2 mM CaCl(2), 0.2 mM ATP, 2 mM Mops, pH 7.5, 5 mM DTT plus protease inhibitor mixture. The actin was then sedimented, as described above, with almost all of the labeled caldesmon remaining in the supernatant along with a small amount of actin. 5 mM EDTA was added to the supernatant to denature the small amount of actin and then dialyzed against actin-denaturing buffer (0.5 M NaCl, 2 mM Mes, 0.2 mM EDTA, pH 6, 1 mM DTT plus protease inhibitor mixture). Denatured actin was sedimented, leaving the Cys-153-labeled caldesmon in the supernatant, which was finally dialyzed against buffer C.

Labeling Specificity

The degree of labeling of rhodamine-labeled caldesmon was determined from the extinction coefficient reported by Taylor et al.(41) . The degree of labeling of the Cys-153-labeled protein was 0.5, consistent with partial labeling of Cys-153. Evidence for the specific labeling of Cys-153 came from performing SDS-PAGE of thrombin-digested Cys-153-labeled caldesmon as performed previously for Cys-153 labeled with a coumarin fluorescent probe(29) . Thrombin cleaves (chicken) caldesmon primarily at residue 483(39, 42) , resulting in a large NH(2)-terminal fragment containing Cys-153 and a small COOH-terminal fragment containing Cys-580. As we found previously for the coumarin probe(29) , only the NH(2)-terminal fragment was fluorescent on a gel of thrombin-digested caldesmon labeled at Cys-153 with the rhodamine probe (data not shown). The labeling ratio of Cys-580-labeled (porcine) caldesmon was close to 1 mol of rhodamine/mol of caldesmon, consistent with full and specific labeling of the only cysteine at or close to residue 580.

The absorption spectrum of BPM has a relatively low extinction coefficient and overlaps with protein absorption, making determination of the degree of labeling difficult and uncertain. Therefore, to confirm that ``Cys-153-labeled'' gizzard caldesmon was singly labeled at Cys-153 with BPM, use was made of the fact that the two cysteines of chicken gizzard caldesmon form intra- and intermolecular disulfide bonds, catalyzed by Cu-(o-phenanthroline)(2), to yield disulfide-cross-linked monomer, dimer, and oligomers(43) . At relatively low caldesmon concentrations, the formation of the intramolecular cross-linked monomer predominates(29, 30, 43) . For single-cysteine caldesmons, like that from porcine stomach, no cross-linked monomer forms, and the dimer is the only cross-linked product(30) . Chicken gizzard caldesmon specifically labeled at Cys-153 is also a single-cysteine caldesmon and should not result in any cross-linked monomer upon oxidation at low caldesmon concentrations. Oxidation was performed as described previously (29) with the additional step of adding excess N-ethylmaleimide, in order to block all free sulfhydryl groups, after the oxidation reaction was stopped with EDTA. As demonstrated in Fig. 1, under conditions where oxidation of unlabeled caldesmon results in a high yield of cross-linked monomer (lanes A and B), Cys-153-labeled caldesmon forms very little cross-linked monomer and some cross-linked dimer, with most of the caldesmon remaining in the uncross-linked state (lanes C and D). This is consistent with most of the caldesmon being singly labeled at Cys-153. If one assumes that the cross-linked monomer represents all of the unlabeled caldesmon present, then densitometry of the gel indicates that 88% of the caldesmon is singly labeled. Although, hypothetically, the labeled caldesmon migrating as the uncross-linked species could also represent doubly labeled caldesmon or caldesmon singly labeled at Cys-580, photocross-linking experiments described under ``Results'' rule this out.


Figure 1: Cu-phenanthroline-catalyzed oxidation of chicken gizzard caldesmon, unlabeled or specifically labeled at Cys-153 with BPM. Shown is the 6.7% SDS-PAGE of unlabeled caldesmon before (lane A) and after (lane B) oxidation and of labeled caldesmon before (lane C) and after (lane D) oxidation. CaD, caldesmon; M, disulfide-cross-linked monomer; D, disulfide-cross-linked dimer. The bands above D are disulfide-cross-linked oligomers(43) . Since lanes A and B and lanes C and D were run on separate gels, there is not an exact match-up between bands of the same species.



Photocross-linking

Photocross-linking was performed in a 4 °C cold room in a Rayonet RPR-100 photochemical reactor equipped with 16 ``3500'' lamps (Southern New England Ultraviolet Co.). During photolysis, air was continuously circulated with a fan, which maintained the solution temperature in the range of 15-20 °C. Photolysis was done in test tubes made of borosilicate glass, which cuts out far-UV radiation, in order to minimize protein photodegradation. Photolysis was carried out in 40 mM NaCl, 2 mM MgCl(2), 5 mM Mops, pH 7.5, 2 mM DTT at molar ratios of actin to caldesmon of 20 and of actin to tropomyosin of 7 and at an actin concentration in the range of 20-40 µM.

Fluorescence Emission and Polarization Spectra

Fluorescence emission and polarization spectra of TRIA-labeled caldesmon were obtained with a Spex Fluorolog 2/2/2 photon-counting fluorometer. Spectra were measured in the ratio mode at an excitation wavelength of 530 nm, and polarization was obtained with Glan-Thompson prisms in L format. Polarization (P) was calculated from the following formula: P = (I - GI)/(I + GI), where G = I/I is the machine correction factor; I and I are the intensities of vertically and horizontally polarized emission, respectively, where the exciting light is vertically polarized; and I and I are the intensities of vertically and horizontally polarized emission, respectively, where the exciting light is horizontally polarized(44) . Spectra were recorded at 20 °C at molar ratios of actin to caldesmon of 20 and of actin to tropomyosin of 7 and at caldesmon concentrations between 0.2 and 0.5 µM in a solution containing 40 mM NaCl, 2 mM MgCl(2), 5 mM Mops, pH 7.5.


RESULTS

In the experiments described below, actin refers to rabbit skeletal muscle actin, and caldesmon refers to that prepared with a heat treatment step, except where otherwise specified. Cross-linking and fluorescence experiments were performed at relatively high actin/caldesmon molar ratios (14-20 for disulfide cross-linking and 20 for photocross-linking and fluorescence) in order to minimize any possible interference between caldesmon molecules on the actin filament, which might reduce the interaction between the caldesmon NH(2)-terminal domain and actin. The molar ratio of actin to caldesmon in native thin filaments has been reported to be 16:1(45) , although much lower values were found in reconstituted thin filaments saturated with caldesmon (see discussion in (29) ).

Disulfide Cross-linking

Reaction between actin modified at Cys-374 with Nbs(2) and gizzard caldesmon results in an almost complete disulfide cross-linking of caldesmon, via Cys-580 in the COOH-terminal domain, to actin Cys-374 in 2 min (see Fig. 4of (29) and Fig. 2A of this work). Upon SDS-PAGE, the cross-linked product, which is a 1:1 molar complex of caldesmon and actin, migrates slower than caldesmon and is referred to as the X band (Fig. 2A)(29) . Further incubation of the reaction mixture over a period of 60 min, as described under ``Experimental Procedures,'' resulted in a slow decrease in the X band and a concomitant increase in a higher molecular weight and well resolved band, referred to as X1, and in a number of unresolved, even higher molecular weight cross-linked species (Fig. 2A). The additional presence of tropomyosin had no significant effect on the yield of X1 (Fig. 2A and Fig. 3A).


Figure 3: Salt and temperature dependence of X1 formation and actin binding of chicken gizzard caldesmon. A, SDS-PAGE Coomassie Blue staining intensity ratio of X1 to X after a 60-min reaction between caldesmon (CaD) and Nbs(2)-modified actin. Triangles, 40 °C; squares, room temperature; circles, 0 °C; open symbols, without tropomyosin; closed symbols, with tropomyosin. Also present were 5 mM Mops and 2 mM MgCl(2), pH 7.5. B, binding of caldesmon to actin under identical conditions and with the same symbols as described for A. At 40 mM NaCl, almost all of the caldesmon was bound to actin, whereas at 500 mM NaCl, very little was bound, at all temperatures (±tropomyosin); thus, at these salt concentrations, there is only a single closed circle.



Since gizzard caldesmon contains a second cysteine residue at position 153 in the NH(2)-terminal domain, I suspected that the X1 product was due to a second cross-link between Cys-153 and another Nbs(2)-modified actin monomer by disulfide exchange. Strong support for this came from cutting out and rerunning the X and X1 bands on SDS-PAGE in the presence of DTT, which reduces disulfide bonds (Fig. 2B). Reduction of both X and X1 resulted in the formation of a caldesmon and an actin band (Fig. 2B) with the actin/caldesmon intensity ratio of the X1 band twice that of the X band, as determined by densitometry, showing that the X1 band is due to a 2:1 molar complex of actin and caldesmon. The migration of X1 on SDS-PAGE also is consistent with a 2:1 actin/caldesmon product, which should have an apparent M(r) of 224,000 (M(r) of actin = 42,000; M(r) of caldesmon = 140,000, which is anomalous(46) ). Indeed, X1 migrates (Fig. 2A) between the X band, which has an M(r) of 182,000, and the D band, which has an M(r) of 280,000. For the remainder of this work, the nature and formation of the unresolved, higher molecular weight species will not be considered, and I will focus on the X1 species, which is well resolved and thus amenable to study.

To gain a better understanding of the disulfide cross-linking of the NH(2)-terminal domain of caldesmon to actin, the yield of the X1 product, relative to the X species, was studied as a function of ionic strength at different temperatures as described under ``Experimental Procedures.'' Essentially all of the caldesmon was first converted to the X product by incubation of caldesmon with Nbs(2)-modified actin (±tropomyosin) for 2 min at room temperature in 40 mM NaCl (Fig. 2A). The salt and temperature were then changed or kept the same, and incubation was continued for another 60 min, whereupon the reaction was stopped with N-ethylmaleimide. SDS-PAGE was run, and the X1/X Coomassie Blue staining ratio was determined by densitometry (Fig. 3A). (An X1/X staining ratio of 1 is equivalent to an X1/X molar ratio of 0.8.) To correlate this cross-linking with the caldesmon-actin interaction strength, the actin binding of caldesmon was also studied under the same conditions and protein concentrations as the cross-linking experiments (Fig. 3B). The yield of X1 increased substantially with increasing temperature, but except for a small decrease in X1 with increasing salt concentration, was relatively insensitive to ionic strength and to the presence or absence of tropomyosin (Fig. 3A). In contrast to the yield of X1, the binding of caldesmon changed dramatically, going from 100% to close to 0% over the same salt concentration range (Fig. 3B). The lack of correlation of caldesmon NH(2)-terminal domain cross-linking to actin with the caldesmon-actin binding strength strongly suggests that the NH(2)-terminal domain of caldesmon is largely dissociated from actin, while the COOH-terminal domain is bound and becomes slowly cross-linked to Nbs(2)-modified actin during collisions with the actin filament. Further dissociation of the COOH-terminal domain of caldesmon from actin, except for a disulfide tether, with high salt would be expected to have only a small effect on cross-linking of the already dissociated NH(2)-terminal domain. The increase in cross-linking of the NH(2)-terminal domain to actin with increasing temperature might be due to greater thermal motion and flexibility of the NH(2)-terminal domain and thus a higher collisional frequency with actin. Alternatively, the increase in cross-linking might result from a thermal increase in the rate of the disulfide exchange reaction, possibly due to a change in conformation of caldesmon and/or actin in the region of the involved cysteines.

Photocross-linking

To further investigate the interaction of the COOH- and NH(2)-terminal domains of caldesmon with actin, I studied the photocross-linking to actin of caldesmon specifically labeled at either Cys-580 or Cys-153 with a sulfhydryl-specific photocross-linker, BPM, as described under ``Experimental Procedures.'' Benzophenone cross-linking has a distinct advantage over disulfide cross-linking in that benzophenone will cross-link to almost any carbon-hydrogen bond (see (47) for a review), which results in highly nonspecific cross-linking, whereas disulfide cross-linking necessitates the proximity of two specific (cysteine) residues. This allowed us to probe the interaction of the two cysteine regions of caldesmon with whichever regions of actin they come in contact with.

It has been shown that photolysis of actin plus chicken gizzard caldesmon, nonspecifically labeled at both cysteines with BPM, results in one major product identified as a 1:1 complex of actin and caldesmon by its apparent molecular weight on SDS-PAGE(48) . I confirmed this finding by also observing one major cross-linked product (Fig. 4A, lane 1), which was identified as a 1:1 complex of actin and caldesmon by the fact that this product comigrated with the X band, the 1:1 caldesmon-actin disulfide-cross-linked complex, on SDS-PAGE (data not shown). When BPM-labeled porcine stomach caldesmon, which contains a single cysteine at/or near position 580(30) , was photolyzed in the presence of (skeletal or smooth muscle) actin (±tropomyosin), similar results were obtained (Fig. 4, A (lanes 6-9) and B (lanes 5 and 6)).

The fact that caldesmon labeled at both cysteines yields about the same cross-linking results as compared to when only Cys-580 is labeled suggests the possibility that the extent of the Cys-153 label cross-linking to actin is low. To test this directly, I studied the photocross-linking of chicken gizzard caldesmon specifically labeled at Cys-153 with BPM. The photolysis of BPM/Cys-153-labeled caldesmon plus actin resulted in only a very faint cross-linked product (±tropomyosin) (Fig. 4A, lanes 2-5). (The faint band cannot be seen in the gel photographs.) The same results were obtained when actin from chicken gizzard muscle was used (Fig. 4B, lanes 1-4) and when caldesmon prepared without a heat treatment step, i.e. native caldesmon, was used with gizzard muscle (lanes 7 and 8) or skeletal muscle (data not shown) actin. (The fact that there is no cross-linking indicates that the caldesmon is neither doubly labeled nor singly labeled at Cys-580 with BPM (see ``Experimental Procedures'').) Thus, the BPM label at Cys-153, in the NH(2)-terminal domain of caldesmon, does not cross-link to actin. The BPM/Cys-153-labeled caldesmon was fully bound to actin since all of the labeled caldesmon cosedimented with actin under the conditions of the photocross-linking experiments. Thus, the lack of cross-linking is not due to a lack of binding. The absence of cross-linking of BPM at Cys-153, together with a high yield of cross-linking of the label at Cys-580, is consistent with the conclusion, drawn above, that the NH(2)-terminal domain of caldesmon binds weakly, if at all, to actin, while the COOH-terminal domain binds strongly.

Caldesmon specifically labeled with BPM at either Cys-580 or Cys-153 was photolyzed in the absence of actin as controls (data not shown). Very little, if any, cross-linked products were obtained with the label at Cys-580. However, for the Cys-153-labeled caldesmon, there was a significant drop in the intensity of the caldesmon SDS-PAGE band with a corresponding increase in the amounts of many higher molecular weight species (which migrated slower than the cross-linked product between caldesmon and actin). For the longest photolysis time (120 min), there was also some cross-linked product that did not enter the stacking gel. These slower migrating species most likely correspond to oligomeric cross-linked caldesmon formed upon very weak interactions between caldesmon molecules. These higher molecular weight bands were not observed when actin was present since the caldesmon is bound to actin and is less likely to interact with other caldesmon molecules. These control experiments indicate that the cross-linked product formed (with Cys-580-labeled caldesmon) in the presence of actin is not due to caldesmon alone (negative control), and they demonstrate that although the BPM label at Cys-153 of caldesmon does not significantly cross-link to actin, it can cross-link to another protein (in this case itself) when it comes in contact with it (positive control).

Fluorescence

To provide information complementary to the cross-linking results, I measured the fluorescence intensity and polarization of caldesmon specifically labeled at either Cys-580 or Cys-153 with the sulfhydryl-specific fluorescent probe tetramethylrhodamine iodoacetamide. The emission intensity and polarization monitor two separate properties of the probe, which increases the possibility of detecting interaction with actin. The fluorescence intensity is sensitive to the probe's environment, and the fluorescence polarization is, for the most part, a measure of the probe's mobility. These properties could change either by a direct interaction of actin with the region of caldesmon that contains the probe or indirectly by actin inducing a conformational change in the part of the protein to which the probe is attached. Since fluorescence is sensitive to conformational change, it is more likely than photocross-linking to sense an interaction at some distance from the probe attachment site. Cross-linking necessitates direct contact between the probe and the interacting protein. Furthermore, in general, if the fluorescent probe is rigidly attached to a protein, then the mobility of the probe reflects the mobility of the protein to which it is attached and could thus be sensitive to the binding of another protein some distance from it.

The fluorescence emission and polarization of the rhodamine probe at Cys-580 were very sensitive to interaction with actin (±tropomyosin). The fluorescence intensity decreased 28% upon interaction with actin and 26% with actin + tropomyosin (Fig. 5A), indicating an interaction between the COOH-terminal domain of caldesmon and actin. The polarization of rhodamine at Cys-580 changed from a value of 0.21 for free caldesmon to 0.38 in the presence of actin and 0.40 with the additional presence of tropomyosin (Fig. 6A). Fluorescence polarization is sensitive to changes in both probe mobility and fluorescence intensity(44) . From the decrease in rhodamine fluorescence intensity upon the addition of actin + tropomyosin, an increase in polarization of 15% is expected (44) . That the polarization increased by 100% to 0.4, which is the maximum observable polarization for this rhodamine probe(49) , indicates a dramatic decrease in the mobility of the probe upon interaction of the COOH-terminal domain of caldesmon with actin. In contrast, the emission intensity (Fig. 5B) and polarization (Fig. 6B) of the rhodamine probe at Cys-153, in the NH(2)-terminal domain of caldesmon, were unresponsive to the binding of actin (±tropomyosin), consistent with the NH(2)-terminal domain being dissociated from actin. The actin binding of the two labeled caldesmons was measured under the same conditions as used for the fluorescence measurements. Essentially 100% of both labeled caldesmons was bound to actin, indicating that the fluorescence changes observed were representative of the fully bound protein.


Figure 5: Fluorescence emission spectra of the rhodamine probe attached to caldesmon at either cysteine 580 (A) or cysteine 153 (B). The excitation wavelength was 530 nm. Circles, caldesmon; triangles, caldesmon + actin; squares, caldesmon + actin + tropomyosin.




Figure 6: Fluorescence polarization spectra of the rhodamine probe attached to caldesmon at either cysteine 580 (A) or cysteine 153 (B). The excitation wavelength was 530 nm. Circles, caldesmon; triangles, caldesmon + actin; squares, caldesmon + actin + tropomyosin.




DISCUSSION

We have shown that, for intact caldesmon, the COOH-terminal domain is very responsive to the binding of actin ± tropomyosin, whereas the NH(2)-terminal domain is almost completely insensitive in reconstituted thin filaments. The COOH-terminal domain can be disulfide-cross-linked to actin (±tropomyosin) much more rapidly and with much higher yield than the NH(2)-terminal domain. Furthermore, the small amount of NH(2)-terminal domain cross-linking, which is already disulfide-cross-linked to actin via Cys-580, is relatively insensitive to increases in ionic strength, which greatly reduces the caldesmon-actin interaction. Benzophenone attached to Cys-580 in the COOH-terminal domain of caldesmon can be photocross-linked to actin (±tropomyosin) with good yield, whereas there is almost no cross-linking of the probe attached to Cys-153 in the NH(2)-terminal domain, results found for native or heat-treated caldesmon and with actin from striated or smooth muscle. Finally, the fluorescence emission and polarization of a rhodamine probe attached to caldesmon Cys-580 are very sensitive to the binding of actin (±tropomyosin), whereas the fluorescence of the probe at Cys-153 does not change. These findings, together with studies that have shown that COOH-terminal fragments of caldesmon bind to actin ± tropomyosin and that NH(2)-terminal fragments do not(23, 24, 25, 26, 27) , strongly suggest that in reconstituted thin filaments, the COOH-terminal domain of caldesmon is bound to actin, while the NH(2)-terminal portion is, for the most part, dissociated. If this interpretation of the results is correct, then the extent of the NH(2)-terminal portion of caldesmon dissociated from actin must extend somewhat beyond Cys-153, but how much beyond cannot be decided from this work.

Tropomyosin, which binds to caldesmon in the absence of actin(31, 50, 51, 52, 53, 54) and increases the actin binding of caldesmon(33, 55) , has no effect on the cross-linking or fluorescence of the NH(2)-terminal domain of actin-bound caldesmon. This suggests that actin-bound tropomyosin neither binds to the NH(2)-terminal domain of caldesmon nor promotes the actin binding of the NH(2)-terminal domain. This is in contrast to studies conducted in the absence of actin that show an interaction between tropomyosin and the NH(2)-terminal domain of caldesmon(25, 56, 57) . Thus, any interaction between actin-bound tropomyosin and caldesmon most likely involves caldesmon's COOH-terminal domain, which can also interact with tropomyosin in the absence of actin(26, 27, 51, 57, 58, 59, 60) and which contains sequence homologies to troponin T(58, 59, 61, 62) , the tropomyosin-binding subunit of skeletal muscle troponin.

Three microscopy studies have addressed the question of the arrangement of the NH(2)-terminal domain of caldesmon in reconstituted and native thin filaments. For native thin filaments, all three studies found no visual evidence for caldesmon projections(38, 63, 64) , projections that might be expected for a dissociated NH(2)-terminal domain. (However, a recent publication has reported unpublished observations of NH(2)-terminal domain projections of caldesmon in native thin filaments(20) .) Similarly for reconstituted thin filaments, two of the studies, using negative stain electron microscopy (63) or x-ray diffraction microscopy(38) , concluded that there are no (38) or occasional (63) molecules projecting out from the filament. On the contrary, one study on reconstituted thin filaments, performed with rotary shadow immunoelectron microscopy(64) , observed that greater than two-thirds of the caldesmon was bound to actin with either its NH(2)- or COOH-terminal end, with the opposite end projecting out away from the filament. About 30% of the projecting molecules were bound via their NH(2)-terminal end, suggesting significant interaction between the NH(2)-terminal domain of caldesmon and actin, even in the absence of COOH-terminal domain binding.

The basis for these discrepancies is presently unclear. First of all, how do I reconcile my conclusion of the caldesmon NH(2)-terminal domain dissociated from actin with the microscopy studies not observing caldesmon projections. One might expect projections if a significant portion of the NH(2)-terminal part of caldesmon was not interacting with actin. However, the part of the NH(2)-terminal domain dissociated from actin may not extend much beyond Cys-153, and it may not be possible to observe projections of such a small part of the caldesmon molecule. Even if a larger part of the NH(2)-terminal part of caldesmon was dissociated from actin, only a small part of this might project out, or the entire dissociated part of caldesmon might be confined to a region close to the filament and might not project enough to be seen, all depending on the degree and region of flexibility of bound caldesmon. Thus, the NH(2)-terminal domain could be dissociated, but not fully projecting out from the actin filament. On the other hand, since one study (64) found a large difference in caldesmon arrangement between native and reconstituted thin filaments and another study (63) finds some difference, caldesmon may bind differently in both types of filaments, with a dissociated NH(2)-terminal domain more prevalent in reconstituted filaments. Since we have provided evidence suggesting that the COOH-terminal domain of caldesmon assembles similarly in native and reconstituted thin filaments(30) , any difference in assembly is most likely confined to the rest of the molecule. Finally, the arrangement of caldesmon on actin might depend on the very different sample conditions and treatments used for each study. Specifically, this work was performed on dilute thin filament solutions, x-ray microscopy was carried out on very concentrated thin filament sols, and electron microscopy was done with fixed thin filament samples. More work is necessary to resolve these differences and uncertainties.

The dissociation of the NH(2)-terminal domain of caldesmon from actin in reconstituted thin filaments is important, regardless of whether or not it is dissociated in vivo. If it is not dissociated in vivo, then knowledge of why it is dissociated in vitro will give a deeper understanding of the caldesmon-actin interaction. If the myosin-binding NH(2)-terminal domain of caldesmon does exist in the dissociated state in vivo, it might easily link the actin filament to the myosin filament, a link for which there is in vitro(19, 20, 21) and in situ(22) evidence. Since caldesmon interacts with the neck (i.e. S2 region) of myosin(16, 65) , a region of myosin that is not thought to come in contact with the actin filament, it might be necessary for the caldesmon NH(2)-terminal domain to be dissociated from actin in order to reach the S2 part of the myosin molecule. It has been proposed that a caldesmon link between myosin and actin might enhance a productive interaction between myosin and actin during contraction (19) or might aid in the assembly of myosin filaments in the vicinity of actin filaments(20) . It has also been suggested (15, 16, 17, 18) that such a static connection between filaments might possibly be a molecular basis for the latch state(6) , in which tension is maintained at the expense of very little energy.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR-30917 and AR-41637. A preliminary report of this work was presented at the 37th Annual Biophysical Society Meeting, Washington, D. C., February 14-18, 1993(1) . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Muscle Research Group, Boston Biomedical Research Inst., 20 Staniford St., Boston, MA 02114. Tel.: 617-742-2010 (ext. 367); Fax: 617-523-6649; graceffa@bbri.harvard.edu.

(^1)
The abbreviations used are: Nbs(2), 5,5`-dithiobis(2-nitrobenzoic acid); Mops, 3-(N-morpholino)propanesulfonic acid; Mes, 4-morpholineethanesulfonic; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; BPM, benzophenonemaleimide; TRIA, tetramethylrhodamine 5-iodoacetamide.


ACKNOWLEDGEMENTS

I thank Adelaida Carlos for the preparation of proteins and Drs. John Gergely, Terence Tao, and Albert Wang for very helpful discussions.


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