INTRODUCTION

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Actin is one of the most ubiquitous and abundant proteins in nature. It is one of the main constituents of the cell cytoskeleton, and its interaction with the myosin motor coupled with the hydrolysis of ATP is the molecular basis of muscle contraction. Actin exists in monomer (G) and polymer (F) forms. According to crystallographic data, the monomer of actin consists of four subdomains (Kabsch et al., 1990). As shown by limited proteolysis (Strzelecka-Golaszewska et al., 1993) and fluorescence labeling (Frieden et al., 1980; Frieden and Patane, 1985), the structure of G-actin is rather dynamic, and it changes upon Ca-Mg and ATP-ADP exchange. Normal mode analysis has also indicated significant movements of subdomains and loops in the actin structure (Tirion et al., 1995). Until now conformational changes in actin were studied primarily in its subdomain 2 and at the C-terminus because of their easy accessibility to proteases and chemical reagents (Frieden et al., 1980; Hegyi et al., 1974; Strzelecka-Golaszewska et al., 1993).

F-actin has been assumed to be a passive element in the myosin-powered cross-bridge cycle of contraction. Recently, however, the accumulating evidence about the dynamic nature of actin filaments (Drummond et al. 1990; Prochniewicz and Yanagida, 1990; Prochniewicz et al., 1996a; Orlova and Egelman, 1995; Orlova et al., 1995; Kim et al., 1998) has indicated an active role of actin in the molecular mechanism of muscle contraction. Several biochemical and structural studies have indicated that actin filament exists in different conformations, depending on bound cation, nucleotides, and proteins. It has been shown by image reconstruction of electron micrographs that a bridge of density, which has been suggested to arise from a major structural shift at the C-terminus, exists between the two strands of the actin filament in Ca-F-actin and is absent in Mg-F-actin (Orlova and Egelman, 1995). The nature of the tightly bound divalent cation and the addition of KCl influence the proteolytic susceptibility of F-actin at subdomain 2 and near the C-terminus (Strzelecka-Golaszewska et al., 1996). Phosphate (P_i) and its analogs, like beryllium fluoride, influence the three-dimensional structure of the filament (Orlova and Egelman, 1992) and, according to our results (Muhlrad et al., 1994), induce conformational changes in F-actin that are highly cooperative and extend over several monomers. A single gelsolin molecule nucleating a new filament affects in a cooperative manner the conformation of the whole actin filament (Orlova et al., 1995; Prochniewicz et al., 1996b). Myosin subfragment 1 (S1) has a significant effect on the structure of the actin filament: it increases the filament flexibility (Menetret et al., 1991) and rotational motion (Ng and Ludescher, 1994), immobilizes internal motion (Thomas et al., 1979), and decreases the distance between two domains of the actin monomer, resulting in a more compact molecule (Miki and Kouyama, 1994).

Despite the important role of subdomain 1 in actin in interactions with myosin and several actin-binding proteins, little is known about conformational changes that occur on it. This is due to the lack of an accessible residue that could be labeled with an environment sensitive probe. The finding of Drewes and Faulstich (1991) that Cys¹⁰ on subdomain 1 can be selectively modified in Cys³⁷⁴-blocked MgADP-G-actin appears to offer such a choice. We explored this possibility and labeled Cys¹⁰ with three different environment-sensitive fluorescent reagents. The specificity of the labeling and the effect of the modification of Cys¹⁰ on actin structure and function were examined in this work. The results show that while the modification perturbs the structure of subdomain 1, actin nevertheless remains fully functional after the labeling. Spectroscopic evidence is provided for conformational changes in the vicinity of Cys¹⁰ due to actin polymerization and the binding of S1. A preliminary report of this study was presented at a Biophysical Society Meeting (Eligula et al., 1998).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents

N-(Iodoacetyl)N'-(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), and monobromo bimane (MBB) were purchased from Molecular Probes (Eugene, OR). ATP, ADP, dithioerythritol (DTE), N-ethyl maleimide (NEM), subtilisin, and phenylmethylsulfonyl fluoride (PMSF) were from Sigma Chemical Co. (St. Louis, MO).

Proteins

Myosin and actin were prepared from rabbit back and leg muscles by the methods of Tonomura et al. (1966) and Spudich and Watt (1971), respectively. S1 and heavy meromyosin (HMM) were obtained by digestion of myosin filaments with chymotrypsin, following the procedures of Weeds and Taylor (1975) and Margossian and Lowey (1982), respectively. Protein concentrations were estimated from their absorption by using an A^1% at 280 nm of 7.5 cm¹ and 6.5 cm¹ for S1 and HMM, respectively, and an A^1% at 290 nm of 6.3 cm¹ for actin. Whenever appropriate, light scattering corrections were applied. Molecular masses were assumed to be 115, 350, and 42.3 kDa for S1, HMM, and actin monomers, respectively.

Chemical modification

Essentially, the procedure of Drewes and Faulstich (1991) was followed in the selective modification of Cys¹⁰. CaATP-G-actin (100-120 µM) in G-buffer (0.1 mM CaCl₂, 0.2 mM ATP, 0.5 mM -mercaptoethanol, 1.0 mM NaN₃, 1.0 mM NaHCO₃, pH 7.6) was incubated with 3.0 mM NEM on ice for 2 h to block Cys³⁷⁴. The reaction was terminated by adding 3.0 mM DTE. The NEM-labeled actin was filtered through a Sephadex G-50 spin column equilibrated with 50 µM MgCl₂ and 5.0 mM Tris-HCl (pH 8.0). The filtered actin was polymerized by the addition of 0.2 mM EGTA and 2.0 mM MgCl₂ and incubation at room temperature for 30 min. F-actin was pelleted by centrifugation at 40,000 rpm at 4°C for 2 h and resuspended in 1.0 mM ADP, 50 µM MgCl₂, 5.0 mM Tris-HCl (pH 8.4). After overnight incubation on ice, actin was spin again at 40,000 rpm at 4°C for 2 h. The supernatant contained Cys³⁷⁴-blocked MgADP-G-actin, which was modified at Cys¹⁰ with one of the three SH reagents, CPM, IAEDANS, or MBB. CPM, IAEDANS, and MBB were added in 1.5, 4.0, and 4.0 molar excesses over actin, respectively, and incubated on ice for 4 h. The reactions were terminated by adding 1.5 mM DTE and 0.5 mM ATP, and the actin was filtered through a Sephadex G-50 spin column equilibrated with 0.5 mM ATP, 50 µM MgCl₂, and 5.0 mM Tris-HCl at pH 7.8 (MgATP-G-actin buffer) to transform MgADP-G- to MgATP-G-actin. Actin was kept overnight in the MgATP-G-actin buffer before any further treatment to fully restore its structure (Drewes and Faulstich, 1991). The labeling stoichiometry of G-actin was determined spectrophotometrically by using the molar extinction coefficients of the three reagents (CPM _384 nm = 16,450, IAEDANS _336 nm = 5700, MBB _396 nm = 5300). Actin labeling was 40-60% with IAEDANS, 60-90% with MBB, and ~100% with CPM. The concentrations of the Cys¹⁰-labeled actins were calculated by taking into account the absorbance of the attached labels at 290 nm according to the following formulas:

CPM-actin (mg/ml) = (O.D.₂₉₀  0.213 × O.D.₃₈₄)/0.63

IAEDANS-actin (mg/ml) = (O.D.₂₉₀ 0.313 × O.D.₃₃₆)/0.63

MBB-actin (mg/ml) = (O.D.₂₉₀  0.38 × O.D.₃₉₆)/0.63

Subtilisin digestion

Labeled MgATP-G-actin and F-actin were digested with subtilisin at 1000:1 and 500:1 ratios (w/w) of actin to protease, respectively, at 25°C in the corresponding G- and F-buffers (1.0 mM MgCl₂, 20 mM Tris-HCl, pH 7.8). The digestion was terminated at different times with 1.0 mM PMSF and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal Edman sequence analysis was performed on subtilisin-cleaved, labeled F-actin.

Fluorescence measurements

All fluorescence measurements were made in a PTI spectrofluorometer (Photon Technology Industries Co., South Brunswick, NJ) at 25°C. Excitation wavelengths for CPM-, IAEDANS-, and MBB-labeled actins were set at 390, 334, and 380 nm, respectively.

Fluorescence quenching

Fluorescence quenching measurements were performed on labeled actin samples in 0.1 mM MgCl₂, 0.4 mM ATP, and 5.0 Tris-HCl (pH 7.8) at 25°C by adding nitromethane or acrylamide (in 5-40-µM consecutive steps) and averaging the fluorescence signal for 30 s. Emission wavelengths for CPM-, IAEDANS-, and MBB-labeled actin were set at 460, 485, and 475 nm, respectively (for excitation wavelengths see Fluorescence Measurements, above). Stern-Volmer constants (K_SV) were calculated from the plots of F_o/F (F_o and F, fluorescence intensities in the absence and presence of quencher, respectively) against quencher concentration.

ATP-ATP exchange

Labeled and unlabeled MgATP-G-actin (100 µM) was filtered through a Sephadex G-50 spin column equilibrated with 50 µM MgCl₂ and 5.0 mM Tris-HCl (pH 7.8). ATP was added at a 1.5-fold molar excess over filtered actin, and the sample was incubated on ice for 1 h. Immediately before the measurement, actin was diluted to 3.0 µM. The sample was transferred to a thermostatted spectrofluorometer cell (25°C), and the change in fluorescence was recorded after the addition of 200 µM ATP. Excitation and emission wavelengths were set at 340 nm and 410 nm, respectively.

Actin ATPase

Actin ATPase was assayed on labeled and unlabeled 10 µM MgATP-G-actin samples in 0.1 mM MgCl₂, 2.0 mM DTE, 0.4 mM ATP, 5.0 mM Tris-HCl (pH 7.6) at 25°C. At given time intervals 250-µl aliquots were withdrawn and the reaction was terminated by adding equal amounts of 0.6 M perchloric acid. Activity (micromoles of phosphate per micromole of actin per hour) was calculated from the produced inorganic phosphate as measured by the malachite green dye method (Kodama et al., 1986).

Actin-activated S1 ATPase

Actin-activated S1ATPase activity (micromoles of phosphate per micromole of S1 per second) was calculated from the inorganic phosphate produced, measured according to the method of Fiske and Subbarow (1925). The reaction was carried out at 25°C on 1-ml aliquots taken at various time intervals. Incubation times were chosen such that no more than 15% of the ATP was hydrolyzed. The assay contained 0.1 µM S1 and between 2.5 and 40 µM F-actin in 2.0 mM MgCl₂, 20 mM HEPES buffer (pH 7.4), and 2.0 mM ATP. The ATPase data were fitted directly to the Michaelis-Menten equation to obtain the K_M and V_max values.

Actin polymerization

Polymerization of 10 µM MgATP-G-actin in 0.1 mM MgCl₂, 0.4 mM ATP, and 5.0 mM Tris-HCl (pH 7.8) was initiated by adding 2.0 mM MgCl_2. Polymerization was followed by light scattering at 25°C in a PTI spectrofluorometer. Both the excitation and emission wavelengths were set at 360 nm. Actin polymerization was also tested by pelleting the polymerized mixture at 75,000 rpm for 45 min in a Beckman TL-100 ultracentrifuge and subsequent analysis of the resulting pellet and supernatant by SDS-PAGE.

In vitro actin motility assay

This assay was performed at 25°C as described previously (Miller et al., 1996). HMM (300 µg/ml) was adsorbed to the nitrocellulose-coated coverslips. ATP-desensitized HMM was removed from the stock solution by pelleting HMM in the presence of actin and ATP. The assay solution was composed of 25 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4), 25 mM KCl, 2.0 mM MgCl_2, 2.0 mM EGTA, 1.0 mM ATP, and the glucose-oxidase-catalase system to slow photobleaching. Methylcellulose (0.4%) was present in all solutions. Actin filaments were labeled by rhodamine phalloidin as described by Miller et al. (1996). Sliding speeds of actin filaments were determined using the Expertvision System (Motion Analysis, Santa Rosa, CA).

	RESULTS

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Specificity of Cys¹⁰ labeling and the perturbation of G-actin structure

Three fluorescent thiol reagents, CPM, IAEDANS, and MBB, were used for labeling Cys¹⁰ on Cys³⁷⁴-blocked MgADP-actin. Because MgADP-G-actin easily undergoes spontaneous denaturation (Gershman et al., 1989), the labeled actin was transformed to the more stable MgATP-G-actin by incubating it in an excess of ATP for at least 12 h (Drewes and Faulstich, 1991). We verified, using both Cys³⁷⁴-blocked G-actin that had been subjected to MgADP-MgATP transformation and control Cys³⁷⁴-blocked MgATP-G-actin, that the above procedure of Cys¹⁰ labeling does not impair the main properties of actin, including polymerization, sedimentation, activation of myosin ATPase, and sliding of filaments in the in vitro motility assay. Thus, in accordance with Drewes and Faulstich (1991), we found that the incubation in MgATP-containing solution restores the native structure of MgATP-G-actin.

The specificity of Cys¹⁰ labeling was checked by subtilisin cleavage. Subtilisin cleaves G-actin between Met⁴⁷ and Gly⁴⁸ in the DNase 1 binding loop of subdomain 2. Of the two products of this cleavage, fragment 1-47 contains a single thiol, Cys¹⁰, while the large C-terminal (48-375) fragment has the other actin thiols. In Fig. 1, A and B, are the electrophoretograms of MgATP-G-actin labeled with AEDANS on Cys¹⁰, before and after subtilisin digestion. It can be seen that the undigested actin (Fig. 1 A, lane a) is highly fluorescent, while the 48-375 C-terminal proteolytic fragment, which is produced during the digestion (Fig. 1 A, lanes b and c) is not fluorescent (the small 1-47 fragment cannot be seen on the gel). The absence of a label on the 48-375 fragment, which contains the remaining actin thiols, indicates that Cys¹⁰ has been selectively modified. As a control, Fig. 1 shows that after the subtilisin digestion of MgATP-G-actin labeled by AEDANS on Cys³⁷⁴, both the intact actin and its 48-375 fragment are fluorescent (Fig. 1, C and D). Notably, in the Cys¹⁰-labeled actin, fluorescence is absent not only in the C-terminal fragment but also in the upper, apparently undigested actin band (Fig. 1, A and B, lanes b and c). The loss of fluorescence from the "intact" actin band was also observed when AEDANS-Cys¹⁰-F-actin, which is rather resistant to subtilisin digestion (Vahdat et al., 1995), was digested at a 500:1 (w/w) ratio of actin to subtilisin for 5 min (results not shown). The same loss of fluorescence was also observed with actin labeled with CPM and MBB (results not shown). We assumed that the fluorescence loss from the actin band is due to a cleavage in the N-terminal region of actin, which apparently does not influence the electrophoretic mobility of the protein. To test this assumption, N-terminal Edman degradation was performed on subtilisin-digested AEDANS-Cys¹⁰-F-actin. Two sequences were obtained in the analysis. Because the N-terminus of actin is blocked, these sequences identify the locations of the subtilisin cuts. The major and minor sequences were Gly-Phe-Ala-Gly-Asp-Asp and Gly-Gln-Lys-Asp, respectively. The major sequence unambiguously assigns the location of the new subtilisin cleavage site to the region between Ala¹⁹ and Gly²⁰, near the N-terminus of actin. The minor sequence represents the known subtilisin cut between Met⁴⁷ and Gly⁴⁸ (Schwyter et al., 1989). The Ala¹⁹-Gly²⁰ site becomes available for subtilisin cleavage because of the perturbation of actin's structure by Cys¹⁰ labeling. This site is on a -sheet that runs antiparallel relative to the -sheet on which Cys¹⁰ is located (Fig. 2). The distance from the peptide bond between Ala¹⁹ and Gly²⁰ to the sulfur atom of Cys¹⁰ is 6.5 Å (Kabsch et al., 1990).

View larger version (43K): [in this window] [in a new window]   FIGURE 1   Subtilisin digestion of MgATP-G-actin labeled with IAEDANS on Cys10 or Cys374. Actin was digested with subtilisin as described in Materials and Methods. (A and B) Cys10-labeled actin. (C and D) Cys374-labeled actin. A and C are fluorescent; B and D are Coomassie blue-stained electrophoretograms. Lanes a and d: Undigested actin; lanes b, c, e-h: actin digested for 5 min (lanes b and e), 10 min (lanes c and f), 30 min (lane g), and 60 min (lane h). Labeling stoichiometries for Cys10 and Cys374 labeling were 0.46 and 0.82 AEDANS/actin, respectively.

View larger version (186K): [in this window] [in a new window]   FIGURE 2   G-actin structure according to Kabsch et al. (1990). Full columns are -helices, arrows are -sheets. Light and dark spheres label Cys10 and Ala19, respectively.

Energy transfer from tryptophans to fluorescent probes on Cys¹⁰

Excitation of CPM-Cys¹⁰-MgATP-G-actin at 297 nm produced two emission bands, a tryptophan band with a maximum at 330 nm and a CPM band with _max at 464 nm (Fig. 3 A). In comparison with the tryptophan emission of the unmodified MgATP-G-actin recorded under identical experimental conditions, the intensity of the tryptophan band of CPM-Cys¹⁰-MgATP-G-actin is substantially decreased. This indicates fluorescence resonance energy transfer from the actin's tryptophans to the Cys¹⁰-attached CPM probe. A decrease in fluorescence intensity of the tryptophan band relative to the unmodified MgATP-G-actin was also observed for AEDANS- and MBB-MgATP-G-actins (Fig. 3, B and C). According to the atomic structure of G-actin (Kabsch et al., 1990), the distances from the four actin tryptophans (79, 86, 340, and 356) to the sulfur atom of Cys¹⁰ are 12.0, 5.3, 6.9, and 11.6 Å, respectively. Because of the relatively short distances, all actin tryptophans may contribute to the observed energy transfer.

View larger version (15K): [in this window] [in a new window]   FIGURE 3   Emission spectra of MgATP-G-actin and Cys10-modified MgATP-G-actins excited at 297 nm. , G-actin; ·····, Cys10-labeled actins. (A) CPM-labeled; (B) AEDANS-labeled; (C) MBB-labeled G-actin. Labeling stoichiometries of CPM-, AEDANS-, and MBB-G-actin were 1.0, 0.41, and 0.9 probe/actin, respectively.

Characterization of Cys¹⁰-modified actin

The ATPase activity of MgATP-G-actin was significantly decreased after Cys¹⁰ modification (Table 1). The larger decreases in ATPase were found with AEDANS- and MBB-modified actins, while the decrease with CPM-Cys¹⁰-MgATP-G-actin was somewhat smaller. The rate of ATP-ATP exchange from MgATP-G-actin increased slightly as a result of the modification (Table 1). The order of the probe-dependent increase was AEDANS > CPM > MBB. These results support the conclusion derived from the appearance of a new subtilisin cleavage site, i.e., the modification of Cys¹⁰ causes some perturbations in the structure of actin.

The rates and extents of polymerization of Cys¹⁰-labeled-MgATP-G-actins, measured by either light scattering or sedimentation, were similar to those of unmodified MgATP-G-actin (data not shown). The polymerized Cys¹⁰-modified actins activated the Mg-modulated ATPase activity of S1 with only minor probe-dependent variations in the K_M and V_max values (Table 1). MBB-labeled F-actin activated S1 ATPase in a manner similar to that of its activation of native F-actin. The kinetic parameters of the ATPase activity were slightly affected when IAEDANS and CPM were used for modification (Table 1). In the in vitro motility assays no significant difference was found between the MBB-Cys¹⁰-F-actin and unmodified F-actin filaments. The mean velocities for the control and the MBB-Cys¹⁰-F-actin filaments were 4.02 ± 0.81 and 4.03 ± 0.89 µm/s, respectively.

Effect of actin polymerization and S1 binding on the spectral characteristics of the Cys¹⁰-attached probes

Subdomain 1 of actin, where Cys¹⁰ is located, has a key role in intermolecular interactions with actin and myosin. Therefore, it was of interest to examine the effect of actin polymerization and S1 binding on the Cys¹⁰ environment. Fig. 4A shows the emission spectra of AEDANS-MgATP-Cys¹⁰-G-actin and F-actin recorded using an excitation at 297 nm. The intensity of the tryptophan band centered at 330 nm decreased, while the intensity on the AEDANS band, _max 486 nm, increased and blue shifted as a result of the polymerization. The tryptophan fluorescence decrease is comparable to that normally observed upon polymerization of unlabeled actin (Selden et al., 1994). The increase in the AEDANS band could be detected also upon direct excitation of the probe at the _max wavelength for its absorption (Fig. 4 B). In addition, the emission spectrum of AEDANS-Cys¹⁰-F-actin (Fig. 4 B) was blue shifted ( _max 479 nm) relative to the spectrum of G-actin (_max 484 nm). Thus, the results shown in Figs. 4A and 4B indicate conformational changes in the vicinity of Cys¹⁰ but not alterations in the fluorescence energy transfer between actin tryptophans and Cys¹⁰-AEDANS following actin polymerization. The fluorescence intensity of the MBB and CPM probes attached to Cys¹⁰ decreased following the polymerization of actin (Fig. 4 C and D). These results support the conclusion about conformational changes that take place in the vicinity of Cys¹⁰ during actin polymerization. Binding of S1 also affects the actin structure as manifested in the intensity decrease of the emission spectra of all probes attached to Cys¹⁰ on F-actin (Fig. 4).

View larger version (24K): [in this window] [in a new window]   FIGURE 4   Effect of polymerization and S1 on the emission spectra of Cys10-labeled MgATP-G-actins. (A) Emission spectra for AEDANS-labeled MgATP-G-actin and F-actin excited at 297 nm. (B-D) AEDANS- (B), CPM- (C), and MBB-actin (D) with excitation set at 334, 390, and 380 nm, respectively. Spectra: a, G-actin; b, F-actin; c, F-actin plus S1. For conditions of the measurements see Materials and Methods. Labeling stoichiometries of AEDANS-, CPM-, and MBB-actin were 0.51, 0.98, and 0.91 probe/actin, respectively.

The effect of polymerization and S1 binding on the accessibility of the probes on Cys¹⁰ was studied by collisional quenching of fluorescence with nitromethane and acrylamide (Table 2). Nitromethane quenches the fluorescence of all three probes while acrylamide is an effective quencher only for IAEDANS and CPM. The Stern-Volmer quenching constants (K_SV) were calculated by plotting F_o/F against the quencher concentration as described in MATERIALS AND METHODS. All plots were linear indicating the dynamic nature of the quenching. The probes on Cys¹⁰ are all partially shielded from the quenchers since their K_SV values are substantially smaller than those of free probes. The K_SV values obtained for F-actin were lower than those for G-actin but not for all combinations of probes and quenchers. On the other hand, addition of S1 significantly reduced the accessibility of probes to quenchers in all cases except the acrylamide quenching of AEDANS-Cys¹⁰-F-actin. The quenching results substantiate the conclusion that actin polymerization and the binding of S1 induce conformational changes in the vicinity of Cys¹⁰ on subdomain 1 of actin.

	DISCUSSION

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Because of its reactivity and accessibility to reagents Cys³⁷⁴ on the actin C-terminus has been the main site for attachment of fluorescent and spin probes on -actin. Other sites of actin labeling, Lys-61 and Tyr-69, although useful for fluorescence energy transfer (FRET) measurements (Barden et al., 1987; Miki et al., 1992), are involved in actin polymerization (Miki, 1987; Holmes et al., 1990; Chantler and Gratzer, 1975) and their derivatization affects actin properties. The labeling of Gln-41 on actin with a dansyl probe appears to have no effect on actomyosin interactions (Kim et al., 1998), but the transglutaminase mediated reaction does not offer the same labeling flexibility and choices as cysteine modifications do. These considerations prompted the attempts to label Cys¹⁰ on actin with reporter groups for spectroscopic studies. Specific modification of this residue appeared to be achieved on Cys³⁷⁴ pre-blocked G-actin in the presence of 2.0 M urea (Barden et al., 1987). However, the distances determined by FRET between Cys¹⁰ and other sites of actin were inconsistent with the atomic structure of G-actin suggesting that the exposure to urea might have altered its structure irreversibly (O'Donoghue et al., 1992). An alternative procedure for attacking Cys¹⁰ in the structurally labile MgADP-G-actin (with a pre-blocked Cys³⁷⁴) was developed by Drewes and Faulstich (1991). According to these authors, the restoration of native G-actin structure is achieved upon transformation of the labeled MgADP-G- actin into MgATP-actin, following its long incubation with ATP.

The results of this study, with three different thiol regents, confirm the previous observation on the feasibility of a specific Cys¹⁰ modification and the reversal of the ADP-induced structural destabilization of actin (Drewes and Faulstich, 1991). Moreover this work documents that three key functional properties of actin are affected only marginally by, CPM, MBB, and AEDANS probes attached to Cys¹⁰. The polymerization of labeled actin, its in vitro motility over HMM, and the kinetic parameters V_max and K_M of acto-S1 ATPase are similar to those of control actin. Obviously, our results show also that the fluorescent probes attached to Cys¹⁰ do induce probe-dependent, local changes in the nucleotide cleft and in loop 18-29. Importantly, these structural perturbations of actin by Cys¹⁰ probes have little impact on its polymerization and interactions with myosin. This indicates that the local perturbations caused by Cys-labeling either do not spread to functionally important sites on actin or are inconsequential to their action. Conversely, both polymerization of actin and the binding of S1 do cause small but distinct changes in the fluorescence emission of the probes. Because Cys¹⁰ is not located at the binding interface with S1 or with other actin protomers in F-actin, the above spectral transitions reflect allosteric changes in the environment of the probe, which are transmitted from other sites on actin. Thus, the main result of this study is the addition of the Cys¹⁰ site to the growing number of structural elements of actin that show dynamic changes upon S1 binding and actin-actin interaction. On the basis of the tests carried out in this work, Cys¹⁰ probes should provide appropriate spectroscopic tool for monitoring such dynamic changes in actin via FRET measurements.