A Fluorescent Probe for Conformational Changes in Skeletal Muscle G-Actin*

Actin from rabbit skeletal muscle has been modified with the fluorescent label N-iodoacetyl-N"(5-sulf0-1-naphthy1)ethylenediamine (1,5-I-AEDANS). Under con- ditions where the actin is in the unpolymerized form (G-actin), the addition of M$+ or KC1 results in en- hancement of the fluorescence. Titration of the labeled G-actin with Mg2+ at varying concentrations of CaCL gives, by extrapolation, a value for the dissociation constant for Mg' of 35 PM in the absence of Ca" and a calculated value of 10 PM for CaZ+ in the absence of M$+. The two metal ions compete with each other. The fluorescence enhancement induced by Mg2+ is reversed by the addition of CaZ+ and both processes are time-dependent, indicating a reversible conformational change of G-actin as a consequence of addition of di- valent metal. KC1 also enhances the fluorescence of the labeled G-actin but does not appear to compete with the divalent metal ion. The enhancement of the fluorescence is very rapid and any conformational change induced by KC1 is probably different from that induced by divalent metal ions. Finally, it is shown that loss of fluorescence of the labeled G-actin may be associated with inactivation of the actin.

At concentrations greater than the so-called critical concentration, the addition of MgClz and/or KC1 to G-actin induces a polymerization to fibrous or filamentous F-actin. The polymerization process is usually regarded as involving at least two steps: a nucleation step in which several G-actin monomers aggregate, followed by a highly cooperative elongation reaction (1,2). There have been a number of studies which indicate that actin changes conformation during the G to F transformation (3)(4)(5), but little information has been presented to indicate whether conformational changes occur within the Gactin itself or as a consequence of the formation of the doublestranded polymer. In one study of this question, Rich and Estes (6) showed that the addition of KC1 to actin at concentrations below those required to form a polymer decreased the rate at which the actin was proteolytically degraded. Since the rate of proteolysis induced by KC1 was similar to that of F-actin, these authors postulated a new form of actin which they termed F-actin monomer.
* This work was supported in part by Grant AM 13332 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In this paper, we have labeled G-actin with N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine (1,5-I-AEDANS) ' and have observed that the addition of Mg2+ or KC1 will enhance the fluorescence at sufficiently low concentrations of actin where no polymerization occurs. Under these conditions, it is possible to obtain the dissociation constants of Mg", Ca2+, and KC1 for G-actin. Further, the Mg'+-induced fluorescence enhancement is, in part, time-dependent and therefore presumably represents a conformational change after binding of this cation. Finally, we find that the fluorescence of 1,s-AEDANS-G-actin is decreased when the actin is inactivated and this decrease can be used as a monitor of inactivation.

MATERIALS AND METHODS
G-actin was extracted from rabbit skeletal muscle acetone powder as described by Spudich and Watt (7). However, 30 rnin after adding 100 mM KC1 and 2 mM MgC12 to induce polymerization, KC1 was added to 0.8 M (rather than 0.6 M) and the solution was stirred at 4OC for 30 min prior to centrifugation. Actin was labeled with 1.5-1-AEDANS by a procedure similar to that of Tawada et al. (8). G-actin (1 mg/ml) in 2 mM Tris/Cl, pH 8, 0.2 mM ATP, 0.2 mM CaCb, and 0.01% NaN:r was treated with an equimolar amount of the dye (dissolved in dimethylformamide and serially diluted with buffer to give a final concentration of dimethylformamide of less than 0.5%). The reaction was allowed to proceed for 50 h at 4°C in the dark.l The labeled F-actin was centrifuged at 160,000 X g for 2 b and suspended in 2 mM Tris/Cl, pH 8, containing 0.2 m~ ATP, 0.2 mM CaCh, and 0.01% NaN:r and dialyzed extensively against this buffer to depolymerize the actin. Before use, the labeled G-actin was clarified by centrifugation at 160,000 X g for 90 min. The concentration of label was determined from the absorbance at 337 nm using an extinction coefficient of 6,000 M" cm" (8). Actin concentration was measured by the absorbance at 290 nm using an A&";"/"' = 0.63 and corrected for absorbance due to the dye by subtracting 0.21 An:n from A~w (5,9). The molar ratio of dye to actin in several preparations was 0.8 f 0.1.
Fluorescence studies were performed on a Spex Fluorolog spectrofluorometer using excitation and emission wavelengths of 340 and 460 nm, respectively, for maximal fluorescence enhancement. The fluorometer was used in the E/R mode which corrects for any changes in light intensity during the experiment. Fluorometer titrations were performed by the addition of small volumes of ligand with correction for any dilution (total dilution was maintained at less than 10%). Titration curves (as shown in Fig. 1, for example) were completed within 40 min of dilution of labeled G-actin into the cuvette. All titrations were performed a t 20°C.
All nucleotides used were obtained from Sigma Chemical Co. 1,5-I-AEDANS was obtained from Aldrich and ultrapure metals were obtained from Ventron Corp. (Alpha Division).

RESULTS
1,5-I-AEDANS is believed to specifically label cysteine-373 (9) of actin and the labeled G-actin is fully capable of undergoing polymerization. The experiments reported below, however, were performed under conditions where no polymerization occurs since the concentration of actin used (10 pug/&) was below the critical concentration. Thus, at the end of the experiments, the labeled actin remained in the supernatant fluid after centrifugation at 160,000 X g for 2 h. Also, as shown below, the G-actin was not inactivated during the time course of the experiments.  and pH 8, in 2 m~ Tris/Cl buffer? In each experiment, the ATP concentration was adjusted to give a free concentration of ATP, prior to the titration, of 4 p~. For these determinations, the MgATP dissociation constant under these conditions was assumed to be 3 p~ (10) and that for CaATP was assumed to be about 2-fold larger (6 p~) (11). The ATP level in these experiments was sufficiently low that differences between total and free concentrations of Mg'+ were small even at the lower concentration of Mg2+ used. Scatchard plots of the raw data shown in Fig. 1  In most experiments, titrations were performed using ultrapure MgSO,. However, identical results were observed with reagent grade MgCL as titrant.
Some nonlinearity in Scatchard plots would be expected since the addition of Mg'+ to the G-actin solution containing Cas+ and ATP will release Ca'+ from the Ca-ATP complex. Thus, the concentration of Ca2+ (which competes with Mg") will increase somewhat during the titration. However, computer simulation of the titration data under the conditions given in Fig. 1 show that deviation from linearity in the Scatchard plot will occur only near the start of the titration. The data shown in the inset of Fig. 1 therefore are plotted as a function of the total Ca2+ concentration rather than the free concentration.
It may also be seen from Fig. 2 that the addition of Ca2+ reverses the fluorescence enhancement induced by Mg2+. Return of the fluorescence to its original value is also a timedependent process and is also independent of actin concentration (data not shown).
As discussed below, both of these changes probably reflect metal-induced conformational changes.
Titration of G-Actin with KCI-The addition of KC1 to 1,5-AEDANS-labeled G-actin also results in an enhancement of the fluorescence either in the presence or absence of Mg".  fluorescence to about 70% of its initial value. A t concentrations of G-actin of 10 pg/ml (0.23 p~) and Ca"' and ATP concentrations of about 1 p~, the tip for the loss of fluorescence at 20°C is about 20 min. After the loss of fluorescence, addition of Mg2+ does not enhance the remaining fluorescence. The same extent of loss of fluorescence occurs in less than 5 min in the presence of EDTA, in agreement with observations that EDTA enhances inactivation (5). Low concentrations (10 p~) of either Ca2+ or ATP and higher concentrations of Mg2+ (ie. 1 mM) stabilize the G-actin against the loss of fluorescence and presumably, therefore, the inactivation, so that no inactivation occurs in the time course of the experiments. It should be noted that the fluorescence change observed here is in the opposite direction from that induced by Mg2+ or KC1. The titration experiments described in Figs. 1 and 3 were performed under conditions where no loss of fluorescence occurs, since inactivation is prevented by ATP or Ca'+.

DISCUSSION
There have been several reports related to the use of 1,5-I-AEDANS-labeled actin (8,9,12). Tao and Cho (9) concluded, on the basis of fluorescence lifetime studies, that the major labeling site was cysteine-373 with labeling of other sites being considerably less. Studies which have utilized this label have done so primarily with respect to polymerization of the actin or with respect to interaction with other proteins. Clearly, however, the fluorescence changes reported in this paper are not associated with polymerization, since the concentration of G-actin used is below the critical concentration and the changes which occur are rapid. Furthermore, no further change in fluorescence is observed when higher concentrations of labeled G-actin undergo polymerization. The observed fluorescence changes allow one to determine the absolute dissociation constant of G-actin for Mg2+ and Ca2+ and perhaps other metals as well and to show that these metals directly compete for a particular binding site. This site may be the socalled tight-binding site for metal, A value of -10 p~ for the dissociation constant of Ca2+ to G-actin is in agreement with estimates of 1 to 10 p~ presented in the literature (13-15). In addition, inactivation of the actin may be measured as a loss in fluorescence. Thus, 1,5-AEDANS-labeled actin may be a convenient probe for several properties of G-actin.
Evidence for a KC1-induced conformational change has been reported by Rich and Estes (6). Their conclusion was based on the observation that in the presence of KC1, G-actin was degraded by proteolytic enzymes at a slower rate than in the absence of KCl. These authors termed this form the F-ATP-actin monomer. However, it is quite possible that there are a number of different conformational forms of G-actin. Our results, for example, suggest that the change induced by KC1 is different from that induced by divalent metals, since I) the former is rapid and the latter is slow; 2) the addition of (although to a smaller extent); and 3) KC1 and Mi'+ do not appear to compete with each other.
Of particular interest is the time dependence of the Mg2'induced fluorescence change and its time-dependent reversal on addition of Ca" (Fig. 2). Since the dissociation constants for CaZ+ and Mg'+ are 10 and 35 p~, respectively, it would be expected that dissociation of either metal from the metalbinding site should be very rapid. For example, assuming a diffusion-controlled on-rate constant of lo7 s" mol-', the offrate may be calculated as greater than 100 s" (t1/2 < 0.007 s).
Thus, the slow changes observed in Fig. 2 are not the consequence of a slow off-rate of the metal ion, but rather a reflection of conformational changes which occur on replacing one metal by the other. That the rate of the fluorescence change is independent of the actin concentration reinforces this view (data not shown). Furthermore, this conformational change is reversible, since the addition of Ca"' returns the Mg"-induced fluorescence enhancement to the original value. The data suggest the following basic scheme: where G . ATP (G-actin with ATP bound) may exist in at least two conformational forms, G.ATP and G'-ATP, and the conversion between them is reversible. To our knowledge, the results presented here represent the first direct evidence for a metal ion-induced conformational change of G-actin. We are unable to say, at present, whether the conformational change induced by Mg2' is an essential one for a given mode of actin polymerization.