- Email: [email protected]
The helix content of tropomyosin and the interaction between tropomyosin and F-actin under various conditions
556
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 36222
T H E H E L I X CONTENT OF TROPOMYOSIN AND T H E I N T E R A C T I O N B E T W E E N TROPOMYOSIN AND F-ACTIN U N D E R VARIOUS CONDITIONS
H I D E H I R O TANAKA
Department of Physics, l:aculty of Science, Nago~,a University Chikusa-hu, Nagoya 464 (Japau) (Received May ioth, 1972)
SUMMARY
The interaction between tropomyosin and F-actin was studied by means of flow birefringence and optical rotatory dispersion methods. An analysis of the results suggests that the conformation of tropomyosin plays an important role in its binding to F-actin, i.e. there exists a threshold value for the helix content of tropomyosin necessary for binding. Investigations of the binding under various conditions also showed that (I) the lowering of pH and the addition of ethanol induced, as a primary effect, the stabilization of the helical structure of tropomyosin, while (2) the addition of small amounts of divalent cations made the polymer structure of l:-actin more rigid. Both conditions made the binding of tropomyosin to Y-actin stronger. Urea affected both F-actin and tropomyosin, making their structures more flexible or looser, thus causing a weakening of the binding. The effects of pH, ethanol, urea and divalent cations on structure of the proteins and on their interactions are discussed from the point of view of the conformational change of protein structure. 1;inally, the effect of salt (KC1) on this interaction is also discussed.
INTRODUCTION
At low shear rates F-actin shows flow birefringence and tropomyosin does not. However, when mixed with F-actin, tropomyosin can increase the degree of flow birefringence of F-actin by binding to F-actin. Therefore, the flow birefringence measurement of tropomyosin F-actin mixtures is useful for the study of the binding of troponlyosin to F-actin under various conditions l& In a previous paper 2, it was concluded that tropomyosin binds to F-actin within a certain range of environmental conditions. At physiological salt concentrations, tropomyosin binds to F-actin at room temperature, but it dissociates at higher temperatures. This dissociation temperature is dependent on the solvent condition. It is made higher by lowering pH or addition of ethanol. On the other hand, it becomes lower when a small amount of urea is added or when the salt condition deviates from the physiological one. These analyses have suggested some correlation between the dissociation temperature and the a-helix content of tropomyosin. Biochim. t3iophys, dcta, 278 (I972) 550 560
COMPLEX OF TROPOMYOSIN AND F-ACTIN
557
Therefore, we examined the effects of pH, ethanol, urea, mono and divalent cations on the dissociation temperature between tropomyosin and F-actin, and on the thermostability of the a-helical structure of tropomyosin. MATERIALS
A cti~z Acetone-dried powder was prepared from rabbit skeletal muscle by the method of Straub 3. G-actin was extracted at low temperature (2 °C) with 20 ml of distilled water per g of the acetone powder. For polymerization, KC1 was added to the extracted solution to 60 rnM. The F-actin solution thus obtained was sedimented by ultracentrifugation at 78 ooo × g for 3 h. Following this procedure, repeated cycles of depolymerization and polymerization were performed.
Tropornyosi~z Tropomyosin was prepared by the method of Ebashi et al?. METHODS
Flow birefringence Flow birefringence was measured at a shear rate of 16 s -1 by a temperaturecontrolled Rao-type home-made apparatus. The degree of flow birefringence (3n) of F-actin is proportional to its concentration 5 (G-actin has no contribution). The degree of flow birefringence is scaled in arbitrary units throughout this paper (An of a I mg/ml F-actin solution is 77 °). The degree of flow birefringence of F-actin increased on the addition of tropomyosin at the physiological condition. This increase was proportional to the amount of bound tropomyosin up to stoichiometric saturation of tropomyosin to F-actin. Further addition of tropomyosin did not contribute to An, as shown in Fig. I (tropomyosin/F-actin is about 1/3 I/4 by weight). The value of An of the mixture is a measure of the binding between tropomyosin and F-actin. When the ratio of added tropomyosin to F-actin is lower than the stoichiometric one, the decrease of An means dissociation. lOO i
80 ~
/o--O--o
60 ° ~ ° ,dn 4O 2O
%'
o!2 0'.4 &
Added tcopomyosin (mg/ml) Fig. I. F l o w b i r e f r i n g e n c e of a m i x t u r e of F - a c t i n a n d t r o p o m y o s i n . S o l v e n t c o n d i t i o n : 70 mM KC1, IO mM T ris-HC1 (pH 8.0) a n d 0.2 mM A T P a t 20 °C. V a r i o u s a m o u n t s of t r o p o m y o s i n w e re a d d e d to a c o n s t a n t a m o u n t of F - a c t i n (0.8 mg/ml).
Biochim. Biophys. dcta, 278 (i972) 556-566
558
H. tANAKA
optical rotatory dispersion The optical rotation of tropomyosin was measured with a temperaturecontrolled Jasco autospectrophotometer, ORD/UV-5. Rotations were expressed in terms of reduced mean residue rotation, Em']z; 3
[m'],,, ~
AI0
!?@.
~- 2 IOO
where Ia]~ is the specific rotation, M 0 is the mean residual weight of tropomyosin and assumed to be 12o (ref. 6) and n is the refractive index determined by a Carl Zeiss interferometer. The concentration of tropomyosin was determined by the method of Ooi 7, using the absorbance value, A~ ~;~, at 277 n m = 2.5. Then, a value of [m']2aa nnl -I I 5o0 was obtained at pH 8.0 and 20 °C. This value is lower than that obtained by Woods s, who used the value of 3.3 for AI ~%. To obtain a well-accepted value of the stoichiometric ratio of the binding of tropomyosin to F-actin, we preferred the above value of A ~ 2~ of tropomyosin. The specific rotation at 233 nm, denoted simply by ira'] throughout this paper, is used as a measure of the helix content of tropomyosin under various conditions.
Ultracentrifugation Ultracentrifugation was performed with a temperature-controlled Hitachi 55P2 preparative ultracentrifuge. The amount of protein sedimented (i.e. the tropomyosin-F-actin complex) was determined by the Biuret method. RESULTS
The interaction between tropomyosin and F-actin Effect of temperature. As published before, the degree of flow birefringence of the mixture of F-actin and tropomyosin decreased with increasing temperature within the temperature range studied (~<55 °C) to the value of F-actin alone and increased reversibly with lowering temperature. However, when the experiment was performed carefully, the critical temperature, where the half dissociation of tropomyosin was observed, was slightly different from the temperature, where the half association of tropomyosin was again observed by lowering temperature. This difference was detected only in the first cycle of the temperature change, as shown in Fig. 2. The dissociation temperature in the first cycle was always higher by about 2 °C than the
zJ
o--o~
30~" , 30
--o--~
4JO Ternpera%ure
Figi 2. T e n l p e r a t u r e dependence of the degree of flow birefringence of a m i x t u r e of troponlyosin and F-actin. F-actin o.52 mg/ml, t r o p o m y o s i n o.17 mg/ml, in 8o mM KC1, io mM T r i s - H C l (pH 7.7) and 0.2 mM ATP. T r o p o m y o s i n and F-actin were mixed at r o o m t e m p e r a t u r e and t h e n t e m p e r a t u r e was changed. ©, in the course of first increase of t e m p e r a t u r e and 0 , first decrease ; [~, second increase of t e m p e r a t u r e and II, second decrease.
Biochim. t~iophys. Acta, 278 (1972) 556-566
559
COMPLEX OF TROPOMYOSIN AND F-ACTtN
association temperature. When the two proteins were mixed at a temperature higher than the dissociation temperature, no hysteresis appeared. Therefore, the delay of the first dissociation may be due to some metastable state of binding. Hereafter we denote by "dissociation temperature" the dissociation (or association) temperature of the second (or the first) cycle of the temperature change. This "dissociation temperature" changed with the solvent condition. Effect of KCl and pH. The effect of salt on the binding between tropomyosin and F-actin at different pH values was investigated at 2o °C. Tropomyosin was added to an F-actin solution in 3o mM KC1 at the weight ratio of tropomyosin to F-actin of about I/3, and then various amounts of KC1 were added to the mixture. Fig. 3a shows that An of F-actin only was almost constant over the range of the salt concentration studied (3o-2oo mM KC1), while that of the mixture depended on the salt concentration. At lower or higher salt concentrations, less tropomyosin bound to F-actin. With lowering pH, the range of the salt concentration where tropomyosin bound to F-actin became wider, although the general feature of binding was the same (Fig. 3a). Fig. 3b shows an example of the dissociation of tropomyosin by increasing temperature at various KC1 concentrations at pH 7.7. The dissociation temperature depended not only on the KC1 concentration but also on pH. The results are summarized in Fig. 3c. The lower the pH of the solution, the higher the dissociation temperature. The salt concentration, where the higher dissociation temperature was observed, shifted to the lower side with lowering pH.
dn 80
120
3b
a
IO0
i:CG:
dn 8O
~
,
,
I t ,,,I
0.05 0.1 KCI ( M )
'
I
,~o
Temperature
o~ /
I I
0.5
. . . .
~O 4 0
P
•
k
60 40'
-o~ o
.~ r',
,
o
,o . a~
1
,
I,l,,I
0.05 0.1
t
t
I I
0.5
KCI ( M )
Fig. 3. a. KC1 concentration dependence of the binding between t r o p o m y o s i n and F-actin. O, p H 8.1 (IO mM Tris-HC1); [Z, p H 6.5 (5 mM p h o s p h a t e ) ; A , p H 8.1 (io mM Tris-HC1 and 15% ethanol). Other conditions: F-actin, i.o m g / m l ; t r o p o m y o s i n , 0.37 m g / m l ; 0.2 mM A T P and 20 °C. Broken line indicates d n of F-actin only. b. The effect of p H and KC1 concentration on the dissociation of t r o p o m y o s i n from F-actin with increasing t e m p e r a t u r e . At p H 7-7, IO mM Tris-HC1, 0.2 mM ATP and O, 60; (]L 90; O, I2o; ID, 200 and ~x, 12o (F-actin only) mM KC1. F-actin, 0.73 m g / m l ; t r o p o m y o s i n , 0. 3 mg/ml, c. Dependence of the dissociation t e m p e r a t u r e on KC1 c o n c e n t r a t i o n at various conditions• Condition is the same as a. except for the various buffers: A , io mM Tris-HC1, p H 8.1; D, " , io mM Tris HCI, p H 7.7 ( I , in the presence of 6.2% ethanol); 0 , IO mM p h o s p h a t e , p H 6.5.
The interaction between tropomyosin and F-actin in the presence of ethanol In the presence of I5% ethanol (v/v) tropomyosin could bind to F-actin at ever), concentration of salt in the range between 30 and 300 mM KC1 (Fig. 3a). Biochim. Biophys. Acta, 278 (1972) 556 566
56o
H. TANAKA
J u s t as in the case w i t h o u t ethanol, A~, of the m i x t u r e increased b y the binding of t r o p o m y o s i n to F - a c t i n a n d decreased with increasing t e m p e r a t u r e . The dissociation t e m p e r a t u r e d e p e n d e d also on the KC1 c o n c e n t r a t i o n (see also Fig. 3c). A t 6.2, 8.o a n d I I . 6 % ethanol, the highest dissociation t e m p e r a t u r e s were a b o u t 43, 48 a n d 50 °C, respectively. The dissociation t e m p e r a t u r e became higher with increasing c o n t e n t of ethanol. However, a b o v e 8.o°/~, e t h a n o l , / b ~ of the m i x t u r e was not recovered b y lowering t e m p e r a t u r e . W h e n the c o n c e n t r a t i o n of e t h a n o l was low, An of the m i x t u r e in i o o mM KC1, IO mM Tris-HC1 (pH 7.7) a n d o.2 mM A T P decreased in two steps, at a b o u t 43 a n d 55 °C. The former means the dissociation of t r o p o m y o s i n from F - a c t i n a n d the l a t t e r the irreversible d e n a t u r a t i o n of actin. H e r e a f t e r we define the t e m p e r a t u r e where the irreversible d e n a t u r a t i o n of actin is induced, as the " d e n a t u r a t i o n t e m p e r a t u r e " (Fig. 4)- W i t h increasing c o n t e n t of ethanol, b o t h the dissociation of t r o p o m y o s i n a n d the d e n a t u r a t i o n of actin occurred simultaneously. I n this case, we could not define the dissociation t e m p e r a t u r e . S u m m a r i z e d , on the a d d i t i o n of e t h a n o l the dissociation t e m p e r a t u r e becomes higher while the d e n a t u r a t i o n t e m p e r a t u r e becomes lower.
9ol-
•
,j~ / o ~ = ¢ ~ , I ~ . .
.
.
.
.
.
.
.
.
_"=o_o o_ _~O.o. o
.
.
.
.
.
/o
o--..o-,
;o
o
""
Temperature Fig. 4. Effect of ethanol on the dissociation of t r o p o m y o s i n fronl F-actin with increasing t e m p e r a ture. 6.2% ethanol, F-actin o.86 mg/ml, t r o p o m y o s i n o. 3 mg/ml, io mM T r i s - H C I (pH 7-7), o.2 mM ATP, and ©, 6o; ID, 75; 0 , 95 and (]), 125 mM KC1. Broken line m e a n s / I n of F-actin alone.
Effect of urea on F-actin and on the interactio~ betwee~ troporn3'osin and F-acti~ Effect of urea o~ F-actin. The degree of flow birefringence of F - a e t i n decreased on the a d d i t i o n of urea. The decrease of d n d e p e n d e d upon the a m o u n t of urea a d d e d (Fig. 5a). I n Fig. 5 b, the a m o u n t s of F - a c t i n (An.) at various concentrations of actin are shown. This figure shows t h a t the a m o u n t of F - a c t i n is p r o p o r t i o n a l to the a m o u n t of actin a b o v e the critical aetin c o n c e n t r a t i o n 9, which is d e t e r m i n e d b y the solvent condition (i.e. the c o n c e n t r a t i o n of urea in this case), a n d all lines are parallel. W h e n urea is absent, almost all the actin exists in a p o l y m e r i z e d state, a n d with increasing c o n c e n t r a t i o n of urea the a m o u n t of F - a c t i n decreases a n d t h a t of G-actin increases. G - F t r a n s f o r m a t i o n of aetin is a k i n d of condensation p h e n o m e n o n similar to gas liquid condensation 9. This p h e n o m e n o n always occurs as long as actin is not d e n a t u r e d , even in the presence of urea. Now, the results of Fig. 5b m e a n t h a t actin in the p o l y m e r i z e d s t a t e c o n t r i b u t e s to zl n in the same way, irrespective of the c o n c e n t r a t i o n of urea. To confirm the existence of F - a c t i n in the presence of urea, aetin solutions at various concentrations of urea were u l t r a e e n t r i f u g e d at I O O o o o × g for 3 h at 20 °C a n d the p r o t e i n concentration of the sediments was d e t e r m i n e d ( ~ - - - - --L~ in Fig. 5a). The a m o u n t of protein s e d i m e n t e d decreased with increasing c o n c e n t r a t i o n of urea, corresponding to the flow birefringence results. Therefore, F - a c t i n has the same p r o p e r t y in the presence a n d absence of urea a n d the degree of flow birefringence is p r o p o r t i o n a l to the c o n c e n t r a t i o n of F - a c t i n . Biochim. Biophys. dcla, 278 (1972) 556-566
COMPLEX OF TROPOMYOSIN AND F-ACT1N
~h
56i
a
100 ~4
dn 100
A-___~.~. A 9.O'& E
b o
6.0 $ E 3.0 tn
"oo | .
° ~
Oh 0
I 1.0
o
..
i ~ o 2.0
0
I" 3.0
4,0
0
05
1.0
I
1.5
Concentration of actin (rng/ml)
Coneentration of urea (M)
Fig. 5. Effect of u r e a on F - a c t i n . a. F - a c t i n u n d e r v a r i o u s c o n c e n t r a t i o n s of urea. F l ow birefringence: An of a c t i n s o l u t i o n was m e a s u r e d 2o rain a f t e r t h e a d d i t i o n of urea. S o l v e n t c o n d i t i o n ; i o o mM KC1, [o mM Tris HCI (pH 8.o) a n d o.2 mM A T P a t 2o °C. C o n c e n t r a t i o n o f a c t i n : A - - A , 1. 3 m g / m l ; A - - , ' ~ , i.o m g / m l ; 0 - - 0 , 0.55 m g / m l ; O - - © , o.26 m g / m l . U l t r a c e n t r i f u g a t i o n : Fa c t i n s o l u t i o n s (4 ml of 1.2 m g / m l ) in v a r i o u s c o n c e n t r a t i o n s of u r e a were c e n t r i f u g e d a t 2o °C a n d t h e a m o u n t of s e d i m e n t was d e t e r m i n e d . S o l v e n t c o n d i t i o n : t h e s a m e as above. ( ~ [~). b. G F e q u i l i b r i u m of actin. This figure is a r e p l o t of a. The c o n c e n t r a t i o n of u r e a : (_3, o M; O, 1.6 M; ~ , 2.0 M; A, 2.6 M; [], 3.2 M.
Binding of tr@omyosin at various concentrations of urea. To m i x t u r e of t r o p o m y o s i n a n d F - a c t i n , various a m o u n t s of urea were a d d e d u n d e r the condition where the b i n d i n g was confirmed in the absence of urea at 2o °C. W h e n the a m o u n t of Vactin itself decreased at high c o n c e n t r a t i o n s of urea, the a m o u n t of t r o p o m y o s i n b o u n d to F - a c t i n was o b t a i n e d b y s u b t r a c t i n g the zln of E-aetin from t h a t of the m i x t u r e (fig. 6a). As shown in Fig. 6a, t r o p o m y o s i n begins to dissociate from F - a c t i n at a b o u t o. 7 M urea, a n d it can h a r d l y b i n d to F - a c t i n a b o v e I.o M urea. A s t u d y using u l t r a c e n t r i f u g a t i o n confirmed this result (Fig. 6b). A t z.o M urea the a m o u n t of s e d i m e n t e d protein of the m i x t u r e was almost the same as t h a t of E-actin alone. Effect of temperature. The a d d i t i o n of a small a m o u n t of urea was v e r y effective ~n
a
b ~5
40
E 3.0
g
20
2.5
7-:h
E co 0 5
D~o~ o
0'5
1!o
1
1.5
Concentration of urea (M)
0 I
o
I
0.5
1'.o
Concentration of
115
urea (M)
Fig. 6. T h e b i n d i n g a t v a r i o u s c o n c e n t r a t i o n s of urea. a. F l o w b i r e f r i n g e n c e : O, m i x t u r e of F - a c t i n (o.39 m g / m l ) a n d t r o p o m y o s i n (o.15 m g / m l ) ; 0 , F - a c t i n (o.39 m g / m l ) ; [B, difference of A n b e t w e e n t h e m i x t u r e a n d F - a c t i n only. S o l v e n t c o n d i t i o n : t h e s a m e as Fig. 5. b. U l t r a c e n t r i f u g a t i o n : o.62 m g / m l F - a c t i n , o.2 m g / m l t r o p o m y o s i n . S o l v e n t c o n d i t i o n a n d s y m b o l s are t he s a m e as above. S a m p l e s o l u t i o n s (4.4 ml) were c e n t r i f u g e d a t 2o °C a n d t h e a m o u n t s of s e d i m e n t s were d e t e r m i n e d .
t?iochim. Biophys. 14cta, 278 (i972) 556-566
562
H. TANAKA
in lowering the dissociation t e m p e r a t u r e . A t 0.25 M urea, the highest dissociation t e m p e r a t u r e was a b o u t 38 °C a n d at 0.5 M urea it was 3o °C. The higher the concent r a t i o n of urea, the lower the dissociation t e m p e r a t u r e . This t e m p e r a t u r e dependence of the b i n d i n g was reversible. However, a b o v e I.O M urea, no b i n d i n g was observed even if the t e m p e r a t u r e was lowered to 2 °C. A l t h o u g h the a d d i t i o n of urea resulted in the lowering of the dissociation t e m p e r a t u r e , the salt c o n c e n t r a t i o n dependence of the binding was still observed, just like in the case of absence of urea. Reversibility of the effect of urea. G-actin formed b y d e p o l y m e r i z a t i o n of F - a c t i n on the a d d i t i o n of 1.6 M urea could polymerize again into F - a c t i n when urea was r e m o v e d b y dialysis. In the case of 3.2 M urea, however, a b o u t one-half of G-actin could not polymerize after r e m o v a l of urea. This m a y be due to p a r t i a l d e n a t u r a t i o n of actin. U r e a of i . o M was a d d e d to a m i x t u r e in IOO mM KC1, IO mM Tris HC1 (pH 8.o) a n d o.2 mM A T P . A f t e r dissociation of t r o p o m y o s i n from F - a c t i n , it was d i l u t e d with the solvent so t h a t b o t h the c o n c e n t r a t i o n of urea a n d proteins decreased. As references, b o t h the m i x t u r e a n d an F - a c t i n solution in the absence of u r e a were also diluted. The degree of flow birefringence of these two references decreased linearly with dilution as e x p e c t e d (U a n d O in Fig. 7). I f the effect of urea on the b i n d i n g be r e m o v e d reversibly, t r o p o m y o s i n m u s t b i n d again to F - a c t i n with lowering concent r a t i o n of urea. Then, ,dn of the m i x t u r e would not decrease linearly with dilution. Fig. 7 (e) clearly shows t h a t t r o p o m y o s i n , once dissociated from F - a c t i n in I.O M urea, could b i n d again to F - a c t i n when the c o n c e n t r a t i o n of urea was lowered. Z~D
200
,
°°
I
~'4
I
2~4
I
3J4
E x t e n t of dilution
Fig. 7. Reversibility of the effect of urea on,the binding. Mixture of F-actin and tropolnyosin in the absence ([~) and presence (O) of i M urea. F-Actin in the absence of urea (O). The three solutions were diluted with the solvent: IOO mM KC1, IO mM Tris-HC1 (pH 8.o) and o.2 mM A T P at 25 °C. Solutions before dilution contain 0.95 m g / m l t r o p o m y o s i n a n d / o r 2. 7 mg/rnl F-actin. The abscissa shows the e x t e n t of dilution.
The effect of divalent cations on the binding between tropomyosin and F-actin G-actin polymerizes into F - a c t i n on a d d i t i o n of KC1, a n d 0.I M KC1 is o p t i m a l for full p o l y m e r i z a t i o n . However, a 2 mM c o n c e n t r a t i o n of d i v a l e n t cations is enough for full p o l y m e r i z a t i o n , even in the absence of KCP. T r o p o m y o s i n c a n n o t b i n d to F actin at low concentrations of salt (say, less t h a n 20 mM KC1), while in 2 mM d i v a l e n t cations (Mg 2+, Ca ~+ or B a 2+) full b i n d i n g occurs, even t h o u g h KC1 is a b s e n t 1. Biochim. I3iophys. Acta, 278 (1972) 556-566
563
COMPLEX OF TROPOMYOSIN AND F-ACTIN
At pH 7.7 in the presence of KCI, the highest dissociation temperature was observed at about 42 °C, 48 °C in 8.0% ethanol and 38 °C in 0.25 M urea. However, the addition of 2.5 mM MgC12 resulted in rise of the dissociation temperature to 48, 52 and 45 °C, respectively. In the presence of MgC12, the salt (KC1) concentration dependence of the dissociation temperature was still observed. In 2.5 mM MgC12, 1.5 M urea cannot dissociate tropomyosin from F-actin at 2o °C (cf. Fig. 6). The same trend was also observed for Ca 2+, but it was less effective than Mg2+.
The helix content of tropomyosin It is well known that the helical structure of tropomyosin becomes more stable at lower pH values or in the presence of ethanol, but more unstable on the addition of urea or at higher temperatures. Thus, the effect of pH, ethanol, urea and temperature on the stability of the helical structure of tropomyosin seems to be similar to the effect of those on the dissociation temperature mentioned above. We measured the helix content of tropomyosin at various temperatures and solvent conditions. With lowering pH or by adding ethanol, the helical structure of tropomyosin became more stable against increasing temperature (Fig. 8a and 8b). The higher the concentration of ethanol, the more stable the helical structure of tropomyosin within the concentration range studied (o-16.7%). On the other hand, the addition of urea resulted in the decrease of the helix content. [m ']
a
13
-12c~°I
[t ~-~._.
-8000
"
o,~,
-4000
I
I
I
30 40 50 Temperature
I
I
I
30 40 50 Temperature
Fig. 8. a. Effect of p H on t h e helix c o n t e n t of t r o p o m y o s i n , i . i m g / m l t r o p o m y o s i n , in 20 m M KCl a n d v a r i o u s buffers (both a t p H 8. 4 a n d 7.8; io m M Tris-HC1, b o t h at p H 7.2 a n d 6.5; io m M p h o s p h a t e ) , p H : O, 6.5; [Z, 7.2; 0 , 7.8; /~, 8.4- b. Effect of e t h a n o l on t h e helix c o n t e n t of t r o p o m y o s i n , i.o m g / m l t r o p o m y o s i n , in io m M Tris-HC1 (pH 8.o), 2o m M KC1 a n d (), o; [~, 8. 3 a n d Q, 16.7% ethanol.
DISCUSSION
Mechanism of interaction As described in the previous paper 2 and this paper, many conditions have to be satisfied for the binding between tropomyosin and F-actin. Tropomyosin bound to Factin dissociates with deviation of the salt concentration from optimal (i.e.o.I M KC1), on the addition of urea, or with increasing temperature. The dissociation ternBiochim. Biophys. Acta, 278 (1972) 556 566
564
H. TANAKA
perature depends on KC1, pH, ethanol, urea and divalent cations. Detailed analyses of the dissociation t e m p e r a t u r e seem to be very useful to elucidate a possible mechanism of the i n t e r a c t i o n between tropomyosin a n d F-actin. For this purpose, we tried to find some properties which change in parallel to the dissociation temperature. From this point of view, a possible correlation between the helical structure of tropomyosin a n d its b i n d i n g to F - a c t i n was investigated. We define the dissociation t e m p e r a t u r e at the o p t i m a l salt (KC1) condition as the "highest" dissociation temt)erature. Fig. 9 a shows the highest dissociation temperatures at various p H values. On the other hand, Fig. 9 b shows the t e m p e r a t u r e - p H relation of t r o p o m y o s i n at c o n s t a n t values of Fm']. As shown in these figures, lowering of the dissociation t e m p e r a t u r e with increasing p H occurred in parallel to the lowering of the t e m p e r a t u r e where [m'] -- ---8ooo. Therefore, we use the t e m p e r a t u r e where [m'] = - - 8 o 0 o as a measure of t e m p e r a t u r e specifying the state of tropomyosin, a n d the highest dissociation t e m p e r a t u r e as a measure of t e m p e r a t u r e related to the interaction between t r o p o m y o s i n a n d F-actin. a
b
cO 5 o
50
45
c° 45
E
¢.
40
%, 35
35
I
6 T
8
pH
\o" - 9 0 0 0
30
pH
Fig. 9- a. pH dependence of the highest dissociation temperature. The highest dissociation temperature at a given pH is replotted from Fig. 3 so that the lowering of the dissociation temperature with pH is clearly seen. b. pH dependence of the helix content of tropomyosin. This figure is reph)tted from the results in Fig. 8a and shows pH dependence of the temperature at a given helix content [m'] : ~, -- 7ooo; O, 8000 and ~ , - -9ooo. Then, the present results can be expressed as a relation between these two temperatures, as shown in Fig. Ioa. This figure suggests t h a t the effects of (i) p H a n d ethanol, (ii) urea (iii) d i v a l e n t cations a n d (iv) salt on the b i n d i n g are different from each other. I n Fig. lob, is shown a schematic representation of these four types of effects on the binding. (i) p H and ethanol. The rise of tile highest dissociation t e m p e r a t u r e with lowering p H or on the addition of ethanol occurs in parallel to tile change of the stability of the helical structure of tropomyosin. The highest dissociation t e m p e r a t u r e s at different p H values a n d different ethanol concentrations lie on a straight line B in Fig. lob. The slope of this line B is a b o u t u n i t y , suggesting the direct correlation between the helix stability of t r o p o m y o s i n a n d its b i n d i n g to F - a e t i n at the o p t i m a l salt condition. I n Fig. Ioa and lob, tile ordinate gives the t e m p e r a t u r e where [m'] = ---8000. If it were scaled b y the t e m p e r a t u r e where [m'] = - - 9 0 0 0 , the result would be expressed in the same way. However, the t e m p e r a t u r e where Ira'3 = - - 7 0 0 0 could not be t a k e n as the measure, because in t h a t t e m p e r a t u r e range no b i n d i n g was ob-
Biochim. Biophys. ,4cta, 278 (1972) 556-566
COMPLEX OF TROPOMYOSIN AND F-ACTIN
565
& 0
°o 50
pH65 7595 5/12~5 ~859013'11,6'30
CO ,i
D 0 o ug
a(80)
•(8,0)
(621
E 40
_
%o N f l o 60
%o%o
20O90 t20 0120 20W30
DNSI
E 30,
I
I
I
30
40
50
Dissociation t e m p e r a t u r e
o 50 O Q
B
,o
CO i
i, r/n
[31
uJ
% 4o
A
2
~7 c
E
3o
I 30
I 40
I 50
Dissociation t e m p e r a t u r e
Fig. IO. Correlation between the dissociation t e m p e r a t u r e and the t e m p e r a t u r e where im'J = --8ooo. a. The ordinate is scaled by the t e m p e r a t u r e where [m'] -- --8o0o and the abscissa b y the dissociation t e m p e r a t u r e . O, at various p H values s h o w n in the figure; [7, at various concentrations of ethanol (concentrations are s h o w n b y n u m e r a l s in brackets) ; ~ , o.'25 M urea and V , °-5 NI urea. N u m e r a l s in the figure (not in brackets) indicate the concentration of KC1 (raM), and the closed symbols represent the presence of 2. 5 mM MgCI 2. p H of the solvent is 7.7, unless otherwise mentioned, b. Schematic r e p r e s e n t a t i o n of the result in Fig. IOa. F o r explanation see legend in a. Capitals in the figure (A, B, B', and C) represent the four types of solvent effect on the binding (see text).
served in the same salt condition. Thus, the threshold condition for the binding of tropomyosin to F-actin is given by the state of tropomyosin, i.e. where [m'] = --8o0o. (ii) Urea. The addition of urea decreases both the stability of the helical structure and the dissociation temperature. The lowering of the dissociation temperature with increasing concentration of urea is faster than the lowering of the stability of helical structure, as shown by line C in Fig. Iob, suggesting some other mechanism for the dissociation. (iii) Divalent cations. The effect of Mg2+ on the binding is the remarkable shift of the highest dissociation temperature. In the presence of Mg2+ the highest dissociation temperature is uniformly raised by about 5 °C at every condition (line B' in Fig. iob). Asai and Tawada 1° reported that in the presence of Mg2+ the denaturation temperature of actin becomes about 5 °C higher (6o °C instead of 55 °C). This suggests strengthening of the structure of F-actin by Mgz+, which may result in the raising of the dissociation temperature and the lowering of the threshold value of the helix Biochi*n. Biophys. dcta, 278 (~97 z) 556-566
566
H. TANAKA
content of tropomyosin required for the binding. On tile other hand, a small amount of urea makes F-actin more flexible or weaker, in addition to the destabilization of the helical structure of tropomyosin, resulting in the dissociation of tropomyosin at lower temperatures. (iv) Salt (KCl). The salt concentration dependence of the dissociation temperature is almost independent of the presence of ethanol, urea or divalent cation. In this sense, the effect of salt (KC1) on the binding is very different from that of the others. Salt ions change the apparent charge of tropomyosin (and actin) which may control their binding.
Physiological implications A possible mechanism of the regulation of muscular contraction has been proposed by Ebashi and Endo n. The information induced by the binding of Ca 2+ to troponin is transmitted to actin via tropomyosin to regulate the interaction between actin and myosin. Regulatory proteins work as mediators of the Ca ~+ information by changing their own structures 12 14. The condition where the helical content of tropomyosin has a direct correlation with the dissociation temperature, is very similar to the physiological one. This means that the binding between tropomyosin and F-actin is in a delicate state at the physiological condition in vivo. Therefore, if some structural effect of Ca 2+ on troponin is assumed to change the stability of the helical structure of tropomyosin, it is most likely that tropomyosin might effectively work as a mediator of the Ca 2+ information to actin by changing its binding state with actin. ACKNOWLEDGEMENTS
The author thanks Dr S. Fujime for his guidance and also Professor F. Oosawa for his encouragement throughout the present work.
REFERENCES K. M a r u y a m a , Arch. Biochem. Biophys., lO 5 (1964) 142. H. T a n a k a a n d F. Oosawa, Biochim. Biophys. Acta, 253 (1971) 274. F. B. S t r a u b , Stud. Inst. Med. Chem. Univ. Szeged., 2 (1942) 3. S. E b a s h i , A. K o d a m a a n d F, E b a s h i , J. Biochem., 64 (1968) 465 . M. Kasai, H. K a w a s h i m a a n d F. Oosawa, J. Polymer Sci., X L I V (196o) 5I. D. R. Ko min z, F. Saad, J. A. G l a n d e r a n d K. Laki, Arch. Biochem. Biophys., 7 ° (1957) 16. T. Ooi, Biochemistry, 6 (1967) 2433. E. F. Wo ods, Int. J. Prot. Res., I (1969) 29. F. Oosawa, S. A s a k u r a , K. H o t t a , N. I m a i a n d T. Ooi, J. Polymer Sci., X X X V I I (1959) 323H. Asai a n d K. T a w a d a , J. Mol. Biol., 2o (1966) 4o3 . S. E b a s h i a n d M. Endo, in J. A. V. B u t l e r a n d D. Noble, Progress in Biophysics and Molecular Biology, Vol. 18, P e r g a m o n Press, Oxford, 1968, p. 123. 12 T. W a k a b a y a s h i a n d S. E b a s h i , J . Biochem. (Tokyo), 61 (1968) 731. 13 Y. T o n o m u r a , S. W a t a n a b e a n d M. Morales, Biochemistry, 8 (1969) 217I. 14 S. l s h i w a t a a n d S. F u j i m e , J. Mol. Biol., 68 (1972) 511. I 2 3 4 5 6 7 8 9 io ii
Biochim. Biophys. Acta, 278 (1972) 556-566
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 36222
T H E H E L I X CONTENT OF TROPOMYOSIN AND T H E I N T E R A C T I O N B E T W E E N TROPOMYOSIN AND F-ACTIN U N D E R VARIOUS CONDITIONS
H I D E H I R O TANAKA
Department of Physics, l:aculty of Science, Nago~,a University Chikusa-hu, Nagoya 464 (Japau) (Received May ioth, 1972)
SUMMARY
The interaction between tropomyosin and F-actin was studied by means of flow birefringence and optical rotatory dispersion methods. An analysis of the results suggests that the conformation of tropomyosin plays an important role in its binding to F-actin, i.e. there exists a threshold value for the helix content of tropomyosin necessary for binding. Investigations of the binding under various conditions also showed that (I) the lowering of pH and the addition of ethanol induced, as a primary effect, the stabilization of the helical structure of tropomyosin, while (2) the addition of small amounts of divalent cations made the polymer structure of l:-actin more rigid. Both conditions made the binding of tropomyosin to Y-actin stronger. Urea affected both F-actin and tropomyosin, making their structures more flexible or looser, thus causing a weakening of the binding. The effects of pH, ethanol, urea and divalent cations on structure of the proteins and on their interactions are discussed from the point of view of the conformational change of protein structure. 1;inally, the effect of salt (KC1) on this interaction is also discussed.
INTRODUCTION
At low shear rates F-actin shows flow birefringence and tropomyosin does not. However, when mixed with F-actin, tropomyosin can increase the degree of flow birefringence of F-actin by binding to F-actin. Therefore, the flow birefringence measurement of tropomyosin F-actin mixtures is useful for the study of the binding of troponlyosin to F-actin under various conditions l& In a previous paper 2, it was concluded that tropomyosin binds to F-actin within a certain range of environmental conditions. At physiological salt concentrations, tropomyosin binds to F-actin at room temperature, but it dissociates at higher temperatures. This dissociation temperature is dependent on the solvent condition. It is made higher by lowering pH or addition of ethanol. On the other hand, it becomes lower when a small amount of urea is added or when the salt condition deviates from the physiological one. These analyses have suggested some correlation between the dissociation temperature and the a-helix content of tropomyosin. Biochim. t3iophys, dcta, 278 (I972) 550 560
COMPLEX OF TROPOMYOSIN AND F-ACTIN
557
Therefore, we examined the effects of pH, ethanol, urea, mono and divalent cations on the dissociation temperature between tropomyosin and F-actin, and on the thermostability of the a-helical structure of tropomyosin. MATERIALS
A cti~z Acetone-dried powder was prepared from rabbit skeletal muscle by the method of Straub 3. G-actin was extracted at low temperature (2 °C) with 20 ml of distilled water per g of the acetone powder. For polymerization, KC1 was added to the extracted solution to 60 rnM. The F-actin solution thus obtained was sedimented by ultracentrifugation at 78 ooo × g for 3 h. Following this procedure, repeated cycles of depolymerization and polymerization were performed.
Tropornyosi~z Tropomyosin was prepared by the method of Ebashi et al?. METHODS
Flow birefringence Flow birefringence was measured at a shear rate of 16 s -1 by a temperaturecontrolled Rao-type home-made apparatus. The degree of flow birefringence (3n) of F-actin is proportional to its concentration 5 (G-actin has no contribution). The degree of flow birefringence is scaled in arbitrary units throughout this paper (An of a I mg/ml F-actin solution is 77 °). The degree of flow birefringence of F-actin increased on the addition of tropomyosin at the physiological condition. This increase was proportional to the amount of bound tropomyosin up to stoichiometric saturation of tropomyosin to F-actin. Further addition of tropomyosin did not contribute to An, as shown in Fig. I (tropomyosin/F-actin is about 1/3 I/4 by weight). The value of An of the mixture is a measure of the binding between tropomyosin and F-actin. When the ratio of added tropomyosin to F-actin is lower than the stoichiometric one, the decrease of An means dissociation. lOO i
80 ~
/o--O--o
60 ° ~ ° ,dn 4O 2O
%'
o!2 0'.4 &
Added tcopomyosin (mg/ml) Fig. I. F l o w b i r e f r i n g e n c e of a m i x t u r e of F - a c t i n a n d t r o p o m y o s i n . S o l v e n t c o n d i t i o n : 70 mM KC1, IO mM T ris-HC1 (pH 8.0) a n d 0.2 mM A T P a t 20 °C. V a r i o u s a m o u n t s of t r o p o m y o s i n w e re a d d e d to a c o n s t a n t a m o u n t of F - a c t i n (0.8 mg/ml).
Biochim. Biophys. dcta, 278 (i972) 556-566
558
H. tANAKA
optical rotatory dispersion The optical rotation of tropomyosin was measured with a temperaturecontrolled Jasco autospectrophotometer, ORD/UV-5. Rotations were expressed in terms of reduced mean residue rotation, Em']z; 3
[m'],,, ~
AI0
!?@.
~- 2 IOO
where Ia]~ is the specific rotation, M 0 is the mean residual weight of tropomyosin and assumed to be 12o (ref. 6) and n is the refractive index determined by a Carl Zeiss interferometer. The concentration of tropomyosin was determined by the method of Ooi 7, using the absorbance value, A~ ~;~, at 277 n m = 2.5. Then, a value of [m']2aa nnl -I I 5o0 was obtained at pH 8.0 and 20 °C. This value is lower than that obtained by Woods s, who used the value of 3.3 for AI ~%. To obtain a well-accepted value of the stoichiometric ratio of the binding of tropomyosin to F-actin, we preferred the above value of A ~ 2~ of tropomyosin. The specific rotation at 233 nm, denoted simply by ira'] throughout this paper, is used as a measure of the helix content of tropomyosin under various conditions.
Ultracentrifugation Ultracentrifugation was performed with a temperature-controlled Hitachi 55P2 preparative ultracentrifuge. The amount of protein sedimented (i.e. the tropomyosin-F-actin complex) was determined by the Biuret method. RESULTS
The interaction between tropomyosin and F-actin Effect of temperature. As published before, the degree of flow birefringence of the mixture of F-actin and tropomyosin decreased with increasing temperature within the temperature range studied (~<55 °C) to the value of F-actin alone and increased reversibly with lowering temperature. However, when the experiment was performed carefully, the critical temperature, where the half dissociation of tropomyosin was observed, was slightly different from the temperature, where the half association of tropomyosin was again observed by lowering temperature. This difference was detected only in the first cycle of the temperature change, as shown in Fig. 2. The dissociation temperature in the first cycle was always higher by about 2 °C than the
zJ
o--o~
30~" , 30
--o--~
4JO Ternpera%ure
Figi 2. T e n l p e r a t u r e dependence of the degree of flow birefringence of a m i x t u r e of troponlyosin and F-actin. F-actin o.52 mg/ml, t r o p o m y o s i n o.17 mg/ml, in 8o mM KC1, io mM T r i s - H C l (pH 7.7) and 0.2 mM ATP. T r o p o m y o s i n and F-actin were mixed at r o o m t e m p e r a t u r e and t h e n t e m p e r a t u r e was changed. ©, in the course of first increase of t e m p e r a t u r e and 0 , first decrease ; [~, second increase of t e m p e r a t u r e and II, second decrease.
Biochim. t~iophys. Acta, 278 (1972) 556-566
559
COMPLEX OF TROPOMYOSIN AND F-ACTtN
association temperature. When the two proteins were mixed at a temperature higher than the dissociation temperature, no hysteresis appeared. Therefore, the delay of the first dissociation may be due to some metastable state of binding. Hereafter we denote by "dissociation temperature" the dissociation (or association) temperature of the second (or the first) cycle of the temperature change. This "dissociation temperature" changed with the solvent condition. Effect of KCl and pH. The effect of salt on the binding between tropomyosin and F-actin at different pH values was investigated at 2o °C. Tropomyosin was added to an F-actin solution in 3o mM KC1 at the weight ratio of tropomyosin to F-actin of about I/3, and then various amounts of KC1 were added to the mixture. Fig. 3a shows that An of F-actin only was almost constant over the range of the salt concentration studied (3o-2oo mM KC1), while that of the mixture depended on the salt concentration. At lower or higher salt concentrations, less tropomyosin bound to F-actin. With lowering pH, the range of the salt concentration where tropomyosin bound to F-actin became wider, although the general feature of binding was the same (Fig. 3a). Fig. 3b shows an example of the dissociation of tropomyosin by increasing temperature at various KC1 concentrations at pH 7.7. The dissociation temperature depended not only on the KC1 concentration but also on pH. The results are summarized in Fig. 3c. The lower the pH of the solution, the higher the dissociation temperature. The salt concentration, where the higher dissociation temperature was observed, shifted to the lower side with lowering pH.
dn 80
120
3b
a
IO0
i:CG:
dn 8O
~
,
,
I t ,,,I
0.05 0.1 KCI ( M )
'
I
,~o
Temperature
o~ /
I I
0.5
. . . .
~O 4 0
P
•
k
60 40'
-o~ o
.~ r',
,
o
,o . a~
1
,
I,l,,I
0.05 0.1
t
t
I I
0.5
KCI ( M )
Fig. 3. a. KC1 concentration dependence of the binding between t r o p o m y o s i n and F-actin. O, p H 8.1 (IO mM Tris-HC1); [Z, p H 6.5 (5 mM p h o s p h a t e ) ; A , p H 8.1 (io mM Tris-HC1 and 15% ethanol). Other conditions: F-actin, i.o m g / m l ; t r o p o m y o s i n , 0.37 m g / m l ; 0.2 mM A T P and 20 °C. Broken line indicates d n of F-actin only. b. The effect of p H and KC1 concentration on the dissociation of t r o p o m y o s i n from F-actin with increasing t e m p e r a t u r e . At p H 7-7, IO mM Tris-HC1, 0.2 mM ATP and O, 60; (]L 90; O, I2o; ID, 200 and ~x, 12o (F-actin only) mM KC1. F-actin, 0.73 m g / m l ; t r o p o m y o s i n , 0. 3 mg/ml, c. Dependence of the dissociation t e m p e r a t u r e on KC1 c o n c e n t r a t i o n at various conditions• Condition is the same as a. except for the various buffers: A , io mM Tris-HC1, p H 8.1; D, " , io mM Tris HCI, p H 7.7 ( I , in the presence of 6.2% ethanol); 0 , IO mM p h o s p h a t e , p H 6.5.
The interaction between tropomyosin and F-actin in the presence of ethanol In the presence of I5% ethanol (v/v) tropomyosin could bind to F-actin at ever), concentration of salt in the range between 30 and 300 mM KC1 (Fig. 3a). Biochim. Biophys. Acta, 278 (1972) 556 566
56o
H. TANAKA
J u s t as in the case w i t h o u t ethanol, A~, of the m i x t u r e increased b y the binding of t r o p o m y o s i n to F - a c t i n a n d decreased with increasing t e m p e r a t u r e . The dissociation t e m p e r a t u r e d e p e n d e d also on the KC1 c o n c e n t r a t i o n (see also Fig. 3c). A t 6.2, 8.o a n d I I . 6 % ethanol, the highest dissociation t e m p e r a t u r e s were a b o u t 43, 48 a n d 50 °C, respectively. The dissociation t e m p e r a t u r e became higher with increasing c o n t e n t of ethanol. However, a b o v e 8.o°/~, e t h a n o l , / b ~ of the m i x t u r e was not recovered b y lowering t e m p e r a t u r e . W h e n the c o n c e n t r a t i o n of e t h a n o l was low, An of the m i x t u r e in i o o mM KC1, IO mM Tris-HC1 (pH 7.7) a n d o.2 mM A T P decreased in two steps, at a b o u t 43 a n d 55 °C. The former means the dissociation of t r o p o m y o s i n from F - a c t i n a n d the l a t t e r the irreversible d e n a t u r a t i o n of actin. H e r e a f t e r we define the t e m p e r a t u r e where the irreversible d e n a t u r a t i o n of actin is induced, as the " d e n a t u r a t i o n t e m p e r a t u r e " (Fig. 4)- W i t h increasing c o n t e n t of ethanol, b o t h the dissociation of t r o p o m y o s i n a n d the d e n a t u r a t i o n of actin occurred simultaneously. I n this case, we could not define the dissociation t e m p e r a t u r e . S u m m a r i z e d , on the a d d i t i o n of e t h a n o l the dissociation t e m p e r a t u r e becomes higher while the d e n a t u r a t i o n t e m p e r a t u r e becomes lower.
9ol-
•
,j~ / o ~ = ¢ ~ , I ~ . .
.
.
.
.
.
.
.
.
_"=o_o o_ _~O.o. o
.
.
.
.
.
/o
o--..o-,
;o
o
""
Temperature Fig. 4. Effect of ethanol on the dissociation of t r o p o m y o s i n fronl F-actin with increasing t e m p e r a ture. 6.2% ethanol, F-actin o.86 mg/ml, t r o p o m y o s i n o. 3 mg/ml, io mM T r i s - H C I (pH 7-7), o.2 mM ATP, and ©, 6o; ID, 75; 0 , 95 and (]), 125 mM KC1. Broken line m e a n s / I n of F-actin alone.
Effect of urea on F-actin and on the interactio~ betwee~ troporn3'osin and F-acti~ Effect of urea o~ F-actin. The degree of flow birefringence of F - a e t i n decreased on the a d d i t i o n of urea. The decrease of d n d e p e n d e d upon the a m o u n t of urea a d d e d (Fig. 5a). I n Fig. 5 b, the a m o u n t s of F - a c t i n (An.) at various concentrations of actin are shown. This figure shows t h a t the a m o u n t of F - a c t i n is p r o p o r t i o n a l to the a m o u n t of actin a b o v e the critical aetin c o n c e n t r a t i o n 9, which is d e t e r m i n e d b y the solvent condition (i.e. the c o n c e n t r a t i o n of urea in this case), a n d all lines are parallel. W h e n urea is absent, almost all the actin exists in a p o l y m e r i z e d state, a n d with increasing c o n c e n t r a t i o n of urea the a m o u n t of F - a c t i n decreases a n d t h a t of G-actin increases. G - F t r a n s f o r m a t i o n of aetin is a k i n d of condensation p h e n o m e n o n similar to gas liquid condensation 9. This p h e n o m e n o n always occurs as long as actin is not d e n a t u r e d , even in the presence of urea. Now, the results of Fig. 5b m e a n t h a t actin in the p o l y m e r i z e d s t a t e c o n t r i b u t e s to zl n in the same way, irrespective of the c o n c e n t r a t i o n of urea. To confirm the existence of F - a c t i n in the presence of urea, aetin solutions at various concentrations of urea were u l t r a e e n t r i f u g e d at I O O o o o × g for 3 h at 20 °C a n d the p r o t e i n concentration of the sediments was d e t e r m i n e d ( ~ - - - - --L~ in Fig. 5a). The a m o u n t of protein s e d i m e n t e d decreased with increasing c o n c e n t r a t i o n of urea, corresponding to the flow birefringence results. Therefore, F - a c t i n has the same p r o p e r t y in the presence a n d absence of urea a n d the degree of flow birefringence is p r o p o r t i o n a l to the c o n c e n t r a t i o n of F - a c t i n . Biochim. Biophys. dcla, 278 (1972) 556-566
COMPLEX OF TROPOMYOSIN AND F-ACT1N
~h
56i
a
100 ~4
dn 100
A-___~.~. A 9.O'& E
b o
6.0 $ E 3.0 tn
"oo | .
° ~
Oh 0
I 1.0
o
..
i ~ o 2.0
0
I" 3.0
4,0
0
05
1.0
I
1.5
Concentration of actin (rng/ml)
Coneentration of urea (M)
Fig. 5. Effect of u r e a on F - a c t i n . a. F - a c t i n u n d e r v a r i o u s c o n c e n t r a t i o n s of urea. F l ow birefringence: An of a c t i n s o l u t i o n was m e a s u r e d 2o rain a f t e r t h e a d d i t i o n of urea. S o l v e n t c o n d i t i o n ; i o o mM KC1, [o mM Tris HCI (pH 8.o) a n d o.2 mM A T P a t 2o °C. C o n c e n t r a t i o n o f a c t i n : A - - A , 1. 3 m g / m l ; A - - , ' ~ , i.o m g / m l ; 0 - - 0 , 0.55 m g / m l ; O - - © , o.26 m g / m l . U l t r a c e n t r i f u g a t i o n : Fa c t i n s o l u t i o n s (4 ml of 1.2 m g / m l ) in v a r i o u s c o n c e n t r a t i o n s of u r e a were c e n t r i f u g e d a t 2o °C a n d t h e a m o u n t of s e d i m e n t was d e t e r m i n e d . S o l v e n t c o n d i t i o n : t h e s a m e as above. ( ~ [~). b. G F e q u i l i b r i u m of actin. This figure is a r e p l o t of a. The c o n c e n t r a t i o n of u r e a : (_3, o M; O, 1.6 M; ~ , 2.0 M; A, 2.6 M; [], 3.2 M.
Binding of tr@omyosin at various concentrations of urea. To m i x t u r e of t r o p o m y o s i n a n d F - a c t i n , various a m o u n t s of urea were a d d e d u n d e r the condition where the b i n d i n g was confirmed in the absence of urea at 2o °C. W h e n the a m o u n t of Vactin itself decreased at high c o n c e n t r a t i o n s of urea, the a m o u n t of t r o p o m y o s i n b o u n d to F - a c t i n was o b t a i n e d b y s u b t r a c t i n g the zln of E-aetin from t h a t of the m i x t u r e (fig. 6a). As shown in Fig. 6a, t r o p o m y o s i n begins to dissociate from F - a c t i n at a b o u t o. 7 M urea, a n d it can h a r d l y b i n d to F - a c t i n a b o v e I.o M urea. A s t u d y using u l t r a c e n t r i f u g a t i o n confirmed this result (Fig. 6b). A t z.o M urea the a m o u n t of s e d i m e n t e d protein of the m i x t u r e was almost the same as t h a t of E-actin alone. Effect of temperature. The a d d i t i o n of a small a m o u n t of urea was v e r y effective ~n
a
b ~5
40
E 3.0
g
20
2.5
7-:h
E co 0 5
D~o~ o
0'5
1!o
1
1.5
Concentration of urea (M)
0 I
o
I
0.5
1'.o
Concentration of
115
urea (M)
Fig. 6. T h e b i n d i n g a t v a r i o u s c o n c e n t r a t i o n s of urea. a. F l o w b i r e f r i n g e n c e : O, m i x t u r e of F - a c t i n (o.39 m g / m l ) a n d t r o p o m y o s i n (o.15 m g / m l ) ; 0 , F - a c t i n (o.39 m g / m l ) ; [B, difference of A n b e t w e e n t h e m i x t u r e a n d F - a c t i n only. S o l v e n t c o n d i t i o n : t h e s a m e as Fig. 5. b. U l t r a c e n t r i f u g a t i o n : o.62 m g / m l F - a c t i n , o.2 m g / m l t r o p o m y o s i n . S o l v e n t c o n d i t i o n a n d s y m b o l s are t he s a m e as above. S a m p l e s o l u t i o n s (4.4 ml) were c e n t r i f u g e d a t 2o °C a n d t h e a m o u n t s of s e d i m e n t s were d e t e r m i n e d .
t?iochim. Biophys. 14cta, 278 (i972) 556-566
562
H. TANAKA
in lowering the dissociation t e m p e r a t u r e . A t 0.25 M urea, the highest dissociation t e m p e r a t u r e was a b o u t 38 °C a n d at 0.5 M urea it was 3o °C. The higher the concent r a t i o n of urea, the lower the dissociation t e m p e r a t u r e . This t e m p e r a t u r e dependence of the b i n d i n g was reversible. However, a b o v e I.O M urea, no b i n d i n g was observed even if the t e m p e r a t u r e was lowered to 2 °C. A l t h o u g h the a d d i t i o n of urea resulted in the lowering of the dissociation t e m p e r a t u r e , the salt c o n c e n t r a t i o n dependence of the binding was still observed, just like in the case of absence of urea. Reversibility of the effect of urea. G-actin formed b y d e p o l y m e r i z a t i o n of F - a c t i n on the a d d i t i o n of 1.6 M urea could polymerize again into F - a c t i n when urea was r e m o v e d b y dialysis. In the case of 3.2 M urea, however, a b o u t one-half of G-actin could not polymerize after r e m o v a l of urea. This m a y be due to p a r t i a l d e n a t u r a t i o n of actin. U r e a of i . o M was a d d e d to a m i x t u r e in IOO mM KC1, IO mM Tris HC1 (pH 8.o) a n d o.2 mM A T P . A f t e r dissociation of t r o p o m y o s i n from F - a c t i n , it was d i l u t e d with the solvent so t h a t b o t h the c o n c e n t r a t i o n of urea a n d proteins decreased. As references, b o t h the m i x t u r e a n d an F - a c t i n solution in the absence of u r e a were also diluted. The degree of flow birefringence of these two references decreased linearly with dilution as e x p e c t e d (U a n d O in Fig. 7). I f the effect of urea on the b i n d i n g be r e m o v e d reversibly, t r o p o m y o s i n m u s t b i n d again to F - a c t i n with lowering concent r a t i o n of urea. Then, ,dn of the m i x t u r e would not decrease linearly with dilution. Fig. 7 (e) clearly shows t h a t t r o p o m y o s i n , once dissociated from F - a c t i n in I.O M urea, could b i n d again to F - a c t i n when the c o n c e n t r a t i o n of urea was lowered. Z~D
200
,
°°
I
~'4
I
2~4
I
3J4
E x t e n t of dilution
Fig. 7. Reversibility of the effect of urea on,the binding. Mixture of F-actin and tropolnyosin in the absence ([~) and presence (O) of i M urea. F-Actin in the absence of urea (O). The three solutions were diluted with the solvent: IOO mM KC1, IO mM Tris-HC1 (pH 8.o) and o.2 mM A T P at 25 °C. Solutions before dilution contain 0.95 m g / m l t r o p o m y o s i n a n d / o r 2. 7 mg/rnl F-actin. The abscissa shows the e x t e n t of dilution.
The effect of divalent cations on the binding between tropomyosin and F-actin G-actin polymerizes into F - a c t i n on a d d i t i o n of KC1, a n d 0.I M KC1 is o p t i m a l for full p o l y m e r i z a t i o n . However, a 2 mM c o n c e n t r a t i o n of d i v a l e n t cations is enough for full p o l y m e r i z a t i o n , even in the absence of KCP. T r o p o m y o s i n c a n n o t b i n d to F actin at low concentrations of salt (say, less t h a n 20 mM KC1), while in 2 mM d i v a l e n t cations (Mg 2+, Ca ~+ or B a 2+) full b i n d i n g occurs, even t h o u g h KC1 is a b s e n t 1. Biochim. I3iophys. Acta, 278 (1972) 556-566
563
COMPLEX OF TROPOMYOSIN AND F-ACTIN
At pH 7.7 in the presence of KCI, the highest dissociation temperature was observed at about 42 °C, 48 °C in 8.0% ethanol and 38 °C in 0.25 M urea. However, the addition of 2.5 mM MgC12 resulted in rise of the dissociation temperature to 48, 52 and 45 °C, respectively. In the presence of MgC12, the salt (KC1) concentration dependence of the dissociation temperature was still observed. In 2.5 mM MgC12, 1.5 M urea cannot dissociate tropomyosin from F-actin at 2o °C (cf. Fig. 6). The same trend was also observed for Ca 2+, but it was less effective than Mg2+.
The helix content of tropomyosin It is well known that the helical structure of tropomyosin becomes more stable at lower pH values or in the presence of ethanol, but more unstable on the addition of urea or at higher temperatures. Thus, the effect of pH, ethanol, urea and temperature on the stability of the helical structure of tropomyosin seems to be similar to the effect of those on the dissociation temperature mentioned above. We measured the helix content of tropomyosin at various temperatures and solvent conditions. With lowering pH or by adding ethanol, the helical structure of tropomyosin became more stable against increasing temperature (Fig. 8a and 8b). The higher the concentration of ethanol, the more stable the helical structure of tropomyosin within the concentration range studied (o-16.7%). On the other hand, the addition of urea resulted in the decrease of the helix content. [m ']
a
13
-12c~°I
[t ~-~._.
-8000
"
o,~,
-4000
I
I
I
30 40 50 Temperature
I
I
I
30 40 50 Temperature
Fig. 8. a. Effect of p H on t h e helix c o n t e n t of t r o p o m y o s i n , i . i m g / m l t r o p o m y o s i n , in 20 m M KCl a n d v a r i o u s buffers (both a t p H 8. 4 a n d 7.8; io m M Tris-HC1, b o t h at p H 7.2 a n d 6.5; io m M p h o s p h a t e ) , p H : O, 6.5; [Z, 7.2; 0 , 7.8; /~, 8.4- b. Effect of e t h a n o l on t h e helix c o n t e n t of t r o p o m y o s i n , i.o m g / m l t r o p o m y o s i n , in io m M Tris-HC1 (pH 8.o), 2o m M KC1 a n d (), o; [~, 8. 3 a n d Q, 16.7% ethanol.
DISCUSSION
Mechanism of interaction As described in the previous paper 2 and this paper, many conditions have to be satisfied for the binding between tropomyosin and F-actin. Tropomyosin bound to Factin dissociates with deviation of the salt concentration from optimal (i.e.o.I M KC1), on the addition of urea, or with increasing temperature. The dissociation ternBiochim. Biophys. Acta, 278 (1972) 556 566
564
H. TANAKA
perature depends on KC1, pH, ethanol, urea and divalent cations. Detailed analyses of the dissociation t e m p e r a t u r e seem to be very useful to elucidate a possible mechanism of the i n t e r a c t i o n between tropomyosin a n d F-actin. For this purpose, we tried to find some properties which change in parallel to the dissociation temperature. From this point of view, a possible correlation between the helical structure of tropomyosin a n d its b i n d i n g to F - a c t i n was investigated. We define the dissociation t e m p e r a t u r e at the o p t i m a l salt (KC1) condition as the "highest" dissociation temt)erature. Fig. 9 a shows the highest dissociation temperatures at various p H values. On the other hand, Fig. 9 b shows the t e m p e r a t u r e - p H relation of t r o p o m y o s i n at c o n s t a n t values of Fm']. As shown in these figures, lowering of the dissociation t e m p e r a t u r e with increasing p H occurred in parallel to the lowering of the t e m p e r a t u r e where [m'] -- ---8ooo. Therefore, we use the t e m p e r a t u r e where [m'] = - - 8 o 0 o as a measure of t e m p e r a t u r e specifying the state of tropomyosin, a n d the highest dissociation t e m p e r a t u r e as a measure of t e m p e r a t u r e related to the interaction between t r o p o m y o s i n a n d F-actin. a
b
cO 5 o
50
45
c° 45
E
¢.
40
%, 35
35
I
6 T
8
pH
\o" - 9 0 0 0
30
pH
Fig. 9- a. pH dependence of the highest dissociation temperature. The highest dissociation temperature at a given pH is replotted from Fig. 3 so that the lowering of the dissociation temperature with pH is clearly seen. b. pH dependence of the helix content of tropomyosin. This figure is reph)tted from the results in Fig. 8a and shows pH dependence of the temperature at a given helix content [m'] : ~, -- 7ooo; O, 8000 and ~ , - -9ooo. Then, the present results can be expressed as a relation between these two temperatures, as shown in Fig. Ioa. This figure suggests t h a t the effects of (i) p H a n d ethanol, (ii) urea (iii) d i v a l e n t cations a n d (iv) salt on the b i n d i n g are different from each other. I n Fig. lob, is shown a schematic representation of these four types of effects on the binding. (i) p H and ethanol. The rise of tile highest dissociation t e m p e r a t u r e with lowering p H or on the addition of ethanol occurs in parallel to tile change of the stability of the helical structure of tropomyosin. The highest dissociation t e m p e r a t u r e s at different p H values a n d different ethanol concentrations lie on a straight line B in Fig. lob. The slope of this line B is a b o u t u n i t y , suggesting the direct correlation between the helix stability of t r o p o m y o s i n a n d its b i n d i n g to F - a e t i n at the o p t i m a l salt condition. I n Fig. Ioa and lob, tile ordinate gives the t e m p e r a t u r e where [m'] = ---8000. If it were scaled b y the t e m p e r a t u r e where [m'] = - - 9 0 0 0 , the result would be expressed in the same way. However, the t e m p e r a t u r e where Ira'3 = - - 7 0 0 0 could not be t a k e n as the measure, because in t h a t t e m p e r a t u r e range no b i n d i n g was ob-
Biochim. Biophys. ,4cta, 278 (1972) 556-566
COMPLEX OF TROPOMYOSIN AND F-ACTIN
565
& 0
°o 50
pH65 7595 5/12~5 ~859013'11,6'30
CO ,i
D 0 o ug
a(80)
•(8,0)
(621
E 40
_
%o N f l o 60
%o%o
20O90 t20 0120 20W30
DNSI
E 30,
I
I
I
30
40
50
Dissociation t e m p e r a t u r e
o 50 O Q
B
,o
CO i
i, r/n
[31
uJ
% 4o
A
2
~7 c
E
3o
I 30
I 40
I 50
Dissociation t e m p e r a t u r e
Fig. IO. Correlation between the dissociation t e m p e r a t u r e and the t e m p e r a t u r e where im'J = --8ooo. a. The ordinate is scaled by the t e m p e r a t u r e where [m'] -- --8o0o and the abscissa b y the dissociation t e m p e r a t u r e . O, at various p H values s h o w n in the figure; [7, at various concentrations of ethanol (concentrations are s h o w n b y n u m e r a l s in brackets) ; ~ , o.'25 M urea and V , °-5 NI urea. N u m e r a l s in the figure (not in brackets) indicate the concentration of KC1 (raM), and the closed symbols represent the presence of 2. 5 mM MgCI 2. p H of the solvent is 7.7, unless otherwise mentioned, b. Schematic r e p r e s e n t a t i o n of the result in Fig. IOa. F o r explanation see legend in a. Capitals in the figure (A, B, B', and C) represent the four types of solvent effect on the binding (see text).
served in the same salt condition. Thus, the threshold condition for the binding of tropomyosin to F-actin is given by the state of tropomyosin, i.e. where [m'] = --8o0o. (ii) Urea. The addition of urea decreases both the stability of the helical structure and the dissociation temperature. The lowering of the dissociation temperature with increasing concentration of urea is faster than the lowering of the stability of helical structure, as shown by line C in Fig. Iob, suggesting some other mechanism for the dissociation. (iii) Divalent cations. The effect of Mg2+ on the binding is the remarkable shift of the highest dissociation temperature. In the presence of Mg2+ the highest dissociation temperature is uniformly raised by about 5 °C at every condition (line B' in Fig. iob). Asai and Tawada 1° reported that in the presence of Mg2+ the denaturation temperature of actin becomes about 5 °C higher (6o °C instead of 55 °C). This suggests strengthening of the structure of F-actin by Mgz+, which may result in the raising of the dissociation temperature and the lowering of the threshold value of the helix Biochi*n. Biophys. dcta, 278 (~97 z) 556-566
566
H. TANAKA
content of tropomyosin required for the binding. On tile other hand, a small amount of urea makes F-actin more flexible or weaker, in addition to the destabilization of the helical structure of tropomyosin, resulting in the dissociation of tropomyosin at lower temperatures. (iv) Salt (KCl). The salt concentration dependence of the dissociation temperature is almost independent of the presence of ethanol, urea or divalent cation. In this sense, the effect of salt (KC1) on the binding is very different from that of the others. Salt ions change the apparent charge of tropomyosin (and actin) which may control their binding.
Physiological implications A possible mechanism of the regulation of muscular contraction has been proposed by Ebashi and Endo n. The information induced by the binding of Ca 2+ to troponin is transmitted to actin via tropomyosin to regulate the interaction between actin and myosin. Regulatory proteins work as mediators of the Ca ~+ information by changing their own structures 12 14. The condition where the helical content of tropomyosin has a direct correlation with the dissociation temperature, is very similar to the physiological one. This means that the binding between tropomyosin and F-actin is in a delicate state at the physiological condition in vivo. Therefore, if some structural effect of Ca 2+ on troponin is assumed to change the stability of the helical structure of tropomyosin, it is most likely that tropomyosin might effectively work as a mediator of the Ca 2+ information to actin by changing its binding state with actin. ACKNOWLEDGEMENTS
The author thanks Dr S. Fujime for his guidance and also Professor F. Oosawa for his encouragement throughout the present work.
REFERENCES K. M a r u y a m a , Arch. Biochem. Biophys., lO 5 (1964) 142. H. T a n a k a a n d F. Oosawa, Biochim. Biophys. Acta, 253 (1971) 274. F. B. S t r a u b , Stud. Inst. Med. Chem. Univ. Szeged., 2 (1942) 3. S. E b a s h i , A. K o d a m a a n d F, E b a s h i , J. Biochem., 64 (1968) 465 . M. Kasai, H. K a w a s h i m a a n d F. Oosawa, J. Polymer Sci., X L I V (196o) 5I. D. R. Ko min z, F. Saad, J. A. G l a n d e r a n d K. Laki, Arch. Biochem. Biophys., 7 ° (1957) 16. T. Ooi, Biochemistry, 6 (1967) 2433. E. F. Wo ods, Int. J. Prot. Res., I (1969) 29. F. Oosawa, S. A s a k u r a , K. H o t t a , N. I m a i a n d T. Ooi, J. Polymer Sci., X X X V I I (1959) 323H. Asai a n d K. T a w a d a , J. Mol. Biol., 2o (1966) 4o3 . S. E b a s h i a n d M. Endo, in J. A. V. B u t l e r a n d D. Noble, Progress in Biophysics and Molecular Biology, Vol. 18, P e r g a m o n Press, Oxford, 1968, p. 123. 12 T. W a k a b a y a s h i a n d S. E b a s h i , J . Biochem. (Tokyo), 61 (1968) 731. 13 Y. T o n o m u r a , S. W a t a n a b e a n d M. Morales, Biochemistry, 8 (1969) 217I. 14 S. l s h i w a t a a n d S. F u j i m e , J. Mol. Biol., 68 (1972) 511. I 2 3 4 5 6 7 8 9 io ii
Biochim. Biophys. Acta, 278 (1972) 556-566