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Effect of the state of oxidation of cysteine 190 of tropomyosin on the assembly of the actin-tropomyosin complex
79
Biochimica et Biophysica Acta, 626 (1980) 79--87 © Elsevier/North-Holland Biomedical Press
BBA 38554
EFFECT OF THE STATE OF OXIDATION OF CYSTEINE 190 OF TROPOMYOSIN ON THE ASSEMBLY OF THE ACTIN-TROPOMYOSIN COMPLEX
TERENCE P. WALSH and ALBRECHT WEGNER
School of Medicine, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19174 (U.S.A.) (Received May 19th, 1980)
Key words: Actin; Tropomyosin; Complex assembly; Cysteine 190; Oxidation state
Summary Tropomyosin, cross-linked at cysteine 190, was found to bind more weakly to actin filaments than uncross-linked tropomyosin. Cross-linking of tropomyosin can cause actin filaments nearly completely covered with tropomyosin to be uncovered almost completely. The critical monomer concentration of actin is not significantly changed by binding of cross-linked or uncross-linked tropomyosin to actin filaments. The binding curves were analyzed quantitatively, thereby taking into account the polar end-to-end contact of tropomyosin molecules bound by actin and the overlap of the seven subunit binding sites along the actin filament. Under the conditions of the experiment (80 mM KC1, 1 mM MgC12, pH 7.5, 38--42°C), the equilibrium constant for isolated binding of tropomyosin to actin filaments is in the range 1 • 103--3 • 103 M -1. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 106 or 108 to 109 M-1, respectively. The equilibrium constants for cross-linked and uncrosslinked tropomyosin differ by a factor of only about two. Owing to the highly cooperative binding, these differences are sufficient so that actin filaments nearly completely covered with uncross-linked tropomyosin are uncovered almost completely by cross-linking tropomyosin at cysteine 190. Introduction Tropomyosin is a long rod-shaped molecule [1] that is found in many types of cells [1--3]. This molecule can form a weak complex with actin filaments Abbreviations: SDS, sodium d o d e c y l sulphate; NBD, 7-chloro-4-nitrobenzo-2-oxa-l.3-diazole.
80 [4--6]. In highly motile membrane ruffles, actin filaments have been found to be free of tropomyosin whereas in immotile cells tropomyosin is present along the actin filament bundles [7]. When isolated from skeletal muscle, tropomyosin binds to actin filaments in a stoichiometric ratio of I : 7 [8--10]. The tropomyosin molecules bound along the actin filament are joined by a polar end-to-end overlap [11--13]. Tropomyosin consists of two polypeptide chains [14,15] that contain cysteine in position 190 [16,17]. The cysteines of the two chains of the dimer can easily be oxidized to form an intramolecular crosslink [18--20]. The two polypeptide chains are largely s-helical [21] and are arranged in a coil [22--24]. The tropomyosin molecule is considered to be flexible with variations in the stability of the coil structure along the length of the molecule. Certain segments of tropomyosin are more readily cleaved by proteolytic enzymes than others [25]. Tropomyosin cross-linked at cysteine 190 is partially unfolded in the 30--40°C range whereas uncross-linked tropomyosin remains largely s-helical under these conditions [26]. It is the purpose of this study to investigate how the assembly of actin filaments with tropomyosin can be regulated. The association of the cross-linked and uncross-linked forms of tropomyosin with actin filaments was measured by light scattering to find out whether small changes in the structure and flexibility of tropomyosin can induce changes in the interaction with actin filaments. Material and Methods
Preparation of tropomyosin. The tropomyosin-troponin complex was extracted from acetone powder according to the method of Spudich and Watt [27]. Tropomyosin was purified by hydroxyapatite chromatography [28]. Tropomyosin was separated by this chromatographic step into its two multiple forms, ~,~- and ~,~-tropomyosin. The fractions specified in Fig. 1 were used. They contained less that 5% ~-tropomyosin chains or 10% ~,~-tropomyosin, as tested by electrophoresis. Tropomyosin which was reduced completely at cysteine 190 (uncross-linked tropomyosin) was prepared by incubation with 20 mM dithiothreitol for 1 h at 40°C [18]. Tropomyosin cross-linked by a disulphide bond at cysteine 190 (cross-linked tropomyosin) was formed by reaction with 5,5'-dithiobis(2-nitrobenzoate) [18]. Excess reagent was removed by chromatography on Sephadex G-25. The state of oxidation of cysteine 190 was tested by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis in the absence of reducing agents [18,29]. Tropomyosin was reacted with 0.1 M iodoacetamide prior to electrophoresis. 0.01 M iodoacetamide was added to all buffers. Uncross-linked tropomyosin was free of cross-linked tropomyosin. Cross-linked tropomyosin contained a small amount (10%) of uncross-linked tropomyosin. The concentration of tropomyosin was determined photometrically at 276 nm using an extinction coefficient of 24 500 M-1 • cm -1 [6]. The weight concentration was calculated assuming a molecular weight of 65 400 [16]. Preparation of actin. Actin was prepared and reacted with N-ethylmaleimide as described earlier [30]. Part of the actin treated with N-ethylmaleimide was labelled with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD) (Pierce Chemical
81
0.1 o
•
c
©
e•e••
eeee, U
co n
•
L
• co
o
.o
••Hoo•
0
eooq
OooOooe• •
0
10 3
Volume of effluent [ m l ] b
Fig. 1. (a) H y d r o x y a p a t i t e c h r o m a t o g r a p h y o f t h e t x o p o n i n - t r o p o m y o s i n c o m p l e x . C o l u m n , 2.5 × 4 0 c m ; g r a d i e n t , linear increase o f p o t a s s i u m p h o s p h a t e f r o m 1 mM t o 0 . 2 M. The f r a c t i o n s d e f i n e d b y the a r r o w s were used f o r the e x p e r i m e n t s a n d c o n t a i n e d m o r e t h a n 90% c~,~-tropomyosin. (b) E l e c t r o p h o r e t i e p a t t e r n o f the t r o p o m y o s i n p r e p a r a t i o n . U p p e r h a n d , ~ - t r o p o m y o s i n chains; l o w e r b a n d , c~-tropomyosin chains.
Co.) at 4°C for 4 h to form a 1 : 1 complex (NBD-actin) [31]. The concentration of actin was determined photometrically by the biuret m e t h o d [32] at 540 nm using an extinction coefficient of 2750 M-1 • cm -1 [6]. NBD-actin was determined by using the m e t h o d of Lowry et al. [33], standardized by the Kjeldahl analysis. For the calculation of the weight concentration, the molecular weight was assumed to be 42 300 [34]. Light scattering. The actin-tropomyosin complex was formed by mixing equal volumes of an actin solution, a tropomyosin solution and a buffer contalning 2.97 mM MgC12, 750 ~M ATP, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5). Actin was dialyzed against 30 ~M MgC12, 750 pM ATP, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5). Cross-linked tropomyosin was dialyzed against 240 mM KC1, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5). Uncross-linked tropomyosin was dialyzed against the same buffer with 6 mM dithiothreitol in addition. Hence, the actin-tropomyosin complex was dissolved in a buffer containing 1 mM MgC12, 80 mM KC1, 500 pM ATP, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5) plus 2 mM dithiothreitol in the case of complexes containing uncross-linked tropomyosin. Before mixing in lightscattering cells, all solutions were centrifuged at 100 000 × g for 1 h to remove dust. The actin/tropomyosin mixture was incubated at room temperature to form the actin-tropomyosin complex. After 1 day, the light-scattering intensity reached a constant value, indicating that equilibrium was reached. The angular dependence of the light-scattering intensity was measured in a Cantow scattering photometer using a vertically polarized incident beam. The 90 ° scattering intensity was measured using a Perkin Elmer MPF3 fluorimeter. All measurements were performed at 546 nm.
82
The light-scattering intensity Ra(O) of long polydisperse rod-like protein aggregates such as actin filaments has been shown to be given by [35] : Ra(tg) = g
)k
4 sin(0/2)
Ma
- - • Ca la
(1)
where 0 is the observation angle, K is an optical constant, Ca is the weight concentration of actin filaments, X is the wavelength of light in the solvent, Ma is the molecular weight of a filament subunit and l a is the length of a filament subunit. Eqn. 1 has been derived for rod-like particles, the length of which exceeds X/(4 sin(0/2)) and the diameter of which is much smaller than X/(4 sin(0/2)). Both conditions have been proved by electron-microscopic studies on synthetic actin filaments [36,37]. When molecules bound along a rod-like filament induce a small inhomogeneity in the mass-to-length ratio such as in the case of actin filaments partially saturated with tropomyosin, the scattering intensity, R t (tg), has been shown to be given b y [6] : Rt(v~) = g
X 4 sin(0/2)
Ma la
(Ca + Ct) 2 Ca
(2)
where Ct is the weight concentration of the tropomyosin molecules b o u n d along the actin filaments. This weight concentration was determined from the ratio of the scattering intensity of the actin-tropomyosin complex and of pure actin using Eqn. 3 which could be obtained by combining Eqn. 1 and 2: R t ( O ) - (Ca + C t ) 2
Ra(0)
C:a
(3)
It can be shown that the scattering intensity of u n b o u n d tropomyosin is so small compared to the scattering intensity of b o u n d tropomyosin that no correction needs to be applied for it. Fluorescence. NBD-actin fluorescence was measured in a fluorimeter (Perkin Elmer MPF3) using an excitation wavelength of 470 nm and monitoring emission at 520 nm through a 500 nm curt-off filter. All readings were standardized against quinine sulphate in 0.1 N H2SO4. The 450 nm fluorescence of a 1/~M solution excited at 360 nm was divided into 100 units. SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide electrophoresis was performed according to the method of Laemmli [29]. The gels were stained with Fast green and were scanned photometrically at 625 nm. SDSpolyacrylamide gel electrophoresis was used to determine the ratio of a- and ~-tropomyosin and the concentration of unpolymerized actin and of tropomyosin n o t bound to actin filaments. The bands of a- and ~-tropomyosin overlapped partially. The band of a-tropomyosin was found to be so narrow that, at the maximum of the ~-tropomyosin band, no a-tropomyosin was present. Therefore, the height of the peaks could be taken to be proportional to the concentration of a- or ~-tropomyosin chains. For the determination of unpolymerized actin and of u n b o u n d tropomyosin, the mixtures were centrifuged at 100 000 × g for 30 min (distance of sedimentation 7 mm). The supernatant was applied to the gels together with a series of standards of known actin and tropomyosin concentration.
83
Results Ligh t-scattering curves
Light-scattering curves of polymeric actin and of actin filaments partially or totally covered with tropomyosin were measured in order to test whether Eqns. 1--3 can be applied to determine the weight concentration of tropomyosin b o u n d to actin. Fig. 2 shows that the reciprocal of the scattering intensity is nearly proportional to the sine of the half observation angle (Eqns. 1 and 2). The continuous lines were calculated using Eqn. 2. At the temperature of the measurements (15°C) the concentration of u n b o u n d tropomyosin was found to be less than 0.05 #M. On account of this small concentration no correction was made for u n b o u n d tropomyosin. The results show that in the range of the observation angles (30--150 ° ) the ratio of the scattering intensities of actin-tropomyosin complexes and of pure actin is practically independent of the observation angle and that the measured ratio corresponds to the ratio calculated using Eqn. 3. Critical m o n o m e r concentration o f actin
The concentration of monomeric actin coexisting with actin filaments (critical m o n o m e r concentration of actin [38]) was measured in the presence
1.5
o
O~
C]O
00
[]
[]
o
el n-
o o
Rt/R a
o
oo
1.0
0
35
sin [8/2]
40
45
Temperature [°C]
Fig. 2. Light-scatterihg curves of aetin filaments and of aetin partially or t ot a l l y covered w i t h tropomyosin. R a ( 9 0 ° ) / R t ( ~ ) , reciprocal of the scattering i nt e ns i t y normalized by the value of actin at an ob° servation angle of 90°; ~, observation angle. A actin filaments (10.3 /~M); o, actin filaments (10.3 /~M) and t r o p o m y o s i n (0.5 #M); a, actin filaments (10.3/~M) and t r o p o m y o s i n (1.0 #M); ~, actin filaments (10.3/~M) and t r o p o m y o s i n (2.0/~M). The continuous lines were calculated. Fig. 3. TemperattLre dependence of the light-scattering i nt e ns i t y of NBD-actin/uncross-linked tropom y o s i n mixtures. R t, light-scattering intensity of the NBD-actin°tropomyusin c o m p l e x ; R a. light-scattering intensity of pure NBD-actin. Total t r o p o m y o s i n concentration, 1.5/~M. o, t o t a l NBD-actin concentration, 3.0 #M; o, total NBD-actin concentration, 6.5 ~zM.
84
C 0 @ 43 C
@ 0 W
~o~ i
o _= U.
0
0.2
0.4
0.6
Concentration of NBD-actin [ p M ] Fig. 4 . P l o t o f t h e t o t a l N B D - actin c o n c e n t r a t i o n vs. fluorescence intensity ( e x c i t a t i o n 4 7 0 rim, e m i s s i o n 5 2 0 n m ) . M o n o m e r i c N B D - a c t i n w a s d i s s o l v e d in 3 0 # M MgC12 , 5 0 0 # M A T P a n d 5 m M triethanolamine h y d r o c h l o r i d e ( p H 7 . 5 ) . All o t h e r s o l u t i o n s c o n t a i n e d 0.9.7 m M MgC12 a n d 8 0 m M KCI i n a d d i t i o n . * (36.7°C), monomeric NBD-actin; • (36.7°C), o (41.9°C), NBD-actin; • (36.7°C), o (41.9°C), NBD-actin a n d 1 /~M u n c r o s s - l i n k e d t r o p o m y o s i n ; • ( 3 6 . 7 ° C ) , A ( 4 1 . 9 ° C ) , N B D - a c t i n a n d 1 /~M c r o s s - l i n k e d t r o p o m y o s i n . T h e f l u o r e s c e n c e i n t e n s i t y w a s c a l i b r a t e d b y d i v i d i n g t h e f l u o r e s c e n c e o f a 1 #aM q u i n i n e sulphate solution into 100 units (excitation 360 nm, emission 450 nm).
and absence of tropomyosin. As the light-scattering intensity in the range of the critical concentration is t o o small to allow an accurate determination, the critical concentration was measured by electrophoresis (see Materials and Methods, SDS-polyacrylamide gel electrophoresis) and by introducing a fluorescence label to monitor polymerization. When measured by electrophoresis the m o n o m e r concentration was found to be in the range of 0.1 ~M. Various concentrations of NBD-actin were incubated with 1.5 ~M uncross-linked tropomyosin. The temperature dependence of the light-scattering intensity of the NBD-actin-tropomyosin complex revealed features similar to the actin-tropomyosin complex under the same conditions (Fig. 3), indicating that the NBD label did not affect the association of tropomy0sin with actin filaments. The critical m o n o m e r concentration was measured in the absence and in the presence of 1 pM cross-linked or uncross-linked tropomyosin. The NBD-actin concentration was varied between 0.05 and 0.6 #M. Plots of the NBD-actin concentration vs. the fluorescence intensity are depicted in Fig. 4. The bends of the curves can be explained by a 2-fold increased fluorescence intensity of polymeric NBD-actin formed above the critical m o n o m e r concentration. The critical concentration was found to be about 0.2 p.lVl and not to be affected by binding of tropomyosin to actin filaments.
Binding isotherms Binding of tropomyosin to actin filaments was measured by means of 90 ° light scattering. The actin concentration was kept constant at a b o u t 5 ~M. The tropomyosin concentrations were varied between 0.3 and 4 #M. When tropomyosin was present in a stoichiometric excess over actin (greater than 0.8 pM), at 25°C the scattering intensities of all samples were about 50% greater than the scattering intensity of pure actin. On heating, the scattering intensities of the samples decreased and at 45°C the scattering intensities of most of the samples were similar to the scattering intensity of pure actin. The scattering
85
117'
117
/ " / /
>, n ¢: @
• I:: ® qD
~t c ,= m m
01 I: m
¢1
O 0
1
2
3
4 JM
Concentration of free tropomyosln
O, 0
1
2
3
4 JM
Concentration of free tropomyosln
Fig. 5. Binding curves of cross-linked t r o p o m y o s i n to actin filaments. 4 38.2°C; m, 39.4°C; e, 40.7°C; *, 41.9°C. Total actin c o n c e n t r a t i o n 5.4 /~M. The con t i nuous fines were calculated using the fitted equifibriurn constants given in Table I. Fig. 6. Binding curves of uncross-linked t r o p o m y o s i n to actin filaments. 4 38.2°C; m 39.4°C; e, 40.7°C; *, 41.9°C. Total actin concentration 5.3 /~M. The con t i nuous fines were calculated using the fitted equilibrium constants given in Table I.
intensity of pure actin was almost temperature independent. At intermediate temperatures (35--45°C) the scattering intensities of the samples depended strongly on the tropomyosin concentration (see also Fig. 3). The results are depicted in Figs. 5 and 6 as plots of the concentration of free tropomyosin vs. the number of bound tropomyosin molecules per actin subunit (binaing density v). The binding density, v, was calculated from the light-scattering intensity as described in Materials and Methods (light scattering). No correction was made for unpolymerized actin as the critical concentration was found to be negligibly small compared to the total concentration. The concentration of free tropomyosin was calculated as the difference of the concentration of the total and the bound tropomyosin. Uncross-linked tropomyosin was found to bind more strongly to actin filaments than cross-linked tropomyosin.
Interpretation of the binding isotherms The binding isotherms (Figs. 5 and 6) are strongly sigmoidal, indicating cooperative binding of tropomyosin. The cooperative binding can be attributed to the polar end-to-end overlap of the termini of tropomyosin molecules contiguously bound along actin filaments. The equilibrium of the actin-tropomyosin interaction is adequately described by a model of cooperative binding of large ligands to a homogeneous lattice with overlapping binding sites. The 2
3
1
Fig. 7. Types of binding site of txopomyosin along the actin filament represented by Hnearly arranged cbewons. 1, isolated binding site;2, singly contiguous binding site;3, doubly contiguous binding site.
86
TABLE I E Q U I L I B R I U M C O N S T A N T S O F B I N D I N G OF C R O S S - L I N K E D A N D U N C R O S S - L I N K E D T R O P O MYOSIN TO ACTIN F I L A M E N T S K 1 , b i n d i n g c o n s t a n t f o r i s o l a t e d b i n d i n g sites; K 2 , b i n d i n g c o n s t a n t for singly c o n t i g u o u s b i n d i n g sites; K 3 , b i n d i n g c o n s t a n t for doubly contiguous binding sites (all values e x p r e s s e d in M - 1).
Temperature
Cross-linked t r o p o m y o s i n
Uncross-linked t r o p o m y o s i n
(°C) K1
K2
K3
K1
K2
K3
2.1 1.5 ---
0.62 0.44 ---
0.18 0.13 ---
3.1 4.6 3.1 1.1
1.37 1.14 0.76 0.50
0.62 0.29 0.19 0.22
(XIO-3) 38.2 39.4 40.7 41.9
(XIO-6)
(XIO-9)
(XIO-3)
(XlO-6)
(XIO-9)
actin filament represents a homogeneous lattice. This type of equilibrium has been treated by McGhee and von Hippel [39] and by Schwarz [40]. Three types of binding site and three corresponding binding constants are conceivable (Fig. 7). (1) Isolated binding sites: Tropomyosin has no overlap with an adjacent tropomyosin molecule (binding constant K1). (2) Singly contiguous binding sites: Tropomyosin has one overlap with an adjacent tropomyosin molecule (binding constant K2). (3) Doubly contiguous binding sites: Tropomyosin has two overlaps with two adjacent tropomyosin molecules (binding constant K3). The three binding constants, K1, K2 and K3, are not independent. The consecutive binding of two tropomyosin molecules to singly contiguous binding sites (K2) may be achieved also by binding of one tropomyosin molecule to an isolated binding site (K1) seven actin subunits away from another ligand and subsequent insertion of a second tropomyosin molecule into the seven-subunit gap (K3). Using the condition, K~2 = K~. K3, the binding constant K3 can be expressed in terms of K1 and K2 : K3 = K~JK1. McGhee and von Hippel have derived an equation that connects the concentration of free tropomyosin (t) and the binding density v with the binding constants K1 and K2 [39] : v t=K'l--7v
. ( 2 ( K 2 / K I - - 1). ( 1 - - 7v) \i2KT~¥1)-(1--7~+v--R
)6. ( 2 ( 1 _ 7v) 12 \I--Sv+R/
where R = x/{ (1 -- 8v): + 4K2/K1 • v • (1 -- 7v)}. The binding constants were fitted by seeking that set of parameters K~ and K2 where the standard deviation from the measured binding density reaches a minimum. The results are summarized in Table I. Discussion
The stability of the actin-tropomyosin complex was found to depend on the state of oxidation of cysteine 190 of tropomyosin. Uncross-linked tropomyosin binds more tightly to actin filaments than cross-linked tropomyosin. Structural differences between cross-linked and uncross-linked tropomyosin have been reported [26]. Cross-linked tropomyosin partially unfolds in the
87 30--40 ° C range whereas uncross-linked tropomyosin does not undergo this transition. The unfolding effect of the interchain disulphide bridge has been attributed to a strain that increases the instability of the molecule in the region of cysteine 190. This study demonstrates that small changes in the structure and flexibility can alter the actin-tropomyosin interaction. Acknowledgements The authors would like to thank Dr. Annemarie Weber for helpful advice during the course of the studies and for facilities to carry out the work. This study was supported by NIH grant HL 15692. A.W. received a reasearch fellowship from the Deutsche Forschungsgemeinschaft. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
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Biochimica et Biophysica Acta, 626 (1980) 79--87 © Elsevier/North-Holland Biomedical Press
BBA 38554
EFFECT OF THE STATE OF OXIDATION OF CYSTEINE 190 OF TROPOMYOSIN ON THE ASSEMBLY OF THE ACTIN-TROPOMYOSIN COMPLEX
TERENCE P. WALSH and ALBRECHT WEGNER
School of Medicine, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19174 (U.S.A.) (Received May 19th, 1980)
Key words: Actin; Tropomyosin; Complex assembly; Cysteine 190; Oxidation state
Summary Tropomyosin, cross-linked at cysteine 190, was found to bind more weakly to actin filaments than uncross-linked tropomyosin. Cross-linking of tropomyosin can cause actin filaments nearly completely covered with tropomyosin to be uncovered almost completely. The critical monomer concentration of actin is not significantly changed by binding of cross-linked or uncross-linked tropomyosin to actin filaments. The binding curves were analyzed quantitatively, thereby taking into account the polar end-to-end contact of tropomyosin molecules bound by actin and the overlap of the seven subunit binding sites along the actin filament. Under the conditions of the experiment (80 mM KC1, 1 mM MgC12, pH 7.5, 38--42°C), the equilibrium constant for isolated binding of tropomyosin to actin filaments is in the range 1 • 103--3 • 103 M -1. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 106 or 108 to 109 M-1, respectively. The equilibrium constants for cross-linked and uncrosslinked tropomyosin differ by a factor of only about two. Owing to the highly cooperative binding, these differences are sufficient so that actin filaments nearly completely covered with uncross-linked tropomyosin are uncovered almost completely by cross-linking tropomyosin at cysteine 190. Introduction Tropomyosin is a long rod-shaped molecule [1] that is found in many types of cells [1--3]. This molecule can form a weak complex with actin filaments Abbreviations: SDS, sodium d o d e c y l sulphate; NBD, 7-chloro-4-nitrobenzo-2-oxa-l.3-diazole.
80 [4--6]. In highly motile membrane ruffles, actin filaments have been found to be free of tropomyosin whereas in immotile cells tropomyosin is present along the actin filament bundles [7]. When isolated from skeletal muscle, tropomyosin binds to actin filaments in a stoichiometric ratio of I : 7 [8--10]. The tropomyosin molecules bound along the actin filament are joined by a polar end-to-end overlap [11--13]. Tropomyosin consists of two polypeptide chains [14,15] that contain cysteine in position 190 [16,17]. The cysteines of the two chains of the dimer can easily be oxidized to form an intramolecular crosslink [18--20]. The two polypeptide chains are largely s-helical [21] and are arranged in a coil [22--24]. The tropomyosin molecule is considered to be flexible with variations in the stability of the coil structure along the length of the molecule. Certain segments of tropomyosin are more readily cleaved by proteolytic enzymes than others [25]. Tropomyosin cross-linked at cysteine 190 is partially unfolded in the 30--40°C range whereas uncross-linked tropomyosin remains largely s-helical under these conditions [26]. It is the purpose of this study to investigate how the assembly of actin filaments with tropomyosin can be regulated. The association of the cross-linked and uncross-linked forms of tropomyosin with actin filaments was measured by light scattering to find out whether small changes in the structure and flexibility of tropomyosin can induce changes in the interaction with actin filaments. Material and Methods
Preparation of tropomyosin. The tropomyosin-troponin complex was extracted from acetone powder according to the method of Spudich and Watt [27]. Tropomyosin was purified by hydroxyapatite chromatography [28]. Tropomyosin was separated by this chromatographic step into its two multiple forms, ~,~- and ~,~-tropomyosin. The fractions specified in Fig. 1 were used. They contained less that 5% ~-tropomyosin chains or 10% ~,~-tropomyosin, as tested by electrophoresis. Tropomyosin which was reduced completely at cysteine 190 (uncross-linked tropomyosin) was prepared by incubation with 20 mM dithiothreitol for 1 h at 40°C [18]. Tropomyosin cross-linked by a disulphide bond at cysteine 190 (cross-linked tropomyosin) was formed by reaction with 5,5'-dithiobis(2-nitrobenzoate) [18]. Excess reagent was removed by chromatography on Sephadex G-25. The state of oxidation of cysteine 190 was tested by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis in the absence of reducing agents [18,29]. Tropomyosin was reacted with 0.1 M iodoacetamide prior to electrophoresis. 0.01 M iodoacetamide was added to all buffers. Uncross-linked tropomyosin was free of cross-linked tropomyosin. Cross-linked tropomyosin contained a small amount (10%) of uncross-linked tropomyosin. The concentration of tropomyosin was determined photometrically at 276 nm using an extinction coefficient of 24 500 M-1 • cm -1 [6]. The weight concentration was calculated assuming a molecular weight of 65 400 [16]. Preparation of actin. Actin was prepared and reacted with N-ethylmaleimide as described earlier [30]. Part of the actin treated with N-ethylmaleimide was labelled with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD) (Pierce Chemical
81
0.1 o
•
c
©
e•e••
eeee, U
co n
•
L
• co
o
.o
••Hoo•
0
eooq
OooOooe• •
0
10 3
Volume of effluent [ m l ] b
Fig. 1. (a) H y d r o x y a p a t i t e c h r o m a t o g r a p h y o f t h e t x o p o n i n - t r o p o m y o s i n c o m p l e x . C o l u m n , 2.5 × 4 0 c m ; g r a d i e n t , linear increase o f p o t a s s i u m p h o s p h a t e f r o m 1 mM t o 0 . 2 M. The f r a c t i o n s d e f i n e d b y the a r r o w s were used f o r the e x p e r i m e n t s a n d c o n t a i n e d m o r e t h a n 90% c~,~-tropomyosin. (b) E l e c t r o p h o r e t i e p a t t e r n o f the t r o p o m y o s i n p r e p a r a t i o n . U p p e r h a n d , ~ - t r o p o m y o s i n chains; l o w e r b a n d , c~-tropomyosin chains.
Co.) at 4°C for 4 h to form a 1 : 1 complex (NBD-actin) [31]. The concentration of actin was determined photometrically by the biuret m e t h o d [32] at 540 nm using an extinction coefficient of 2750 M-1 • cm -1 [6]. NBD-actin was determined by using the m e t h o d of Lowry et al. [33], standardized by the Kjeldahl analysis. For the calculation of the weight concentration, the molecular weight was assumed to be 42 300 [34]. Light scattering. The actin-tropomyosin complex was formed by mixing equal volumes of an actin solution, a tropomyosin solution and a buffer contalning 2.97 mM MgC12, 750 ~M ATP, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5). Actin was dialyzed against 30 ~M MgC12, 750 pM ATP, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5). Cross-linked tropomyosin was dialyzed against 240 mM KC1, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5). Uncross-linked tropomyosin was dialyzed against the same buffer with 6 mM dithiothreitol in addition. Hence, the actin-tropomyosin complex was dissolved in a buffer containing 1 mM MgC12, 80 mM KC1, 500 pM ATP, 200 mg/1 NaN3 and 5 mM triethanolamine hydrochloride (pH 7.5) plus 2 mM dithiothreitol in the case of complexes containing uncross-linked tropomyosin. Before mixing in lightscattering cells, all solutions were centrifuged at 100 000 × g for 1 h to remove dust. The actin/tropomyosin mixture was incubated at room temperature to form the actin-tropomyosin complex. After 1 day, the light-scattering intensity reached a constant value, indicating that equilibrium was reached. The angular dependence of the light-scattering intensity was measured in a Cantow scattering photometer using a vertically polarized incident beam. The 90 ° scattering intensity was measured using a Perkin Elmer MPF3 fluorimeter. All measurements were performed at 546 nm.
82
The light-scattering intensity Ra(O) of long polydisperse rod-like protein aggregates such as actin filaments has been shown to be given by [35] : Ra(tg) = g
)k
4 sin(0/2)
Ma
- - • Ca la
(1)
where 0 is the observation angle, K is an optical constant, Ca is the weight concentration of actin filaments, X is the wavelength of light in the solvent, Ma is the molecular weight of a filament subunit and l a is the length of a filament subunit. Eqn. 1 has been derived for rod-like particles, the length of which exceeds X/(4 sin(0/2)) and the diameter of which is much smaller than X/(4 sin(0/2)). Both conditions have been proved by electron-microscopic studies on synthetic actin filaments [36,37]. When molecules bound along a rod-like filament induce a small inhomogeneity in the mass-to-length ratio such as in the case of actin filaments partially saturated with tropomyosin, the scattering intensity, R t (tg), has been shown to be given b y [6] : Rt(v~) = g
X 4 sin(0/2)
Ma la
(Ca + Ct) 2 Ca
(2)
where Ct is the weight concentration of the tropomyosin molecules b o u n d along the actin filaments. This weight concentration was determined from the ratio of the scattering intensity of the actin-tropomyosin complex and of pure actin using Eqn. 3 which could be obtained by combining Eqn. 1 and 2: R t ( O ) - (Ca + C t ) 2
Ra(0)
C:a
(3)
It can be shown that the scattering intensity of u n b o u n d tropomyosin is so small compared to the scattering intensity of b o u n d tropomyosin that no correction needs to be applied for it. Fluorescence. NBD-actin fluorescence was measured in a fluorimeter (Perkin Elmer MPF3) using an excitation wavelength of 470 nm and monitoring emission at 520 nm through a 500 nm curt-off filter. All readings were standardized against quinine sulphate in 0.1 N H2SO4. The 450 nm fluorescence of a 1/~M solution excited at 360 nm was divided into 100 units. SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide electrophoresis was performed according to the method of Laemmli [29]. The gels were stained with Fast green and were scanned photometrically at 625 nm. SDSpolyacrylamide gel electrophoresis was used to determine the ratio of a- and ~-tropomyosin and the concentration of unpolymerized actin and of tropomyosin n o t bound to actin filaments. The bands of a- and ~-tropomyosin overlapped partially. The band of a-tropomyosin was found to be so narrow that, at the maximum of the ~-tropomyosin band, no a-tropomyosin was present. Therefore, the height of the peaks could be taken to be proportional to the concentration of a- or ~-tropomyosin chains. For the determination of unpolymerized actin and of u n b o u n d tropomyosin, the mixtures were centrifuged at 100 000 × g for 30 min (distance of sedimentation 7 mm). The supernatant was applied to the gels together with a series of standards of known actin and tropomyosin concentration.
83
Results Ligh t-scattering curves
Light-scattering curves of polymeric actin and of actin filaments partially or totally covered with tropomyosin were measured in order to test whether Eqns. 1--3 can be applied to determine the weight concentration of tropomyosin b o u n d to actin. Fig. 2 shows that the reciprocal of the scattering intensity is nearly proportional to the sine of the half observation angle (Eqns. 1 and 2). The continuous lines were calculated using Eqn. 2. At the temperature of the measurements (15°C) the concentration of u n b o u n d tropomyosin was found to be less than 0.05 #M. On account of this small concentration no correction was made for u n b o u n d tropomyosin. The results show that in the range of the observation angles (30--150 ° ) the ratio of the scattering intensities of actin-tropomyosin complexes and of pure actin is practically independent of the observation angle and that the measured ratio corresponds to the ratio calculated using Eqn. 3. Critical m o n o m e r concentration o f actin
The concentration of monomeric actin coexisting with actin filaments (critical m o n o m e r concentration of actin [38]) was measured in the presence
1.5
o
O~
C]O
00
[]
[]
o
el n-
o o
Rt/R a
o
oo
1.0
0
35
sin [8/2]
40
45
Temperature [°C]
Fig. 2. Light-scatterihg curves of aetin filaments and of aetin partially or t ot a l l y covered w i t h tropomyosin. R a ( 9 0 ° ) / R t ( ~ ) , reciprocal of the scattering i nt e ns i t y normalized by the value of actin at an ob° servation angle of 90°; ~, observation angle. A actin filaments (10.3 /~M); o, actin filaments (10.3 /~M) and t r o p o m y o s i n (0.5 #M); a, actin filaments (10.3/~M) and t r o p o m y o s i n (1.0 #M); ~, actin filaments (10.3/~M) and t r o p o m y o s i n (2.0/~M). The continuous lines were calculated. Fig. 3. TemperattLre dependence of the light-scattering i nt e ns i t y of NBD-actin/uncross-linked tropom y o s i n mixtures. R t, light-scattering intensity of the NBD-actin°tropomyusin c o m p l e x ; R a. light-scattering intensity of pure NBD-actin. Total t r o p o m y o s i n concentration, 1.5/~M. o, t o t a l NBD-actin concentration, 3.0 #M; o, total NBD-actin concentration, 6.5 ~zM.
84
C 0 @ 43 C
@ 0 W
~o~ i
o _= U.
0
0.2
0.4
0.6
Concentration of NBD-actin [ p M ] Fig. 4 . P l o t o f t h e t o t a l N B D - actin c o n c e n t r a t i o n vs. fluorescence intensity ( e x c i t a t i o n 4 7 0 rim, e m i s s i o n 5 2 0 n m ) . M o n o m e r i c N B D - a c t i n w a s d i s s o l v e d in 3 0 # M MgC12 , 5 0 0 # M A T P a n d 5 m M triethanolamine h y d r o c h l o r i d e ( p H 7 . 5 ) . All o t h e r s o l u t i o n s c o n t a i n e d 0.9.7 m M MgC12 a n d 8 0 m M KCI i n a d d i t i o n . * (36.7°C), monomeric NBD-actin; • (36.7°C), o (41.9°C), NBD-actin; • (36.7°C), o (41.9°C), NBD-actin a n d 1 /~M u n c r o s s - l i n k e d t r o p o m y o s i n ; • ( 3 6 . 7 ° C ) , A ( 4 1 . 9 ° C ) , N B D - a c t i n a n d 1 /~M c r o s s - l i n k e d t r o p o m y o s i n . T h e f l u o r e s c e n c e i n t e n s i t y w a s c a l i b r a t e d b y d i v i d i n g t h e f l u o r e s c e n c e o f a 1 #aM q u i n i n e sulphate solution into 100 units (excitation 360 nm, emission 450 nm).
and absence of tropomyosin. As the light-scattering intensity in the range of the critical concentration is t o o small to allow an accurate determination, the critical concentration was measured by electrophoresis (see Materials and Methods, SDS-polyacrylamide gel electrophoresis) and by introducing a fluorescence label to monitor polymerization. When measured by electrophoresis the m o n o m e r concentration was found to be in the range of 0.1 ~M. Various concentrations of NBD-actin were incubated with 1.5 ~M uncross-linked tropomyosin. The temperature dependence of the light-scattering intensity of the NBD-actin-tropomyosin complex revealed features similar to the actin-tropomyosin complex under the same conditions (Fig. 3), indicating that the NBD label did not affect the association of tropomy0sin with actin filaments. The critical m o n o m e r concentration was measured in the absence and in the presence of 1 pM cross-linked or uncross-linked tropomyosin. The NBD-actin concentration was varied between 0.05 and 0.6 #M. Plots of the NBD-actin concentration vs. the fluorescence intensity are depicted in Fig. 4. The bends of the curves can be explained by a 2-fold increased fluorescence intensity of polymeric NBD-actin formed above the critical m o n o m e r concentration. The critical concentration was found to be about 0.2 p.lVl and not to be affected by binding of tropomyosin to actin filaments.
Binding isotherms Binding of tropomyosin to actin filaments was measured by means of 90 ° light scattering. The actin concentration was kept constant at a b o u t 5 ~M. The tropomyosin concentrations were varied between 0.3 and 4 #M. When tropomyosin was present in a stoichiometric excess over actin (greater than 0.8 pM), at 25°C the scattering intensities of all samples were about 50% greater than the scattering intensity of pure actin. On heating, the scattering intensities of the samples decreased and at 45°C the scattering intensities of most of the samples were similar to the scattering intensity of pure actin. The scattering
85
117'
117
/ " / /
>, n ¢: @
• I:: ® qD
~t c ,= m m
01 I: m
¢1
O 0
1
2
3
4 JM
Concentration of free tropomyosln
O, 0
1
2
3
4 JM
Concentration of free tropomyosln
Fig. 5. Binding curves of cross-linked t r o p o m y o s i n to actin filaments. 4 38.2°C; m, 39.4°C; e, 40.7°C; *, 41.9°C. Total actin c o n c e n t r a t i o n 5.4 /~M. The con t i nuous fines were calculated using the fitted equifibriurn constants given in Table I. Fig. 6. Binding curves of uncross-linked t r o p o m y o s i n to actin filaments. 4 38.2°C; m 39.4°C; e, 40.7°C; *, 41.9°C. Total actin concentration 5.3 /~M. The con t i nuous fines were calculated using the fitted equilibrium constants given in Table I.
intensity of pure actin was almost temperature independent. At intermediate temperatures (35--45°C) the scattering intensities of the samples depended strongly on the tropomyosin concentration (see also Fig. 3). The results are depicted in Figs. 5 and 6 as plots of the concentration of free tropomyosin vs. the number of bound tropomyosin molecules per actin subunit (binaing density v). The binding density, v, was calculated from the light-scattering intensity as described in Materials and Methods (light scattering). No correction was made for unpolymerized actin as the critical concentration was found to be negligibly small compared to the total concentration. The concentration of free tropomyosin was calculated as the difference of the concentration of the total and the bound tropomyosin. Uncross-linked tropomyosin was found to bind more strongly to actin filaments than cross-linked tropomyosin.
Interpretation of the binding isotherms The binding isotherms (Figs. 5 and 6) are strongly sigmoidal, indicating cooperative binding of tropomyosin. The cooperative binding can be attributed to the polar end-to-end overlap of the termini of tropomyosin molecules contiguously bound along actin filaments. The equilibrium of the actin-tropomyosin interaction is adequately described by a model of cooperative binding of large ligands to a homogeneous lattice with overlapping binding sites. The 2
3
1
Fig. 7. Types of binding site of txopomyosin along the actin filament represented by Hnearly arranged cbewons. 1, isolated binding site;2, singly contiguous binding site;3, doubly contiguous binding site.
86
TABLE I E Q U I L I B R I U M C O N S T A N T S O F B I N D I N G OF C R O S S - L I N K E D A N D U N C R O S S - L I N K E D T R O P O MYOSIN TO ACTIN F I L A M E N T S K 1 , b i n d i n g c o n s t a n t f o r i s o l a t e d b i n d i n g sites; K 2 , b i n d i n g c o n s t a n t for singly c o n t i g u o u s b i n d i n g sites; K 3 , b i n d i n g c o n s t a n t for doubly contiguous binding sites (all values e x p r e s s e d in M - 1).
Temperature
Cross-linked t r o p o m y o s i n
Uncross-linked t r o p o m y o s i n
(°C) K1
K2
K3
K1
K2
K3
2.1 1.5 ---
0.62 0.44 ---
0.18 0.13 ---
3.1 4.6 3.1 1.1
1.37 1.14 0.76 0.50
0.62 0.29 0.19 0.22
(XIO-3) 38.2 39.4 40.7 41.9
(XIO-6)
(XIO-9)
(XIO-3)
(XlO-6)
(XIO-9)
actin filament represents a homogeneous lattice. This type of equilibrium has been treated by McGhee and von Hippel [39] and by Schwarz [40]. Three types of binding site and three corresponding binding constants are conceivable (Fig. 7). (1) Isolated binding sites: Tropomyosin has no overlap with an adjacent tropomyosin molecule (binding constant K1). (2) Singly contiguous binding sites: Tropomyosin has one overlap with an adjacent tropomyosin molecule (binding constant K2). (3) Doubly contiguous binding sites: Tropomyosin has two overlaps with two adjacent tropomyosin molecules (binding constant K3). The three binding constants, K1, K2 and K3, are not independent. The consecutive binding of two tropomyosin molecules to singly contiguous binding sites (K2) may be achieved also by binding of one tropomyosin molecule to an isolated binding site (K1) seven actin subunits away from another ligand and subsequent insertion of a second tropomyosin molecule into the seven-subunit gap (K3). Using the condition, K~2 = K~. K3, the binding constant K3 can be expressed in terms of K1 and K2 : K3 = K~JK1. McGhee and von Hippel have derived an equation that connects the concentration of free tropomyosin (t) and the binding density v with the binding constants K1 and K2 [39] : v t=K'l--7v
. ( 2 ( K 2 / K I - - 1). ( 1 - - 7v) \i2KT~¥1)-(1--7~+v--R
)6. ( 2 ( 1 _ 7v) 12 \I--Sv+R/
where R = x/{ (1 -- 8v): + 4K2/K1 • v • (1 -- 7v)}. The binding constants were fitted by seeking that set of parameters K~ and K2 where the standard deviation from the measured binding density reaches a minimum. The results are summarized in Table I. Discussion
The stability of the actin-tropomyosin complex was found to depend on the state of oxidation of cysteine 190 of tropomyosin. Uncross-linked tropomyosin binds more tightly to actin filaments than cross-linked tropomyosin. Structural differences between cross-linked and uncross-linked tropomyosin have been reported [26]. Cross-linked tropomyosin partially unfolds in the
87 30--40 ° C range whereas uncross-linked tropomyosin does not undergo this transition. The unfolding effect of the interchain disulphide bridge has been attributed to a strain that increases the instability of the molecule in the region of cysteine 190. This study demonstrates that small changes in the structure and flexibility can alter the actin-tropomyosin interaction. Acknowledgements The authors would like to thank Dr. Annemarie Weber for helpful advice during the course of the studies and for facilities to carry out the work. This study was supported by NIH grant HL 15692. A.W. received a reasearch fellowship from the Deutsche Forschungsgemeinschaft. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
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