BIOCHIMIE, 1979, 61, 601-605.
Calcium, magitesium and the conformation of parvalbumin during muscular activity. Jos A. COX, Dennis R. WINGE * and Eric A. STEIN O.
Department of Biochemistry, University of Geneva, Switzerland.
R6sum6.
AbstracL
Les conformations de trois formes de parvalb u m i n e de perche, c'est-b-dire s a n s m6taux, satur6e en calcium, satur6e en m a ~ n 6 s i u m , ont 6t6 6tudi~es p a r q u a t r e t e c h n i q u e s : fluorescence intrins~que, sensibilit6 b la trypsine, r6activit6 d e s thiols et dichroisme circulaire. Les r6sultats montrent q u e la prot6ine satur6e en calcium possbde 1° une structure plus comp a c t e que la prot6ine s a n s m6taux, 2 ° u n e t e n e u r 61ev6e en h61ice a, 3 ° un thiol m a s q u £ La conformation de la p a r v a l b u m i n e satur6e en m a q n 6 s i u m ne se distinque p a s de celle de la prot6ine satur6e en calcium, ce qui sugq~re qu'il n ' y a que p e u ou p a s de modification de conformation lots de l%chanqe r6versible Ca-Mcj qui peut intervenir p e n d a n t l'activit6 musculaire. De plus, des publications r6centes nous d o n n e n t & p e n s e r q u ' a u cours du c y c l e contraction-relaxation, les p a r a m ~ t r e s cin6tiques sont tels q u e la p a r v a l b u m i n e satur6e en m a q n 6 s i u m n ' a p a s toujours le temps de p a s s e r & la forme calcique, qui est pourtant celle que Yon obtient aprbs isolement de cette prot6ine.
The conformation of p e r c h p a r v a l b u m i n in the Ca-, Mq- a n d metal4ree state w a s studied b y intrinsic fluorescence, trypsin susceptibility, thiol titration a n d circular dichroism. The d a t a r e v e a l that C a - p a r v a l b u m i n h a s a more c o m p a c t structure than the metal-free protein, with a hiqh co-helical content a n d a buried thiol. No difference in conformation could be detected b e t w e e n Mq- a n d C a - p a r v a l b u m i n , indicatinq that the Ca-Mq e x c h a n q e that m a y take p l a c e durinq m u s c u l a r activity is a c c o m p a n i e d b y little or no structural c h a n q e s . Furthermore, recently p u b l i s h e d kinetic p a r a m e t e r s c a n n o w be interpreted as m e a r d n q that, durincj the contraction-relaxation cycle, p a r v a l b u m i n often s t a y s in the Mq-form instead of switchinq to the Ca-form w h i c h is p r e d o m i n a n t in v i t r o .
Introduction.
constant of 109 M-1, and M'g2* competes for both Ca-binding sites with an affinity 3 to 4 orders of magnitude lower than that of Ca 2* [4, 5]. In the presence of 2 mM Mg2÷, the apparent Ca-binding constant determined experimentally [6], or cal.cula~ed using competition equation [7], is 107 M-1.
Parvalbumin is a soluble calcium-binding protein first discovered in muscle of lower vertebrates [1], but later also found in the white muscle of higher vertebrates includirtg mammalians [21. The protein binds two calcimn ions [3] with an affinity
* Present address : D e p a r t m e n t of Biochemistry, Duke University Medical Center, Durham, NC 27710,' USA. To w h o m correspondence and requests f o r reprints s h o u l d be sent : D e p a r t m e n t of Biochemistry, P.O. Box 78 Jonction, 1211 Geneva 8, Smitzerland. Abbreviations : TN-C, troponin-C ; EDTA, (ethylenedinitrilo) tetraacetic acid ; Nbs~, 5,5"-dithiobis(2-nitrobenzoic a c i d ) ; NMR, nuclear magnetic resonance.
Key words : protein conformation, parvalbumin, Ca and Mg binding, contraction-relaxation speed.
Previous communications [8-11] have reported a significant effect of calcium on the conformation of parvalbumin. An analogy was drawn with the calcium-binding subunit of troponin [8, 9, 111, which is homologous to parval'bumin in sequence, and where ibe calcium effect upon protein conformation is intrulnental to muscular contraction [12]. On this basis it has been posttrlated that conformational changes induced by uptake and release of calcium occur i n v i v o during muscular contraction
602
J. A. Cox a n d coll.
a n d r e l a x a t i o n , a n d t h a t t h e s~udy of t h e s e c h a n g e s w o u l d eventually reveal the still u n k n o w n funct i o n of parvalbumin [8, 9, 11]. The present report shows that parvalbumin s a t u r a t e d w i t h M~g2+ h a s e s s e n t i a l l y t h e s a m e c o n f o r m a t i o n as C a - p a r v a l b u m i n , i n c o n t r a s t w i t h metal-free parvalbumin which undergoes significant structural changes upon metal depletion. In vivo h o w e v e r , t h e s e c h a n g e s d o n o t o c c u r , as t h e c o n c e n t r a t i o n o f f r e e l~g2+ d u r i n g r e l a x a t i o n a n d of C a 2÷ d u r i n g c o n t r a c t i o n a r e a l w a y s suffic i e n t to ~ e e p p a r v a l b u m i n s a t u r a t e d w i t h d i v a l e n t cations. Interestingly, recent kinetic experiments [13] s u g g e s t t h a t p , a r v a l b u m i n m a y r e m a i n i n t h e Mg 2+ f o r m e v e n d u r i n g c o n t r a c t i o n , at l e a s t w h e n the f r e q u e n c y of c o n t r a c t i o n s a n d r e l a x a t i o n s r e m a i n s m o d e r a t e . T h e r e f o r e , it is p r o p o s e d t h a t n o Ca2+-induced c o n f o r m a t i o n a l c h a n g e s o c c u r in p a r v a l b u m i n d u r i n g m u s c u l a r activity, in cont r a s t to t r o p o n i n - C (TN-C). §
Results. Intrinsic fluorescence. T h e f l u o r e s c e n c e s p e c t r u m of C a - p a r v a l b u m i n s h o w e d a t y p i c a l p h e n y l TABLE I.
Effect of calcium and ~2agnesium on phenylalanine fluorescence of paroalbumin. Initial state
of protein
Additives
Ca~+ Ca ~+ Ca u+ Metal-free Metal-free Metal-free
none 8 M urea 1 p. c e n t S D S none 10 ?M Ca ~+ 2 m M Mg ~+
Relative fluorescence 12 35 38 38 11 12
The 285 nrfi emission peak was recorded at a protein concentration of 0.2 m g / m l in 20 mM Tris-C1, pH 7.7. Results are expressed as relative i n t e n s i t y of fluorescence at 285 nm. Before the experiment, the protein was either saturated in Ca2+ or m a d e m e t a l - f r e e by c h r o m a t o g r a p h y on Sephadex G-25. TABLE II.
Titration of sulfhgdryl groups.
Materials and Methods. P a r v a l b u m i n was p r e p a r e d f r o m perch muscle as described previously [14]. In this study, isoparvalbum i n II was used exclusively. P r o t e i n concentration was d e t e r m i n e d f r o m ultraviolet a b s o r p t i o n using an 1% value of 1.49 at 259 nm, based on dry weight E lem analysis. Calcium-free p a r v a l b u m i n was obtained by a 1-2 h incubation w i t h 10 mM EDTA, pH 7.0, at 4°C, followed by gel filtration on Sephadex G-25 (1.6 X 15 cm) equilibrated and eluted w i t h calcium-free 20 mM Tris-Ct, pH 7.7. The resulting protein was 95 percent free of calcium as d e t e r m i n e d by atomic absorption spectroscopy. Metal r e s t o r a t i o n was achieved by adding cations to the protein solution so t h a t the resulting concent r a t i o n in free m e t a l ions was a p p r o x i m a t e l y 10 ~tmolar for calcium and 1 m m o l a r for magnesium. Under these conditions, the Ca2+ or Mg2+ content of p a r v a l b u m i n is 2 g a t o m s per mole and the metal uptake is very fast [4, 13]. To m i n i m i z e calcium contamination, acid-washed plastic ware and plexiglass columns were used. Buffers were passed t h r o u g h a column of immobilized parvalb u m i n in the m e t a l - f r e e f o r m [4]. Calcium was analyzed by atomic absorption spectroscopy on a P e r k i n - E l m e r model 303 p h o t o m e t e r . Circular dichroism spectra were recorded on a Jasco J-20A s p e c t r o p o l a r i m e t e r with c o n s t a n t nitrogen flushing ; the spectra were interpreted according to Greenfield and F a s m a n I157. Fluorescence m e a s u r e m e n t s were carried out on a Baird Atomic Fluoricord. Sulfhydryls were titrated w i t h Nbs: according to Habeeb [16]. Fluorescamine (Hoffman-La Roche) was used to determine the extent of proteolysis by trypsin.
BIOCH1MIE, 1979, 61, n ° 5-6.
Initial state of protein
Additives
Number of sullhydryls titrated
Ca2+ Ca ~+ Metal-free Metal-free Metal-free
none 3 mM E D T A none 10 ,~M Ca ~÷ 1 m M Mg "2+
0.08 0.90 0.88 0.11 0.20
P a r v a l b u m i n at a concentration of 0.1 mg per ml in 0.1 M Tris-C1, pH 7,7, was incubated w i t h Nbs~ (0.1 m g / m l ) . After 30 min, color development was monitored at 412 nm. TABLE III.
Effect of calcium and magnesium on the trypsin susceptibility of parvalbumin. Initial state of protein
Additives
Trypsin
Ca'~+
none
Metal-free Metal-free
none
---+ -~-
Ca~+ Metal-free
10 ?M Ca ~+ none none
Metal-free
10 ~M Ca ~+
Metal-free
1 mM Mg ~+
+
-~
Relative
fluorescence
1.0 1.4 1.2 1.1 4.6 1.7 1.7
P a r v a l b u m i n (0.1 to 0.25 m g / m l ) in 20 raM sodium borate, pH 8.3, was incubated at 23 ° C for 6 h w i t h t r y p s i n at an enzyme to p a r v a l b u m i n m o l a r ratio of 1 : 200. After addition of 40 ~tg fluorescamine, the fluorescence emiss;on at 475 n m was measured following excitation at 390 nm.
Calcium, m a g n e s i u m a n d the con[ormation of part, a l b u m i n . a l a n i n e e m i s s i o n b a n d 0'm,x = 285 nm) w h e n excited at 259 nm. The p h e n y l a l a n i n e q u a n t u m yield relative to L-ph.enylalanine Was estimated 'to be 0.02 a n d was insensitive to pH changes from 7 to 11. Removal of c a l c i u m +resulted i n a m a r k e d elevation of the q u a n t u m yield as seen in tabl.e I. D e n a t u r a t i o n of the p r o t e i n w i t h 8 M urea or 1 p e r c e n t SDS p r o d u c e d a s i m i l a r effect. R+eaddition of either Ca 2+ or Mg2+ resulted in a fluorescence i d e n t i c a l to that of the native protein.
Thiol reactivity. W h e r e a s the single c y s t e i n e group of p e r c h i s o p a r v a l b u m i n II reacted sluggishly w i t h the thiol reagent Nbs2, the reaction was essentially b r o u g h t to completion w i t h i n 30 m i n w h e n c a l c i u m was removed (table I,I). Restoration of either c a l c i n u m or m a g n e s i u m to the metal-free p r o t e i n r e s u l t e d in a masked thiol group, as i n the native protein. Trypsin susceptibility. C a - p a r v a l b u m i n showed a r e m a r k a b l e stability against t r y p s i n digestion. Even at t r y p s i n to p r o t e i n ratios as high as 1,:40, p a r v a l b u m i n was unaffected over a 24 h incubation period. Control e x p e r i m e n t s (not shown) on t r y p s i n digestion of casein r u l e d out that parva l b u m i n had an i n h e r e n t t r y p s i n i n h i b i t o r y activity. Removal of c a l c i u m led to a m a r k e d susceptibility to proteolysis as d e t e r m i n e d by the reaction of p r i m a r y a m i n o groups w i t h fluorescamine (table III).
605
tuted Ca- or M g - p a r v a l b u m i n m i g r a t e d like the native p r o t e i n , w h e r e a s the metal-free p r o t e i n was digested to such an extent that no b a n d s could be seen any more.
Circular dichroism. X-ray c r y s t a l l o g r a p h y and c i r c u l a r d i c h r o i s m revealed an a-helix c o n t e n t greater t h a n 40 per cent for p a r v a l b u m i n from carp [17, 10], w h i t i n g and pike [8]. Similarly the c i r c u l a r d i c h r o i c s p e c t r u m of p e r c h p a r v a l b u m i n s h o w n in figure 1 c o r r e s p o n d s to a s t r u c t u r e w i t h about 42 per cent a-helix. The residual ellipticity [0] 222 nm is r e d u c e d from - - 15,300 to - - 8,400 deg cm 2 dmole-1 u p o n removal of Ca 2+ by EDTA. I n c u b a t i o n of the p r o t e i n w i t h 8 M urea resulted i n a c i r c u l a r d i c h r o i s m s p e c t r u m c h a r a c t e r i s t i c of a fully r a n d o m i z e d polypeptide. Addition of Ca 2~ or ~g~+ to metal-free p a r v a l b u m i n caused an immediate restoration of ellipticity and a s p e c t r u m i n d i s t i n g u i s h a b l e from that of the native protein. The data show that the helical c o n t e n t of parvalb u m i n d e p e n d s on the presence of Ca ~+ or Mge÷ on the p r o t e i n ; this r e q u i r e m e n t is not very specific, as even T b 3+ [18] has essentially the same effect.
Discussion. Removal of c a l c i u m from p e r c h i s o p a r v a l b u m i n II results in a less c o m p a c t molecule : the single c y s t e i n e residue for i n s t a n c e becomes exposed to the aqueous e n v i r o n m e n t , a n d the p o l y p e p t i d e c h a i n susceptible to digestion b y trypsin. F u r t h e r more, c a l c i u m removal b r i n g s about a 25 p e r cent loss of a-helicity. These m o l e c u l a r alterations are r a p i d l y reversed w h e n c a l c i u m is readded. This is i n general agreement w i t h data r e p o r t e d for p a r v a l b u m i n of pike a n d w h i t i n g [8] a n d for the carp p r o t e i n [9, 10].
~+< - s
~-10
-15
wavelength in
nm
Fro. 1. - - Circular dichroism spectra of perch parvalbumin : 0.2 mg protein in 0.1 M Tris-Cl, pH 7.7.
I n c u b a t i o n of metaNfree p a r v a l b u m i n w i t h Ca 2+ or Mg2+ p r i o r to the a d d i t i o n of t r y p s i n suppressed the sensitivity to proteolysis : in p o l y a c r y l a m i d e disc gel electrophoresis (not shown) the reconsti-
BIOCHIMIE, 1979, 61, n ° 5-6.
The two c a l c i u m - b i n d i n g sites of p a r v a l b u m i n can also accomodate ~g2+ ions a n d are thus Ca-My sites [~]. Since the level of free Mg2÷ in the sarcoplasm is believed to be in the m i l l i m o l a r range [20], p a r v a l b u m i n in the r e s t i n g muscle m a y be better described as b e i n g in the My-form t h a n in the Ca-free form. Moreover, the results p r e s e n t e d here i n d i c a t e that a compact s t r u c t u r e is conferred to p a r v a l b u m i n not only by Ca 2+ but also by Mg2÷ ions. I n d e e d the My- a n d Ca-forms of p a r v a l b u m i n are i n d i s t i n g u i s h a b l e by the four methods used here, and a m i c r o c a l o r i m e t r i c study F19] confirmed that the Mg-Ca exchange in parvalb u m i n displays negligible e n t r o p y change, in contrast to metal b i n d i n g . This does not exclude that
604
J. A . C o x a n d coll.
small c o n f o r m a t i o n a l differences, that m i g h t be accessible b y NMR [21] or X-ray [17] studies, exist in the r e s t r i c t e d e n v i r o n m e n t of the m e t a l - b i n d i n g site. An elegant e x p e r i m e n t p e r f o r m e d b y P e c h b r e et al. [22] has s h o w n t h a t i n a Ca-free system c o n t a i n i n g p a r v a l b u m i n , m y o f i h r i l s and Mg-ATP, the a d d i t i o n of c a ~+ 'hrings a b o u t a fast a c t i v a t i o n of m y o f i b r i l l a r A T ~ a s e f o l l o w e d b y a slow i n a c t i v a t i o n resultin~g f r o m the s l o w u p t a k e of Ca =÷ b y p a r v a l b u m i n . This raises the question w h e t h e r p a r v a l b u m i n can b e c o m e s a t u r a t e d w i t h Ca 2+ d u r i n g the sho.rt time of m u s c u l a r c o n t r a c t i o n . As a m a t t e r of fact, the Ca ~+ t r a n s i e n t d u r i n g r e l a x a t i o n d e c a y s ~with a h a l f t i m e of only 50 msec [23], and the d u r a t i o n of c o n t r a c t i o n a n d relaxation in fast skeletal m u s c l e is as s h o r t as 27 a n d 70 m s e c r e s p e c t i v e l y [2~]. In contrast, the dissociation of l~g ~+ from p a r v a l b u m i n (t~/2 d~ss= 2:60 msec) [13] is r e l a t i v e l y slo~v, so that the M,g2+ content of the p r o t e i n can h a r d l y d i m i n i s h s i g n i f c a n t l y d u r i n g a few m u s c l e twitches. If the m e t a l : b i n d i n g sites of p a r v a l b u m i n , w h i c h r e p r e s e n t a concent r a t i o n of 1 m ~ in p e r c h m u s c l e [14] need not be s a t u r a t e d w i t h Ca2+ d u r i n g each c o n t r a c t i o n , then the t h r e s h o l d of Ca2+ release r e q u i r e d to t r i g g e r c o n t r a c t i o n b e c o m e s lower. The c o n c e n t r a t i o n in m u s c l e of C a - b i n d i n g sites of t r o p o n i n - G is only about 0.3 raM' a n d half of them, i.e. t h e Ca-Mg sites, a p p a r e n t l y do not b e c o m e s a t u r a t e d w i t h Ca 2+ d u r i n g the s h o r t t i m e of c o n t r a c t i o n [25]. The different k i n e t i c b e h a v i o r of the Ca-specific sites (TN-C) and of the C a-Mg m i x e d sites (TN-C and p a r v a l b u m i n ) m a y e x p l a i n w h y muscle c a n contract, although the total c o n t e n t of Ca 2+ is l o w e r (1.1 m ~ ) than the c a p a c i t y of the Ca-sequestering p r o t e i n s in the s a r c o p l a s m and in the m y o f i b r i l s ( > 1,6 mM) [14, 26]. On the o t h e r h a n d , it is l i k e l y t h a t the C a - f o r m of p a r v a l b u m i n p r e d o m i n a t e s w h e n muscle is r e p e a t e d l y s t i m u l a t e d (e.g. in smooth tetanus), i.e. w h e n t h e r e is a significant }nflux of Ca2+ t h r o u g h the s a r c o l e m m a . : F r o m the above, it can be c o n c l u d e d that the c o n f o r m a t i o n of p a r v a l b u m i n is essentially the s a m e w h e t h e r it is in the ~ g - or in the Ca-form, and that p a r v a l b u m i n in vivo i s a l w a y s s a t u r a t e d w i t h Mg2+ (or w i t h Ca2+). T h e r e f o r e one should not e x p e c t the u l t i m a t e f u n c t i o n of this p r o t e i n to be clarified b y s t u d y i n g the s t r u c t u r a l alterations b r o u g h t about by metal depletion. This contrasts s h a r p l y With the o b s e r v a t i o n s m a d e on TN-C, a p r o t e i n w h i c h possesses Ca-specific sites that are i n s t r u m e n t a l in m u s c u l a r c o n t r a c t i o n and that are l a c k i n g on p a r v a l b u m i n . The fact r e m a i n s that p a r v a l b u m i n s have been f o u n d in all verteBIOCHIMIE, 1979, 61, n ° 5-6.
b r a t e s p e c i e s so far investigated, f r o m fish to m a m m a l i a n s [27] and in some i n s t a n c e s in amounts as large as 10 g p e r kg of fresh tissue [28]. P a r v a l b u m i n s have thus t)een c o n s e r v e d t h r o u g h n e a r l y half a b i l l i o n y e a r s of evolution [2] and the f u n d a m e n t a l p r i n c i p l e of m o l e c u l a r e c o n o m y prev a i l i n g in l i v i n g m a t t e r w o u l d stand b e l i e d if these p r o t e i n s w e r e not s e r v i n g some significant p h y siological p u r p o s e , y e t to be elucidated.
Acknowledgements.
This work was carried out in cooperation w i t h Dr E. H. Fischer, D e p a r t m e n t of Biochemistry, University o f W a s h i n g t o n , Seattle. The authors are indebted to Dr W. W n u k f o r s t i m u l a t i n g discussions, to Miss Miehelle Comte f o r s u p p l y i n g pure p a r v a l b u m i n , and to the S w i s s N ~ . F . f o r f i n a n c i a l support (grants No 3.725.72 and 3.237.77).
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Calcium, magnesium and the conformation of parvalbumin. 18. Nelson, D. J., Miller, T. L. ~ Martin, R. B. (1977) Bioinorganic Chemistry, 7, 325-334. 19. Moeschler, H. J. ~ Schaer, J. J. (1979) Experientia, 36, in press. 20. Veloso, D., Guyn, R. W., Oskarson, M. ~ Veech, R. L. (1973) J. Biol. Chem., 248, 4811-4819 21. Drakenberg, R., L i n d m a n , B., Car6, A. ~ Parello, J. (1978) FEBS Lett., 92, 346-350. 22. Pech6re, J. F., Derancourt, J. ~ Haiech, J. (1977) FEBS Left., 75, 111-114. 23. Miledi, R., Parker, I. ~ Schalow, G. (1977) Proc. R. Soe. London. Set. B, 198, 201-210.
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24. Buller, A. J., Eccles, J. C. ~ Eccles, B. M. (1960) J. Physiol., 150, 399-416. 25. J o h n s o n , J. D., Charlton, S. ~ Potter. J. D. (1978) Biophys. Y., 21, 105a. 26. Baron, G., Demaille, J. ~ Dutruge, E. (1975) FEBS Left., 56, 156-160. 27. Pech~re, J. F. (1974) C. R. Acad. Sci. Paris, 278, 2577-2579. 28. Blum, H. E., Lehky, P., Kohler, L., Stein, E. A. Fischer, E. H. (1977) J. Biol. Chem., 252, 28342838.
Calcium, magitesium and the conformation of parvalbumin during muscular activity. Jos A. COX, Dennis R. WINGE * and Eric A. STEIN O.
Department of Biochemistry, University of Geneva, Switzerland.
R6sum6.
AbstracL
Les conformations de trois formes de parvalb u m i n e de perche, c'est-b-dire s a n s m6taux, satur6e en calcium, satur6e en m a ~ n 6 s i u m , ont 6t6 6tudi~es p a r q u a t r e t e c h n i q u e s : fluorescence intrins~que, sensibilit6 b la trypsine, r6activit6 d e s thiols et dichroisme circulaire. Les r6sultats montrent q u e la prot6ine satur6e en calcium possbde 1° une structure plus comp a c t e que la prot6ine s a n s m6taux, 2 ° u n e t e n e u r 61ev6e en h61ice a, 3 ° un thiol m a s q u £ La conformation de la p a r v a l b u m i n e satur6e en m a q n 6 s i u m ne se distinque p a s de celle de la prot6ine satur6e en calcium, ce qui sugq~re qu'il n ' y a que p e u ou p a s de modification de conformation lots de l%chanqe r6versible Ca-Mcj qui peut intervenir p e n d a n t l'activit6 musculaire. De plus, des publications r6centes nous d o n n e n t & p e n s e r q u ' a u cours du c y c l e contraction-relaxation, les p a r a m ~ t r e s cin6tiques sont tels q u e la p a r v a l b u m i n e satur6e en m a q n 6 s i u m n ' a p a s toujours le temps de p a s s e r & la forme calcique, qui est pourtant celle que Yon obtient aprbs isolement de cette prot6ine.
The conformation of p e r c h p a r v a l b u m i n in the Ca-, Mq- a n d metal4ree state w a s studied b y intrinsic fluorescence, trypsin susceptibility, thiol titration a n d circular dichroism. The d a t a r e v e a l that C a - p a r v a l b u m i n h a s a more c o m p a c t structure than the metal-free protein, with a hiqh co-helical content a n d a buried thiol. No difference in conformation could be detected b e t w e e n Mq- a n d C a - p a r v a l b u m i n , indicatinq that the Ca-Mq e x c h a n q e that m a y take p l a c e durinq m u s c u l a r activity is a c c o m p a n i e d b y little or no structural c h a n q e s . Furthermore, recently p u b l i s h e d kinetic p a r a m e t e r s c a n n o w be interpreted as m e a r d n q that, durincj the contraction-relaxation cycle, p a r v a l b u m i n often s t a y s in the Mq-form instead of switchinq to the Ca-form w h i c h is p r e d o m i n a n t in v i t r o .
Introduction.
constant of 109 M-1, and M'g2* competes for both Ca-binding sites with an affinity 3 to 4 orders of magnitude lower than that of Ca 2* [4, 5]. In the presence of 2 mM Mg2÷, the apparent Ca-binding constant determined experimentally [6], or cal.cula~ed using competition equation [7], is 107 M-1.
Parvalbumin is a soluble calcium-binding protein first discovered in muscle of lower vertebrates [1], but later also found in the white muscle of higher vertebrates includirtg mammalians [21. The protein binds two calcimn ions [3] with an affinity
* Present address : D e p a r t m e n t of Biochemistry, Duke University Medical Center, Durham, NC 27710,' USA. To w h o m correspondence and requests f o r reprints s h o u l d be sent : D e p a r t m e n t of Biochemistry, P.O. Box 78 Jonction, 1211 Geneva 8, Smitzerland. Abbreviations : TN-C, troponin-C ; EDTA, (ethylenedinitrilo) tetraacetic acid ; Nbs~, 5,5"-dithiobis(2-nitrobenzoic a c i d ) ; NMR, nuclear magnetic resonance.
Key words : protein conformation, parvalbumin, Ca and Mg binding, contraction-relaxation speed.
Previous communications [8-11] have reported a significant effect of calcium on the conformation of parvalbumin. An analogy was drawn with the calcium-binding subunit of troponin [8, 9, 111, which is homologous to parval'bumin in sequence, and where ibe calcium effect upon protein conformation is intrulnental to muscular contraction [12]. On this basis it has been posttrlated that conformational changes induced by uptake and release of calcium occur i n v i v o during muscular contraction
602
J. A. Cox a n d coll.
a n d r e l a x a t i o n , a n d t h a t t h e s~udy of t h e s e c h a n g e s w o u l d eventually reveal the still u n k n o w n funct i o n of parvalbumin [8, 9, 11]. The present report shows that parvalbumin s a t u r a t e d w i t h M~g2+ h a s e s s e n t i a l l y t h e s a m e c o n f o r m a t i o n as C a - p a r v a l b u m i n , i n c o n t r a s t w i t h metal-free parvalbumin which undergoes significant structural changes upon metal depletion. In vivo h o w e v e r , t h e s e c h a n g e s d o n o t o c c u r , as t h e c o n c e n t r a t i o n o f f r e e l~g2+ d u r i n g r e l a x a t i o n a n d of C a 2÷ d u r i n g c o n t r a c t i o n a r e a l w a y s suffic i e n t to ~ e e p p a r v a l b u m i n s a t u r a t e d w i t h d i v a l e n t cations. Interestingly, recent kinetic experiments [13] s u g g e s t t h a t p , a r v a l b u m i n m a y r e m a i n i n t h e Mg 2+ f o r m e v e n d u r i n g c o n t r a c t i o n , at l e a s t w h e n the f r e q u e n c y of c o n t r a c t i o n s a n d r e l a x a t i o n s r e m a i n s m o d e r a t e . T h e r e f o r e , it is p r o p o s e d t h a t n o Ca2+-induced c o n f o r m a t i o n a l c h a n g e s o c c u r in p a r v a l b u m i n d u r i n g m u s c u l a r activity, in cont r a s t to t r o p o n i n - C (TN-C). §
Results. Intrinsic fluorescence. T h e f l u o r e s c e n c e s p e c t r u m of C a - p a r v a l b u m i n s h o w e d a t y p i c a l p h e n y l TABLE I.
Effect of calcium and ~2agnesium on phenylalanine fluorescence of paroalbumin. Initial state
of protein
Additives
Ca~+ Ca ~+ Ca u+ Metal-free Metal-free Metal-free
none 8 M urea 1 p. c e n t S D S none 10 ?M Ca ~+ 2 m M Mg ~+
Relative fluorescence 12 35 38 38 11 12
The 285 nrfi emission peak was recorded at a protein concentration of 0.2 m g / m l in 20 mM Tris-C1, pH 7.7. Results are expressed as relative i n t e n s i t y of fluorescence at 285 nm. Before the experiment, the protein was either saturated in Ca2+ or m a d e m e t a l - f r e e by c h r o m a t o g r a p h y on Sephadex G-25. TABLE II.
Titration of sulfhgdryl groups.
Materials and Methods. P a r v a l b u m i n was p r e p a r e d f r o m perch muscle as described previously [14]. In this study, isoparvalbum i n II was used exclusively. P r o t e i n concentration was d e t e r m i n e d f r o m ultraviolet a b s o r p t i o n using an 1% value of 1.49 at 259 nm, based on dry weight E lem analysis. Calcium-free p a r v a l b u m i n was obtained by a 1-2 h incubation w i t h 10 mM EDTA, pH 7.0, at 4°C, followed by gel filtration on Sephadex G-25 (1.6 X 15 cm) equilibrated and eluted w i t h calcium-free 20 mM Tris-Ct, pH 7.7. The resulting protein was 95 percent free of calcium as d e t e r m i n e d by atomic absorption spectroscopy. Metal r e s t o r a t i o n was achieved by adding cations to the protein solution so t h a t the resulting concent r a t i o n in free m e t a l ions was a p p r o x i m a t e l y 10 ~tmolar for calcium and 1 m m o l a r for magnesium. Under these conditions, the Ca2+ or Mg2+ content of p a r v a l b u m i n is 2 g a t o m s per mole and the metal uptake is very fast [4, 13]. To m i n i m i z e calcium contamination, acid-washed plastic ware and plexiglass columns were used. Buffers were passed t h r o u g h a column of immobilized parvalb u m i n in the m e t a l - f r e e f o r m [4]. Calcium was analyzed by atomic absorption spectroscopy on a P e r k i n - E l m e r model 303 p h o t o m e t e r . Circular dichroism spectra were recorded on a Jasco J-20A s p e c t r o p o l a r i m e t e r with c o n s t a n t nitrogen flushing ; the spectra were interpreted according to Greenfield and F a s m a n I157. Fluorescence m e a s u r e m e n t s were carried out on a Baird Atomic Fluoricord. Sulfhydryls were titrated w i t h Nbs: according to Habeeb [16]. Fluorescamine (Hoffman-La Roche) was used to determine the extent of proteolysis by trypsin.
BIOCH1MIE, 1979, 61, n ° 5-6.
Initial state of protein
Additives
Number of sullhydryls titrated
Ca2+ Ca ~+ Metal-free Metal-free Metal-free
none 3 mM E D T A none 10 ,~M Ca ~÷ 1 m M Mg "2+
0.08 0.90 0.88 0.11 0.20
P a r v a l b u m i n at a concentration of 0.1 mg per ml in 0.1 M Tris-C1, pH 7,7, was incubated w i t h Nbs~ (0.1 m g / m l ) . After 30 min, color development was monitored at 412 nm. TABLE III.
Effect of calcium and magnesium on the trypsin susceptibility of parvalbumin. Initial state of protein
Additives
Trypsin
Ca'~+
none
Metal-free Metal-free
none
---+ -~-
Ca~+ Metal-free
10 ?M Ca ~+ none none
Metal-free
10 ~M Ca ~+
Metal-free
1 mM Mg ~+
+
-~
Relative
fluorescence
1.0 1.4 1.2 1.1 4.6 1.7 1.7
P a r v a l b u m i n (0.1 to 0.25 m g / m l ) in 20 raM sodium borate, pH 8.3, was incubated at 23 ° C for 6 h w i t h t r y p s i n at an enzyme to p a r v a l b u m i n m o l a r ratio of 1 : 200. After addition of 40 ~tg fluorescamine, the fluorescence emiss;on at 475 n m was measured following excitation at 390 nm.
Calcium, m a g n e s i u m a n d the con[ormation of part, a l b u m i n . a l a n i n e e m i s s i o n b a n d 0'm,x = 285 nm) w h e n excited at 259 nm. The p h e n y l a l a n i n e q u a n t u m yield relative to L-ph.enylalanine Was estimated 'to be 0.02 a n d was insensitive to pH changes from 7 to 11. Removal of c a l c i u m +resulted i n a m a r k e d elevation of the q u a n t u m yield as seen in tabl.e I. D e n a t u r a t i o n of the p r o t e i n w i t h 8 M urea or 1 p e r c e n t SDS p r o d u c e d a s i m i l a r effect. R+eaddition of either Ca 2+ or Mg2+ resulted in a fluorescence i d e n t i c a l to that of the native protein.
Thiol reactivity. W h e r e a s the single c y s t e i n e group of p e r c h i s o p a r v a l b u m i n II reacted sluggishly w i t h the thiol reagent Nbs2, the reaction was essentially b r o u g h t to completion w i t h i n 30 m i n w h e n c a l c i u m was removed (table I,I). Restoration of either c a l c i n u m or m a g n e s i u m to the metal-free p r o t e i n r e s u l t e d in a masked thiol group, as i n the native protein. Trypsin susceptibility. C a - p a r v a l b u m i n showed a r e m a r k a b l e stability against t r y p s i n digestion. Even at t r y p s i n to p r o t e i n ratios as high as 1,:40, p a r v a l b u m i n was unaffected over a 24 h incubation period. Control e x p e r i m e n t s (not shown) on t r y p s i n digestion of casein r u l e d out that parva l b u m i n had an i n h e r e n t t r y p s i n i n h i b i t o r y activity. Removal of c a l c i u m led to a m a r k e d susceptibility to proteolysis as d e t e r m i n e d by the reaction of p r i m a r y a m i n o groups w i t h fluorescamine (table III).
605
tuted Ca- or M g - p a r v a l b u m i n m i g r a t e d like the native p r o t e i n , w h e r e a s the metal-free p r o t e i n was digested to such an extent that no b a n d s could be seen any more.
Circular dichroism. X-ray c r y s t a l l o g r a p h y and c i r c u l a r d i c h r o i s m revealed an a-helix c o n t e n t greater t h a n 40 per cent for p a r v a l b u m i n from carp [17, 10], w h i t i n g and pike [8]. Similarly the c i r c u l a r d i c h r o i c s p e c t r u m of p e r c h p a r v a l b u m i n s h o w n in figure 1 c o r r e s p o n d s to a s t r u c t u r e w i t h about 42 per cent a-helix. The residual ellipticity [0] 222 nm is r e d u c e d from - - 15,300 to - - 8,400 deg cm 2 dmole-1 u p o n removal of Ca 2+ by EDTA. I n c u b a t i o n of the p r o t e i n w i t h 8 M urea resulted i n a c i r c u l a r d i c h r o i s m s p e c t r u m c h a r a c t e r i s t i c of a fully r a n d o m i z e d polypeptide. Addition of Ca 2~ or ~g~+ to metal-free p a r v a l b u m i n caused an immediate restoration of ellipticity and a s p e c t r u m i n d i s t i n g u i s h a b l e from that of the native protein. The data show that the helical c o n t e n t of parvalb u m i n d e p e n d s on the presence of Ca ~+ or Mge÷ on the p r o t e i n ; this r e q u i r e m e n t is not very specific, as even T b 3+ [18] has essentially the same effect.
Discussion. Removal of c a l c i u m from p e r c h i s o p a r v a l b u m i n II results in a less c o m p a c t molecule : the single c y s t e i n e residue for i n s t a n c e becomes exposed to the aqueous e n v i r o n m e n t , a n d the p o l y p e p t i d e c h a i n susceptible to digestion b y trypsin. F u r t h e r more, c a l c i u m removal b r i n g s about a 25 p e r cent loss of a-helicity. These m o l e c u l a r alterations are r a p i d l y reversed w h e n c a l c i u m is readded. This is i n general agreement w i t h data r e p o r t e d for p a r v a l b u m i n of pike a n d w h i t i n g [8] a n d for the carp p r o t e i n [9, 10].
~+< - s
~-10
-15
wavelength in
nm
Fro. 1. - - Circular dichroism spectra of perch parvalbumin : 0.2 mg protein in 0.1 M Tris-Cl, pH 7.7.
I n c u b a t i o n of metaNfree p a r v a l b u m i n w i t h Ca 2+ or Mg2+ p r i o r to the a d d i t i o n of t r y p s i n suppressed the sensitivity to proteolysis : in p o l y a c r y l a m i d e disc gel electrophoresis (not shown) the reconsti-
BIOCHIMIE, 1979, 61, n ° 5-6.
The two c a l c i u m - b i n d i n g sites of p a r v a l b u m i n can also accomodate ~g2+ ions a n d are thus Ca-My sites [~]. Since the level of free Mg2÷ in the sarcoplasm is believed to be in the m i l l i m o l a r range [20], p a r v a l b u m i n in the r e s t i n g muscle m a y be better described as b e i n g in the My-form t h a n in the Ca-free form. Moreover, the results p r e s e n t e d here i n d i c a t e that a compact s t r u c t u r e is conferred to p a r v a l b u m i n not only by Ca 2+ but also by Mg2÷ ions. I n d e e d the My- a n d Ca-forms of p a r v a l b u m i n are i n d i s t i n g u i s h a b l e by the four methods used here, and a m i c r o c a l o r i m e t r i c study F19] confirmed that the Mg-Ca exchange in parvalb u m i n displays negligible e n t r o p y change, in contrast to metal b i n d i n g . This does not exclude that
604
J. A . C o x a n d coll.
small c o n f o r m a t i o n a l differences, that m i g h t be accessible b y NMR [21] or X-ray [17] studies, exist in the r e s t r i c t e d e n v i r o n m e n t of the m e t a l - b i n d i n g site. An elegant e x p e r i m e n t p e r f o r m e d b y P e c h b r e et al. [22] has s h o w n t h a t i n a Ca-free system c o n t a i n i n g p a r v a l b u m i n , m y o f i h r i l s and Mg-ATP, the a d d i t i o n of c a ~+ 'hrings a b o u t a fast a c t i v a t i o n of m y o f i b r i l l a r A T ~ a s e f o l l o w e d b y a slow i n a c t i v a t i o n resultin~g f r o m the s l o w u p t a k e of Ca =÷ b y p a r v a l b u m i n . This raises the question w h e t h e r p a r v a l b u m i n can b e c o m e s a t u r a t e d w i t h Ca 2+ d u r i n g the sho.rt time of m u s c u l a r c o n t r a c t i o n . As a m a t t e r of fact, the Ca ~+ t r a n s i e n t d u r i n g r e l a x a t i o n d e c a y s ~with a h a l f t i m e of only 50 msec [23], and the d u r a t i o n of c o n t r a c t i o n a n d relaxation in fast skeletal m u s c l e is as s h o r t as 27 a n d 70 m s e c r e s p e c t i v e l y [2~]. In contrast, the dissociation of l~g ~+ from p a r v a l b u m i n (t~/2 d~ss= 2:60 msec) [13] is r e l a t i v e l y slo~v, so that the M,g2+ content of the p r o t e i n can h a r d l y d i m i n i s h s i g n i f c a n t l y d u r i n g a few m u s c l e twitches. If the m e t a l : b i n d i n g sites of p a r v a l b u m i n , w h i c h r e p r e s e n t a concent r a t i o n of 1 m ~ in p e r c h m u s c l e [14] need not be s a t u r a t e d w i t h Ca2+ d u r i n g each c o n t r a c t i o n , then the t h r e s h o l d of Ca2+ release r e q u i r e d to t r i g g e r c o n t r a c t i o n b e c o m e s lower. The c o n c e n t r a t i o n in m u s c l e of C a - b i n d i n g sites of t r o p o n i n - G is only about 0.3 raM' a n d half of them, i.e. t h e Ca-Mg sites, a p p a r e n t l y do not b e c o m e s a t u r a t e d w i t h Ca 2+ d u r i n g the s h o r t t i m e of c o n t r a c t i o n [25]. The different k i n e t i c b e h a v i o r of the Ca-specific sites (TN-C) and of the C a-Mg m i x e d sites (TN-C and p a r v a l b u m i n ) m a y e x p l a i n w h y muscle c a n contract, although the total c o n t e n t of Ca 2+ is l o w e r (1.1 m ~ ) than the c a p a c i t y of the Ca-sequestering p r o t e i n s in the s a r c o p l a s m and in the m y o f i b r i l s ( > 1,6 mM) [14, 26]. On the o t h e r h a n d , it is l i k e l y t h a t the C a - f o r m of p a r v a l b u m i n p r e d o m i n a t e s w h e n muscle is r e p e a t e d l y s t i m u l a t e d (e.g. in smooth tetanus), i.e. w h e n t h e r e is a significant }nflux of Ca2+ t h r o u g h the s a r c o l e m m a . : F r o m the above, it can be c o n c l u d e d that the c o n f o r m a t i o n of p a r v a l b u m i n is essentially the s a m e w h e t h e r it is in the ~ g - or in the Ca-form, and that p a r v a l b u m i n in vivo i s a l w a y s s a t u r a t e d w i t h Mg2+ (or w i t h Ca2+). T h e r e f o r e one should not e x p e c t the u l t i m a t e f u n c t i o n of this p r o t e i n to be clarified b y s t u d y i n g the s t r u c t u r a l alterations b r o u g h t about by metal depletion. This contrasts s h a r p l y With the o b s e r v a t i o n s m a d e on TN-C, a p r o t e i n w h i c h possesses Ca-specific sites that are i n s t r u m e n t a l in m u s c u l a r c o n t r a c t i o n and that are l a c k i n g on p a r v a l b u m i n . The fact r e m a i n s that p a r v a l b u m i n s have been f o u n d in all verteBIOCHIMIE, 1979, 61, n ° 5-6.
b r a t e s p e c i e s so far investigated, f r o m fish to m a m m a l i a n s [27] and in some i n s t a n c e s in amounts as large as 10 g p e r kg of fresh tissue [28]. P a r v a l b u m i n s have thus t)een c o n s e r v e d t h r o u g h n e a r l y half a b i l l i o n y e a r s of evolution [2] and the f u n d a m e n t a l p r i n c i p l e of m o l e c u l a r e c o n o m y prev a i l i n g in l i v i n g m a t t e r w o u l d stand b e l i e d if these p r o t e i n s w e r e not s e r v i n g some significant p h y siological p u r p o s e , y e t to be elucidated.
Acknowledgements.
This work was carried out in cooperation w i t h Dr E. H. Fischer, D e p a r t m e n t of Biochemistry, University o f W a s h i n g t o n , Seattle. The authors are indebted to Dr W. W n u k f o r s t i m u l a t i n g discussions, to Miss Miehelle Comte f o r s u p p l y i n g pure p a r v a l b u m i n , and to the S w i s s N ~ . F . f o r f i n a n c i a l support (grants No 3.725.72 and 3.237.77).
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