Terbium replacement of calcium in parvalbumin

.I. MoE. Bid.

(1978) 124, 123-132

Terbium Replacement of Calcium in Parvalbumin JANUSZSOWADSKI~,GARY

CORNICKAND

ROBERT H. KRETSINGER

Department of Biology University of Virginia Charlottesville, VA 22901, U.S.A. (Received 10 January

1978)

crystallized in 2.9 M-ammonium C’arp muscle calcium binding parvalbumin, sulfate, can bind two Tb3+ ions, which displace the two Ca2+ ions normally in the loop between the E and the F a-helices is present. The Ca2 + co-ordinated ; whereas the Ca2 + at the CD site is replaced displaced at low Tb3 + concentrations There is not a third Tb3+ site as had been only at higher Tb3+ concentration. experiments performed without, suggested in interpretations of Tb 3+ fluorescence ammonium sulfate. A third electron density peak in the difference Fourier maps is tentatively assigned to a sulfate ion co-ordinating the EF site Tb3 + ion.

1. Introduction Calcium, functioning as a second messenger, plays a crucial role in the control of most 1977). Further, it, if not all eukaryotic cells (Rasmussen et aE., 1972; Kretsinger, stabilizes many viruses and extracellular proteins (review by Kretsinger, 1976). HOWever, since the electronic configuration of Ca2+ is 21s 22~ 62p 23s ‘3p, like that of the noble gas argon, it is spectrally inert, except for X-ray absorption. Hence various scientists have done spectroscopic experiments in which the Ca2+ of a protein is replaced by a lanthanide ion (review by Reuben, 1975). In such experiments it, is important to know the localization of lanthanide binding site(s) as well as any changes in the lanthanide protein relative to the native calcium protein. Terbium is a particularly attractive replacement for calcium because it, alone among t,he lanthanides, displays a significant fluorescence when the liganding protein is irradiated in the region of its phenylalanyl (259 nm), tyrosyl (280 nm), or tryptophanyl (295 nm) absorption bands. Brittain et aZ. (1976) observed Tb3+ mediated fluorescence in 36 of 40 tested proteins, representing over 15 different homolop families. In a sense this was an unexpected result in that one might not have anticipated that so many proteins would have an aromatic ring near their Ca2 + (Tb3 +) binding site(s). However, it does establish the general applicability of terbium fluorescence spectroscopy to the study of proteins. Of most direct relevance to our work, all except one of the proteins show increasing fluorescence with increasing Tb3 +/protein molar ratios. However, when Tb3 + is added parvalbumin. the fluorescence rises almost linearly, to native (Caz+ containing) reaches a maximum at a Tb3+/parvalbumin ratio of 1.5. and then decreases t,o So,,, t Prevent New Haven,

address: Department Corm. 06620, U.S.A.

of Molecular

Riophysics

and

Biochemistry,

Yale

Univwsit

y.

123 0022~-2836/78/250123-j-10 $02.00/O

0

1978 Academic

Press

Inc.

(London)

Lttl.

124

J. SOWADSKI,

G. CORNICK

AND

R. H. KRETSINGER

of the peak value at a molar ratio of 3-O (Donato & Martin, 1974; Nelson et al., 1977). This is a unique and intriguing result because it suggests a quenching mechanism, unprecedented in protein studies. Before exploring this mechanism we describe parvalbumin and its importance as a model system for studying calcium-modulated proteins. Muscle calcium-binding parvalbumin is found in the white muscle of most, if not all, vertebrates and in particularly high concentration (0.1 to 0.8 mmol/kg wet weight) in fish. Although its exact function remains unknown, there is inferential evidence that parvalbumin can act as a soluble relaxing factor (Gerday & Gillis, 1976; Gillis & Gerday, 1977; Pech&re et al., 1977). Its Ca 2 + binding affinity and rate may be matched to those of troponin and of the sarcoplasmic reticulum to provide the desired intensity and duration of contraction (PeehAre, 1977). The amino acid sequences of at least 14 different isotypes from ten different species have been determined. The crystal structure of carp parvalbumin (isotype 3, p1 4.2) has been determined and refined using 1.9 A resolution X-ray diffraction data (Moews & Kretsinger, 1975a). Parvalbumin is a valuable model system since the following calcium modulated proteins, calcium-binding component of troponin, various myosin light chains, calcium dependent regulator of adenylate cyclase and/or cyclic nucleotide phosphodiesterase, each contain four EF homolog regions (Kretsinger, 1977). Also, vitamin D-induced calcium-binding protein contains two EF homolog regions. These homology results have been deduced from amino acid sequence determinations. One variation on this basic evolutionary pattern is seen in the EF region of parvalbumin. Residue 98, the ninth residue in the EF loop, is glycine and can provide no side-chain oxygen to co-ordinate the EF Ca2 + ion. Water co-ordinat.es the EF Ca2 + ion at that position. In contrast the CD Ca2+ ion is completely surrounded by protein ligands and is not co-ordinated by solvent molecules (Fig. 1). The basic spectroscopic results of Donato & Mart’in (1974) were confirmed and extended by Nelson et al. (1977) as shown in Figure 2. In contrast to all other proteins examined, on Tb3+ addition, the Tb 3+ fluorescence decreases after a maximum corresponding to a 1.5 Tb3 + /parvalbumin ratio. The tentative interpretation offered by Donato & Martin and elaborated by Nelson et al. is outlined. (1) As Tb 3 + is added to native parvalbumin, the Tb3 + ions displace the Ca2+ ions at both the EF and the CD sites. (2) Further, Tb3 + binds to a third site postulated to be near the EF site and involving the EF site water as one of its ligands. (3) The affinities of the three sites are greater for Tb 3 + than for Ca2 + . Their bindings are non-co-operative. (4) The EF Tb 3 + ion fluoresces intensely. The CD Tb3 + ion neither fluoresces nor quenches. The third site Tb 3 + ion does not fluoresce but does quench the fluorescence of the EF Tb3 + . In parallel with the original spectroscopic studies of Donato & Martin, Moews & by difference Fourier Kretsinger (1975b) studied the binding of Tb 3 + to parvalbumin projections using hO1, or centric, data. They found that Tb3+ replaces the EF site Ca2 + . They found no evidence of Tb3 + at either the CD site or at the putative third site. Because of the increasing importance of parvalbumin as a model system for the calcium-modulated proteins and because of the uniqueness of this fluorescence quenching phenomenon, we decided to extend the crystallographic studies. We

TERBIUM

REPLACEMENT

OF

CALCIUM

IN

PARVALBUMIK

125

Glu 60

Gly 95 L Glu 59

X(51) 0 Gly 56

Pro. 1. The 2 Ca2 + binding regions of parvalbumin are viewed from the surface of the molecule looking down the intramolecular approximate 2.fold axis. The CD Ca2+ ion, to the right, is octahedrally co-ordinated. Residues Asp51, Asp53, Ser56, Glu69 and Glu62 each co-ordinate the CD2’ ion with a side-chain oxygen atom. Phe67 co-ordinates at the - Y vertex with it,s carbonyl oxygen atom; its phenyl ring approaches the EF Ca 2+ ion. The EF CaZ+ ion, to the left, is co-ordinated in a homologous manner. Asp92 and GlulOl co-ordinate with both oxygen atoms of their carboxylate groups. Residue 98 is glycine, homologous to Glu59. Water co-ordinates the EF Ca2+ ion at the -X vertex. The number @ designates t,he position of the third peak appearing in the difference Fourier maps (see Fig. 4).

1

Pm. 2. The relative intensity (heavy line) of Tb 3+ fluorescence at 545 nm is shown for the addition of TbCl, to native (adapted from Nelson et ul., 1977). The maximum Ca 2+-pa~valbumin intensity, defined as 100, occurs at a Tb3+/protein molar ratio of about 1.5. The fluorescence calculated for our 2.site, non-co-operative model is shown by the lighter line.

126

J. SOWADSKI,

G. CORNICK

ASI)

K.

H.

KRETSINGER

obtained a titration curve in which each point corresponds to an h0Zdifference Fourier projection. At higher Tb3+/parvalbumin ratios t’hc CD site binds a ‘l’b” + ion. Based on these results we chose a Tb3 +/prot,ein rat,io at’ which b&h EP and CD sitt,s ~YIY: occupied and calculated a three-dimensional difference Fourier map. The Tb3 + peaks at the CD and EF sites are confirmed. We find no third (quenching) Tb3+ site.

2. Materials and Methods The carp muscle calcium-binding parvalbumin (isotype 3, p1 4.2) came from the same concenlyophilized stock as used in the Moews & Kretsinger (19758) study. Parvalbumin trations were determined from the weight of lyophilized material used and confirmed by absorbance measurements (A,,o “,,, = 2.02 x IO3 III-~; Pechere et al., 1971). Crystals isomorphous with those used in the original structure determination (space group C2, a = 28.2 A, b = 61.0 A, c = 54.1 A, /3 = 95.0”) were grown at 4°C by dialyzing a known volume of parvalbumin dissolved in water, 2.2 x 10 - 4 M, against a known volume of 73% (w/v) saturated ammonium sulfate (about 2.9 in) buffered to pH 7.0 with 0.2 Mtrishydroxymethylaminomethane. TbCl, was added to a specified Tb3+/parvalbumin molar ratio. A knowledge of this ratio does not tell us the exact molar ratio within the crystal but we can reproducibly grow crystals under these defined conditions. One zone precession photographs of nominal resolution I.83 A were taken using Ni-filtered CuKor radiation. Reflection intensities were measured on a rotating drum densitometer and integrated using programs described by Nockolds & Kretsinger (1970). Two-dimensional difference Fourier h0Z projections and the 3-dimensional difference Fourier synthesis were calculated using the so-called F-6h phases from the refined structure (Moews & Kretsinger, 1975a). The 3-dimensional difference Patterson map and the anomalous dispersion Patterson map were calculated as usual, using [P(parvalbumin + Tb3 + ) - P(parvalbumin)lz and [P(parvalbumm + Tb3+) +,, - F(parvalbumin + Tb3 +) _ J2 as coefficients. An anomalous dispersion difference Fourier map was calculated using the expression derived by Kraut (1968) AP

lmaginary(4= + 2 [P(h) - P(--)l*sin(C(h) - 277h*d.

Moews & Kretsinger (1975a) placed the native parvalbumin data on-a (near) absolute scale. In our present work we scaled our data by the same procedure..As we varied the Tb3 +/parvalbumin ratio in our crystallization vials we were able to grow crystals yielding lower and higher Tb3+ occupancies, in h01 projections, than that found by Moews & Kretsinger (19753). We chose crystallization conditions for our 3-dimensional studies based on our 2-dimensional titration results. All of the crystals used in the S-dimensional study were grown in one batch. From that batch we obtained a plane of h0Z data and calculated a difference Fourier projection map. That h0Z difference Fourier projection map is very similar to one obtained from data from crystals grown at a nominal Tb3+/protein ratio of 1-o. Several refinement methods were used to evaluate the occupancies; they all gave corresponding results. The occupancies and temperature factors of the CD and the EF sites were first refined by least-squares analyses. The 2 parameters are closely coupled. Based on our least-squares refinement results and on the temperature factors assigned to the calcium ions in the Moews & Kretsinger (1975a) structure refinement, we assigned a constant temperature factor of B = 12 to the Tb 3+ ions in all subsequent calculations. The occupancies of the sites in various maps were then determined by calculating difference, difference Fourier maps in which the Fourier amplitudes are : [P(parvalbumin

+ Tb3+(obs.))

- (P(parvalbumin

(obs.) + w * P(Tb3+ (talc)))].

The fractional occupancy (w) was adjusted so that the electron density at the peak position is zero. Complete occupancy (w = 1-O) is considered to be 62(Tb3+) - 18(Ca2+) = 44 electrons.

TERBIUM

REPLACEMENT

OF CALCIUM

TN PARVALBUMIX

127

3. Results We calculated difference Fourier maps for crystals grown at five different Tb3 + / parvalbumin ratios (Table 1). The map derived from 0.5 Tb3+/parvalbumin crystals (Fig. 3(a)) is nearly identical to the map of Moews & Kretsinger and shows significant) Tb3 + occupancy at only the EF site. However, at a 1-O Tb3 +/parvalbumin ratio. the peak corresponding to the CD site is as large as the EF site peak (Fig. 3(b)). Maps calculated at 4.0 and 8-O Tb3+/parvalbumin ratios do not show increased CD or EF site occupancy, but the general noise level increases slightly. On the basis of these two-dimensional maps, we decided to grow a large batch of crystals from a Tb3 ‘, parvalbumin solution near 1.0. We anticipated t,hat these conditions would yield maximal occupancy with minimal noise. All of the crystals used for three-dimensional data collection were grown in the same batch and were mount,ed and sealed in quartz capillaries at the same time. In order to assure ourselves of t’he Tb”+ occupancy for t’his batch of crystals, we calculated the h0Z difference Fourier map. It) is similar to that shown in Figure 3(b) (also see Table 1). From the three-dimensional data we calculated four complementary and internall) consistent maps: difference Fourier (AF), difference Patt,erson (AF’), anomalous dispersion difference Fourier (UF), and anomalous dispersion difference Patterson (idF2). Peak heights and co-ordinates for all four maps are summarized in Table 2. Comparable sections about the CD site and the EF site are shown in Figure 4(a) for t,he F map and in Figure 4(b) for the idF map. The entire AF map contains only three peaks whose height, exceeds 4.0 sigma. The idF map lacks the third-largest positive peak. Ltre interpret t,he three-dimensional maps as follows.

TABLE

1

Tb3+ occupancies at the CL) and the EF sites

I3

Jlap h01 Moews 8: K&singer h01 Tb/MCBP 0.50 0.76

1.oo 4.00 8.00 h0Z subset of 3-dimensional

:+-dimensional

total

(1975b)

37 42 34 32 35 32 30 30

CD 20 N 0.30 0.34 0.31

0.19 0.25

EF P

u

P

Sigma

0.7

0.24

4.9

1.9

0.23

4.1

110 14’

4.3 3.3 2.9 2.8 4.3 15.0

100 125 116 134 126 -

0.0 3.3 3.3 2.9 2.5 12.4

0.20 0.28 0.27 0.25 0.27 0.37

411 data used to calculate the indicated h0Z difference Fourier maps were placed on a common scalr. I< is the temperature factor calculated from the standard Wilson plot. Sigma, the standard deviation of the electron density of all points in the asymmetric unit, is taken as a measure of the relative noise level of the maps. All the data sets are of similar quality. zu refers to the frectional Tbs+ occupancy, which flattens the peak in a difference, difference Fourier map. p refers tn the peak height, in terms of sigma levels for that particular map. It, is notoriously difficult to place X-ray diffraction data on an absolute scale. We are confident, that, our calculations yield reliable values of relative occupancies from map to map; however, t,hr derived values of the occupancies (w) could easily be in error by a fact,or of 4. Note that the increase of the Tb3+/parvalbumin ratio from 1.0 to 8.0 does not increase the Tb3+ occupancy. This apparent saturation suggests that the actual occupancies are significantly greater than those derived from our calculations. MCBP, muscle calcium-binding parvalbumin.

Ab iAF IVlw~vs S.5Kwtsingc~r

(1975(/)

0.3126

0~3086

0.4063

12.4

0.5938

0.3187

0.3078

0.4062

11.1

0.6031

0.1922 0.1922

0.3114

0.3125

0.41 10

0.5’814

0.1975

13cst (‘I)

Rest

0.3219 0.3172

15.0 12.6

0.6156 0~6000

0.2000 0~2109

0.3158 (‘I)-

ELI’

Al-

0.3 140

0~4030

0~6000

0.3 190

0.3750

04000

iAl?”

0.3188

0~4000

0.5937

0.3120

0.3625

04000

Thr co-ordinates of the larger peaks in the :S-dimensional difference Yourior maps (Fig. 4) me tabulat co-ordinates are listed from the Mocws & liretsinger (19750) refinement for comparison. The positimr: peaks and the wrll as the dcrivr(I best positions for the (‘1) and the EF Th3+ ions. Tho 2 nest, highest

i 2

P

.,’

Nrgativr ?I z

‘4~x344

4.4

0.5313

0.1875

0~2969

b395R

04

0~5313

0.1875

0~2813

Srxt, .I’

I-’ -4.4

hlyhrst,

peak

(1

Next P

2

lowest .v

peak 2

P

0.468ti

lbl7l~J

lbi’J69

3.6

0.7188

0.1563

1~~1181 -~ 4.0

,5.x 0.5625

lb1406

0~1094

3.8

0~7813

0~1094

0.0156

PI( as fractions of the unit cell. Prak height,, p, arc’ cited in terms of standard dwiations. of tho self vectors, CD-CD and EF-EF, and of the crow vectors, CD-EF and EF-CD, nrxt Iowwt peak in the LIP and thv idFZ maps rtw 5.3. 4.7. --4.‘? and 3.7, 3.3, 4.0.

--x.9

The calcium are listed as (4’1,f,rw ,I. 1271

128

J. SOWADSKI,

G. CORNICK 3a/4

a/4

(..

AND

R. H. KRETSINGER 3a/4

a/4

, .

I

EF

I

2

(b)

Fro. 3. Centric difference Fourier projections of Tb 3 +-parvalbumin were calculated using F-6h phases from the refined (native) Ca2+ -parvalbumin. They are contoured at levels of 1.6 o. The interval of z from a/4 to 3n/4 is shown in order that the CD Ca2+ site and the EF C&2+ site (designated by crosses) be within the same molecule. The area enclosed by broken lines is shown in the 3.dimensional difference Fourier maps of Fig. 4. (a) This map is calculated from data from crystals grown at a nominal Tb3+/parvalbumin ratio of 0.5. It is very similar to the original map calculated by Moews & Kretsinger (19766). Only the EF site is occupied by Tb3 +. (b) In this map, calculated at a Tb3+/parvalbumin ratio of 1.0, both the CD and the EF sites are occupied. There is significant electron density at site t’hree (see Fig. 4).

(1) All four maps indicate that both the CD site and the EF site contain Tb3 + ions. (2) The CD Tb3 + ion is between 0.4 a (dF map) and O-7 d (idF2 map) of the refined position of the CD Ca2+ site. The EF Tb3+ ion lies between 0.2 J%(idF2 map) and O-6 A (dF map) of the EF Ca2+ site. The error in the positioning of the Ca2+ sites is about 0.15 A. There is a slightly larger error associated with determining the Tb3 + ion positions. The possible shift (Ca2+ to Tb3 +) at either the CD or the EF site cannot be considered to be significant. Conversely, we cannot exclude some departure from isomorphism. (3) Site three is significant in the dF map; however, this map, per se, tells us nothing of its atomic composition. The only element present in these crystals with a significant anomalous diffraction with CuKa radiation is terbium. Peak three does not appear in the iAF map and is not due to Tb3 + . (4) The negative peak does not lie at the position of any protein atom. It does not give rise to any peak in the AF2 map as would be expected if an atom left its position in the protein structure as a result of Tb 3+ binding. It is usual for a large positGivepeak to be surrounded by a negative trough. This artifact is due primarily to termination

TERBIUM 80132

REPLACEMENT

OF CALCIUM 23002

I

IN

PARVALBUMIK

W32 I7c/64

,’I. ,::+

29d64

129 230132 I

1

FIG. 4. (a) The 3-dimensional difference Fourier (dF) maps are contoured at levels of 3.0 D m the area ranging from x = k/32 to 23a/32 and from z = 17c/64 to 29c/64 of the unit-cell. For comparison this same area is enclosed by broken lines in the 2-dimensional projection of Fig. 3(a). The 2 planes at the CD site are at levels y = 19 and 206/64 with 20 being nearer the viewer. The planes at the EF site are on levels 12, 13, and 14, the third site at 12, 13 and the negative site at ?/ = 126/64 of t’he unit-cell. The following positions are indicated on the map: Ca2+ CD site Ca2+ EF site Lys96 carbonyl 0 Asp94 carbosylate &h= t--W Phe57 Cy C&l C% Cfl a2 Cl (b) The same region of the anomalous 3.0 O. No terbium is present at the third

(- I’) 0 (+ Z)

dispersion site.

n/32 10.0 19.0 16.8 16.7 19.4 16.5 16.0 16.3 17.6 17.6 18.0 difference

b/64 19.6 12.6 13.8 11.0 12.3 15.0 14.2 14.8 13.3 13.9 13.3 Fourier

c/64 26.4 “0.1 19.4 19.9 23.0 26.4 25.2 27.4 25.3 28.2 26.7 (idF)

map is contoured

at.

of series in the Fourier summation. Bot,h the CD and t’he EF peaks are surrounded, in all three directions, by negative troughs. We do not understand why the trough. shown in Figure 4, is slightly deeper. It does not appear to be due t,o any struct,ural alteration in the protein. (5) The third positive peak of the AF map (Fig. 4(a)) cannot be attributed to a movement of the protein side-chains. It may be due to the replacement of the water that co-ordinates the EF Ca2+ ion at the -X vertex by a sulfate anion.

4. Discussion The two main conclusions are unambiguous. Both the EF and the CD Ca2+ ions can be displaced by Tb3+ at higher Tb3+/parvalbumin ratios. Second, there is not a third Tb3 + binding site under the conditions of our experiments. A third sik: appears on the AF map but not on the iAF map; it cannot be att,ributed to an anomalous scatterer. Our results appear to contradict the suggestion of Nelson et al. (1977) t’hat the fluorescence quenching is due to a Tb3 + ion bound to a third site. It seems appropriate to outline the spectroscopic observatjions which are relevant to the evaluation of this suggestion. (1) Tb3 f titration in D,O produces greater relative emission intensities. Since water provides a better radiationless mechanism for dissipation of energy, a water molecule is presumably involved in the quenching action by Tb3 + (Nelson et al., 1977).

130

J. SOWADSKI,

G. CORNICK

AND

R. H.

KRETSINGER

(2) When Tb 3+ is added t0 Ca2+ -parvalbumin the fluorescence is maximal at a Tb3 +lparvalbumin ratio of 1.5 ; here the fluorescence is arbitrarily defined as 100. At a Tb”+/parvalbumin ratio of 3.0 the fluorescence intensity drops to 55. If then the Tb3 f-Ca2+-parvalbumin solution is didyzed against an excess of O-1 M-KCl, piperazine buffer (pH 6.5) for 16 hours, the fluorescence intensity rises to 140 (Nelson et al., 1977). This increase can be due to the removal of the third Tb3 + which quenches the fluorescence of the Tb3 + EF site. Unfortunately, these experiments do not allow one to establish whether 140 represents a plateau or a maximal value. Of greater concern, the Tb3 + content was not determined at this 140 fluorescence level. The Ca3+ /parvalbumin molar ratio was determined to be 0.15 from similar experiments. When Ca2+ is added to the “140 fluorescing” Tb3 + -parvalbumin solution, the fluorescence decreases. Based on the magnitude of the decrease, and theoretical improbability of Ca2+ quenching, Nelson et al. (1977) argued that Ca2+ decreases Tb3 + fluorescence by competing for the EF site. Thus, there is apparently a significant competition between two ions, and t’he reason that the peak intensity in the forward Tb3+/parvalbumin titration does not exceed 100 appears to be that Ca2+ competes with Tb3+ for the EF site. We consider five interpretations. (1) It is conceivable that a third Tb3 + ion binds to parvalbumin under the conclitions of the spectroscopic experiments, 0.1 M-KCl, piperazine buffer (pH 6.5), but not under the conditions of the crystallographic experiments (0.2 M-Tris, pH 7.0, 2.9 Mammonium sulfate). (2) c a2+ displaced from the parvalbumin or inevitably present in the solution as a low level cbntaminant, may bind to the water molecule that co-ordinates the Tbii ion as Nelson et aE. (1977) suggested a third Tb3 + ion might do. This seems improbable as there are no protein oxygen atoms near enough to co-ordinate this putative third site Ca2+ ion. (3) The side-chain of Phe57 is the only region of the protein, main-chain or siclechain, that could move to site three without greatly distorting the structure of the protein. The phenyl ring could not reach site three with rotations about only the Ca-Cs and the C&$, single bonds. Further, there is no negative electron density indicating movement of either the main-chain or the side-chain of Phe57. (4) The H,OEF may be displaced by an HSO; or SO:- ion. The Tb3 + ion carries one more positive charge than does the Ca2+ ion. However, the EF site was not originally electrically neutral since it has four carboxylate groups and one Ca2+ ion. If one oxygen atom of a sulfate anion occupies the water position the remainder of the anion could occupy the third site. The third site, H,O,, site, and EF site lie on a straight line with distances from the third site to the water site l-5 A and to the EF site 3.8 A. The phenyl ring would have to shift about O-5 A (Figs 1 and 4). The distance from the third site to C-5 of Phe57 is 2.5 A. There would be no other unacceptable van cler Waal’s contacts. However, a sulfate ion would not be expected to quench terbium fluorescence. Under the conditions of the spectroscopic experiments, 86% of the piperazine (HN(CH2),NHCH2CH2, p1 = 5*68,9.82) would be in the monoanionic form. It is conceivable, though we feel improbable, that t*he piperazine anion is bound to the third site. (5) We made the following calculations t’o test whether a simple two-site, non-cooperative model might explain the spectroscopic results of Figure 2. In the spectroscopic experiment parvalbumin, co-ordinating two calcium ions, is titrated with

TERBIUM

REPLACEMENT

Tb3+. The metallo-protein represented by :

OF

CALCIUM

complexes potentially

IN

FARVALBUMIN

available

131

in t’he experiment

are

In this notation PCT refers to parvalbumin with Tb3 + at the EF site and Ca2 + at the CD site; in PO0 there is no metal at either site. The total fluorescence is postulated to be proportional to the concentration of the parvalbumin containing Tb3+ at, the EF site only (i.e. PCT and POT). The CD site Tb 3+ does not fluoresce (i.e. PTC and PTO). Further PTT is postulated to display no fluorescence either because the CD site Tb3 + quenches the potential fluorescence or because the CD Tb3 + captures the initial absorbed energy allowing none to reach the EF Tb3 + . The postulate of nonco-operativity means that the relative concentrations of all nine molecular species are determined by four dissociation constants: K,,,, KCEF, K,,, and K,,,. The values of both K,,, and K,,, were taken from Benzonana et aZ. (1972) as 2 x 10e7 31. The total concentration of parvalbumin used in the experiment was 3 x 10M4 and of Ca2+ 6 x 1O-4 M. i.e. a Ca2+/parvalbumin ratio of 2.0. We obt,ained the best, leastsquares fit to the observed fluorescence at K,,, = 6.3 x 10 7 and K,,, = 14 ,< 10 ’ ht. As shown in Figure 2 this simple, two-sit’e, non-co-operat,ive model predicts a fluorescence curve with a maximum followed by a plat)eau. The fit could be improved by incorporating more parameters into a model having co-operat,ivity between t,he CD and EF sites. We emphasize that this model calculation does not reveal the quenching mechanism. It suggests that a third quenching sibe need not be p&ulat,cd to explain the observed results. In summary, although the involvement of a “third site” in the observed Tb” r fluorescence quenching cannot be excluded, under t)he conditions of our experiments a third Tb3 + ion is not bound to parvalbumin. REFERENCES

Benzonana,

G., Capony,

J.-P. & Pechere, J.-F.

(1972). B&him.

Biophys.

110-116. Brittain, H. G., Richardson, F. S. & Martin, R. B. (1976). ,I. Amer. Ch,em. Sot. 8260. Donato, H. & Martin, R. B. (1974). Biochemistry, 13,4575-4579. Gerday, C. & Gillis, J. M. (1976). J. Phyeiol. 258, 96-97. Gillis, J. M. & Gerday, C. (1977). In Calcium Binding Proteins and Calcium

dcta. 278. 98,

8255

Function

(Wasserman, R., Corradino, R., Carafoli, E., Kretsinger, R. H., MacLennan, D. KSiegel, F., eds), pp. 6193-6196, Elsevier North-Holland, New York. Kraut, J. (1968). J. Mol. Biol. 35, 511-512. Kretsinger, R. H. (1976). Annu. Rev. Biochem. 45, 239-266. Kretsinger, R. H. In Calcium Binding Proteins and Cal&m Function (Wasserman, It.. (lorradino, R., Carafoli, E., Kretsinger, R. H., pp. 63--72, Elsevier North-Holland, New York.

MacLennan,

D. & Siegel.

F.. eds),

132

J. SOWADSKI,

G. CORNICK

AND

R. H. KRETSINGER

Moews, P. C. & Krctsinger, R. H. (1975a). J. Mol. BioE. 91, 201-228. Moews, P. C. 8: Kretsinger, R. H. (197.55). J. Mol. Biol. 91, 229-232. Nelson, D. J., Miller, T. L. & Martin, R. B. (1977). Bioinorg. Chem. 7, 325-334. Nockolds, C. E. & Kretsinger, R. H. (1970). J. Phys. E. Sci. In&r. 3, 842-846. Pechere, J.-F. (1977). InCalcium Binding Proteins alzdCalcium Function (Wasserman, R., Corradino, R., Carafoli, E., Kretsinger, R. H., MacLennan. D. & Siegel, F., eds), pp. 2 13322 1, Elsevier North-Holland, New York. Pechere, J.-F., Capony, J.-P. & Ryden, L. (1971). Eur. J. Biochem. 23, 421-428. Pechere, J.-F., Derancourt, J. & Haiech, T. (1977). FEBS Letters, 75, 111-114. Rasmussen, H., Goodman, D. B. P. & Tenehouse, A. (1972). CRC Grit. Rev. Biochem. 1, 95-148.

Reuben, J. (1975). Naturwiasenschaften,

62, 172-178.