Non-cooperative Ca(II) removal and terbium(III) substitution in carp muscle calcium binding parvalbumin

BIOINORGANKCHEMSTRY

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(1977)

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Non-cooperative Ca(I1) Removal and Tetbium(II1) Substitution in Carp Muscle Calcium Binding Parvalbumin”

DONALD J. NELSONP, THEODORE L. MILLER: and R_ BRUCE MARTIN Chemistry Departmenr, University of Virginia, Charlottesville, VA 22901

ABSTRACT

Close correlation of atomic absorption measurements for Ca(H) contents indicates that from pH 5.8-7.4 a twentyfold excess of EGTA’ removes but one of two Ca(H) from carp parvalbumin. Thus binding of the two Ca(II) appears to be noncooperative- The maximum in emission intensity observed at a nonintegral l-4-1.7 equivs of added Tb(iII) is shown to be due to quenching by excess Tb
INTRODUCTION Parvalbumins, derived from the white muscle of fish such as carp, are small proteins of molecular weight - 11,500 which tightly bind two Ca(II) ions [I] . Muscle calcium binding parvalbumin from carp contains a single cysteine, no tryptophan, and ten phenylalanine residues and no tyrosine (isotypes 3 and 5) [2] _ The crystal structure of isotype 3 or B has been solved and refined to 1.9 A resolution [3-S] _ There are six helical regions, designated helices A through F. One calcium ion is 6-coordinated by protein ligands in the polypeptide loop between helices C and D, while the second calcium ion, located in the EF loop, j-Present address: Chemistry Department, Clark University, Worcester, Massachusetts 01610. @resent address: Chemistry Department, Ohio Wesleyan University, Delaware, Ohio. ’ Ethylene glycol his@-aminoethyl ether)N,N’-tetraacetate. 0 Elsevier North-Holland, Inc., 1977

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is Scoordim+ted, with one coordination site occupied by a solvent water molecule_ We designate these calcium ion sites as Ca(CD) and Ca(EF), respectivelyBoth cooperative and non-cooperative Ca(I1) binding to parvalbumins have been suggested_ Cooperative binding was originally proposed on the basis of similar binding constants for both Ca(I1) and an indication of curvature given by a singIe point in one of three Scatchard plots [l] . Cooperative Ca(iI) binding has also been postulated in a sequence of conformational events leading to Ca(II) reiease [6] _ Features of the proton magnetic resonance spectra upon loss of all but 0.4 equivalents of Ca(II) have been interpreted on the basis of a mixture of two Ca(I1) and zero Ca(I1) protein [7] _ On the other hand, several diverse lines of evidence indicate that binding of the two Ca(I1) to parvalbumins is not cooperative_ The Ca(II) more easily removed by EGTA was suggested to be Ca(EF) [S]. This Ca(I1) was also the only one to be replaced by Tb(IIl) upon long standing in a crystal grown for an X-ray diffraction study [P] _ Modification of Arg-75 reduced the Ca(I1) binding capability by about one-half [ lo]_ Finally, release of about one Ca(I1) resuhs in loss of only one of two unusually shifted carbonyl carbon-13 magnetic resonance signals, consistent with differential loss of one of the two Ca(II) [ I1 ] _ Since the above experiments were performed on several parvalbumins at a variety of pH vahres, it is possible that apparent coopperativity occurs under some circumstances and not others. The effect of the Ca(I1) binding ligand EGTA upon addition to parvalbumin containing sohrtions has been found to be pH dependent [8] _ At pH 6 even a IOO-fold excess of EGTA produced no change in the circular dischroism spectrum at 224 nm. At pH 8.5 a reduction in the 224 nm magnitude levels off at a 20-fold excess of EGTA. Addition of Ca(II) to saturate the EGTA reverses the effects. In “&is paper the effects of added EGTA from pH 5.8-7.4 is correlated with the Ca(I1) content of the protein as monitored by atomic absorption spectroscopy. Additional support for the noncooperativity of Ca(Il) binding is obtained_ Terbium(III), of simiIar ionic radius to Ca(II), undergoes a dramatic enhancement of its characteristic green emission upon substitution into parvalbumin and irradiation of the protein at 259 nm [8 J _ The emission emanates from intramoIecular energy transfer between the aromatic ring of phenylalanine-57 and the terbium ion bound at the EF metal ion binding site, which is located 4.6 A away_ Energy transfer between the aromatic ring of phenylalanine47 and a terbium ion in the CD metal ion binding site is remote since a distance of 6.7 A is involved. The carbonyl oxygen of Phe-57 serves as a donor atom to the CD calcium SO that this residue interacts with both metal ion sites. Due to &&ity of the EF site the Tb(III) emission is also circularly polarized [S] . A peculiarity of the Tb(Il1) emission is that its intensity peaked upon addition of between I and 2 equivalents of Tb(III) rather than at an integer ratio_ This phenomenon is explored further in this paper and an explanation proposed_ Of more than thirty

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proteins that display enhanced Tb(II1) emission [ 12 3 , only parvalbumin exhibits an intensity maximum. The single sulfhydryl group of carp parvalbumin isotype 5 has been labeled with a trifluoroacetonyl group for lg F nuclear magnetic resonance studies to be reported by D. 3. Nelson. In this paper a comparison is made between the effects of added Tb(II1) upon the emission of native and fluorine labeled parvalbumin.

METHODS Carp muscle calcium binding parvalbumin was isolated from the white muscle of common mirror carp (Cyprinus carpio) [ 13]_ Purified isotype 5 or A was employed for suIfhydry1 group labeling by the trifluoroacetonyl group [14] _ Isotypes 3 and 5 were both used in the emission experiments. 2-Chloromercuri4nitrophenol, used to quantitate the extent of the sulfhydryl group labeling, was obtained from Eastman Organic Chemicals. All other chemicals were high grade commercial products_ Protein concentrations were determined by ultraviolet absorbance at the 259 nm phenylalanine maximum (e = 2000) on a Gary 14 recording spectrophotometer. The terbium(III)-parvalbumin emission studies were conducted at pH 6.5 in 0.1 M KCI-piperazine buffer. Protein concentrations in these studies were typically 0.3 mM_ Relative emission intensities are concentration corrected. Emission spectra were recorded on a Perkin-Elmer MPF-3 spectrometer and the emission titration data was obtained on an Aminco SPF-125 fluorometer with the sample temperature thermostatically controlled at 25.1”C. Circularly polarized emission experiments were preformed on an apparatus designed and built in this department by F. S. Richardson and C. K. Luk [ 15]_ Circular dichroism spectra were obtained on a Jasco J10 B instrument_ Protein bound calcium was determined on a Perkin-Elmer Model 303 atomic absorption spectrophotometer with a DCRl concentration readout_ Each protein sample was analyzed by both the routine working curve procedure and the method of additions. The atomic absorption results reported in this paper are much more precise than those of Donato and Martin [83, in which the protein samples (pH = 8.2) were dialyzed against rather than incubated with EGTA. RESULTS The effects of added increments of Tb(II1) at pH 6.5 on the relative emission intensity at 545 nm, when both native and fluorine-labeled parvalbumin are irradiated at 259 nm, are shown in Fig_ 1. Both titration curves exhibit essentially the same shape and maximum intensity. The latter maximum is displaced to a slightly higher [Tb,,,] /protein] ratio due to the presence of a small

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80

60

4ot

f F /I t

/

P

I

I

2.0

3.0

[Tb+ot]/[

I

4.0

Protein]

FIG. I_ Relative intensity of Tb(III) emission for addition of Tb(III) to native (solid circles) and tritluoracetonylated (open circles) parvalbumin as a function of the number of equivs of added Tb(Il1).

residual amount of EGTA from the preparation_ Addition of more EGTA results in further displacement of the curve to higher ratios. Consequently, EGTA binds Tb(II1) more strongly than parvalbumin at this pH and protein titration occurs only after all the EGTA has become complexed. Native protein subjected to .sirniIarpreparative treatment without labeling also shows a displacement in the maximum

to .a higher [Tb,,,]/protein] ration. The curve for the native protein in Fig. 1 is similar to that reported previously [8] except that the maximum occurs at a slightly lower ratio. The position of the maximum occurred from 1.4- 1.7 equivs of Tb(III) for three different preparations (both isotypes 3 and 5) and appears to be a function of sample preparation. When the sulfhydryE group is Iabeied with 2-cNoromercuric4nitrophenol no luminescence appears on Tb(II1) addition_ This larger sulfhydryl group reagent evidently disrupts conformational features essential for luminescence. Due to its asymmetric environment in the EF metal ion binding site, the emission from the Tb-parvalbumin complex is circularly polarized. The shapes of the circularly polarized emission spectra from 530 nm to 560 nm for isotypes 3 and 5, and F-labeled parvalbumin are the same as that reported by Donato and Martin [8] for isotype 3_ However, the intensity signs reported in that paper should be reversed. The correct spectrum is identical to that illustrated for

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120 z v, 100 5 g 80 n-l 2

60

5 = a

40 20 0

0

‘*O

4.0

FIG. 2. Relative intensity (on the same scale as Fig. 1) for Tb(III) emission upon addition of Ca(II) and trivalent lanthanide ions to parvalbumin containing Tb(ilU in both Ca(Ii) binding sites. substituted rabbit muscle troponin-C [ 161 _ The shape of the parvalbumin spectrum is unaffected by the number of equivalents of Tb(lII) from 12-S-6.

Tb(III)

When a solution of either native or lgF-labeled parvalbumin that had undergone TB(II1) titration is dialyzed against buffer, the emission intensity increases. This phenomenon was examined by additional emission experiments with native parvalbumin When the Tb(III) titration maximum occurred at 1.4 equivalents and the addition was stopped at 2.8 equivalents, the relative emission intensity was 60 on the scale of Fig. 1. Dialysis was conducted against 1 liter of 0.1 M KCl-piperazine buffer at pII 6.5. After 1 h the intensity was 68; after 3% h against a second buffer solution, 93; and after 16 additional h against the latter buffer solution the emission intensity increased to 140. Addition of successive amounts of Tb(lll) after dialysis quenched the emission as illustrated in Fig. 2. Subsequent dialysis again increased the relative intensity which was quenched by the additional Tb(III), so the phenomenon is reversible_ Simple standing of a parvalbumin solution containing Tb(IiI) results in no change in emission intensity so that Tb(II1) is identified as the quencher_ Other metal ions were studied for their ability to reduce the emission with Tb(II1) in both metal binding sites. After addition of 4 equivaIents of Tb(II1)

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TABLE

AND R. B. MARTIN

1

Calcium Contents of Parvalbumin Exp_ No_ I 2 3 4 5 6 7

Conditions Following preparation With 20-fold molar excess EGTA With 8 &I urea and 0.05 M EGTA With 1.8 equivs EGTA With 1.6 equivs Tb(III) With 3.7 equivs Tb(III) With 30 equivs Tb(II1)

ICa]/[Protein] 1.9 I.10 0.06 1.78 0.23 0.15 0.05

+ 0.1 f 0.08 2 0.02 -i-0.06 k 0.02 + 0.04 t 0.02

and dialysis against excess buffer at pH 6.5 for about 20 h, the initial relative intensity was approximately 140_ The decrease in intensity upon addition of Ca(I1) and other lanthanides is shown in Fig. 2_ When solutions containing Tb-parvalbumin and about 4.5 equivs of Eu(III), Er(III), or Gd(II1) were dialyzed against buffer at pH 6.5, the intensities of the first two solutions increase about 50% more than that for Gd(III). These results suggest Eu(II1) and Er(II1) quenching of the Tb(III) emission. Emission does not appear from 630-630 nm, a normal Eu(III) emission region, upon addition of Eu(III) to Tb-protein, after subsequent dialysis, or when Eu(II1) is added to a solution of native protein. Tb(II1) titration in D20 produces greater relative emission intensities throughout the curve found in Figure I ; 22% greater at the maximum and about 30% greater at 4 equivalents. Since Hz0 provides better radiationless mechanisms for dissipation of energy than D,O, this result suggests that water is involved in the quenching action by Tb(II1). Atomic absorption analyses of the Ca(I1) contents of carp parvalbumin were performed under a variety of conditions with added EGTA or Tb(II1) in order to correlate the Ca(II) content with these added substances. The results of the atomic absorption measurements appear in Table 1_ An average value of I .9 cakium ions per molecule was found for freshly purified isotype 5_ Protein samples from the same preparation were then subjected to a 20-fold molar excess of EGTA. The samples were added to the EGTAwith the pH controlled at ?A while the solution was stirred at room temperature for either 3 or 8 hours. After incubation the reaction mixture was transferred to a dialysis sac and the soIution exhaustively dialyzed against deionized distilled water at pH 5.8 or 7.5. Great care was exercised during the dialysis to prevent calcium contamination. At the end of the dialysis, a 3-fold molar excess of TbCl3 was added to the solutions to prevent calcium contamination from the glass apparatus used in sample preparation for the atomic absorption analysis_

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Since terbium binds to parvalbumin more strongly than calcium, contamination after the dialysis is eIiminated and any calcium substituted by the excess terbium treatment will remain in solution and will be detected in the atomic absorption analysis_ Experiments with TbCla added to either the calcium stock solution or to protein solution show that terbium does not interfere in the calcium analysis. Results of 12 determinations over the range of conditions are reported as experiment 2 in Table 1_ An average value of 1.1 calcium ions per protein molecule was found for the samples treated with a 20-fold molar excess of EGTA. EGTA binds to the protein when present in a 20-fold molar excess @. J. Nelson, unpublished observations)_ However, it is unlikely that the Ca-EGTA complex would bind to the protein. To examine this hypothesis, native protein subjected to the same EGTA treatment was dialyzed against deionized distilled water at different controlled pH’s. Under different electrostatic environments (pH 5.8 and 7.5) the proteinbound calcium contents were similar. This result suggests that the Ca-EGTA complex does not bind to the protein. Similar atomic absorption results for different incubation periods with EGTA at pH 7.4 as well as sharp lsF magnetic resonance signals from data accumulation over a three hour period in experiments which progressively removed bound Ca(I1) from F-labeled parvalbumin with EGTA additions, indicate that the EGTA-protein reaction is not significantly time dependent_ The atomic absorption results demonstrate that a single calcium ion is removed by excess EGTA from pH 5.8-7.4. However, when parvalbumin is incubated under harsher conditions (8 M urea and 0.05 M EGTA at PI-I 89 for 2 h) both calcium ions are removed (exp. 3, Table 1). In order to determine whether Ca(I1) is removed from parvalbumin in a progressive manner, atomic absorption analysis was performed on a solution to which 1.8 equivalents of EGTA had been added. After dialysis the Ca(I1) content was reduced to l-78 equivalents (exp. 4, Table 1) indicating the removal of about 0.10 equivalents of Ca(lI). This result is consistent with progressive removal of Ca(I1) by EGTA and the need for a 20 fold excess to effect the removal of one equivaient of Ca(II). All these results with EGTA are indicative of the noncooperative binding of the two Ca(I1) of carp parvalbumin from pH 5 -8-7 -4. Atomic absorption results were also obtained in several experiments after addition of Tb(II1) and dialysis against excess 0.1 M KC1 with a final dialysis against 0.05 M KCl. Solutions to which 3.7 and 30 equivalents of Tb(IlI) had been added showed only 0.15 and 0.05 Ca(lI), respectively (exps. 6 and 7, Table 1). Thus both the CD and EF calcium ions are replaced upon addition of only modest amounts of Tb(ll1). The relative emission intensities of these solutions containing two Tb(II1) per protein were 140 on the scale of Fig. 1 and 2. Even when only 1.6 equivalents of Tb(III), which corresponds to about the maximum in Fig. 2 for this sample, are added and the solution dialyzed, only a

332

D. J. NELSON, T. L. MILLER

AND R. B. MARTLN

minimum amount of Ca(i1) is retained by the protein (exp. 5, Table I). The relative emission intensity of sample 5 after dialysis is 85% of the I40 value in Figure 2 indicating 0.85 equivalents of Tb(III) in the EF site.

DI!XU!WON The results described in the previous section provide an explanation for the maximum occurring at a non-integral number of equivaIents of added Tb(III) in the emission titration curve of Fig. 1. Both Ca(CD) and Ca@F) are replaced by only modest amounts of Tb(III). Addition of 3.7 equivs of Tb(III) displaces virtually all the Ca(I1) and the magnitude of the CD minimum at 222 mn is unaffected indicating that the approximately 50% helicaI content of the native protein is maintained upon substitution of both Ca(R) by Tb(II1). This two terbium parvalbumin, after dialysis against buffer, exhibits a 40% greater relative emission intensity than observed at the maximum in Fig. 1. Thus Tb(CD) does not quench rhe emission Tb(EF), even though Tb(CD) is coordinated to the carbonyl oxygen of Phe-57 and Tb(EF) is juxtaposed to the Phe-57 aromatic ring. Figure 2 shows that emission of lb-parvalbumin is quenched by excess Tb(III)_ Therefore in the titration of Fig. 1, added Tb(II1) is replacing Ca(I1) in both sites and also reducing the emission of the Tb(EF) by association at a third site. The maximum in the curves of Fig 1 is accounted for by the balance between Tb(III) substitution for Ca(EF) and quenching by some Tb(II1) not bound in the CD or EF sites. The reduction is emission intensity by excess Tb(II1) may occur by two mechanisms: association with the protein which wouId result in indirect action on the conformation to alter the critical spatial requirement for intramolecular energy transfer between the aromatic side chain of Phe-57 and Tb(EF), or association at the water molecule that is bound to Tb(EF) and radiationless energy dissipation from Tb(EF) through the bound water to associated Tb(II1) which is higbIy hydrated_ The greater emission observed in D20 suggests that water is involved in the quenching process [173 _ The latter interpretation receives some support from the quenching action of several metal ions reported in Fig_ 2 The curves of Fig. 2 may be accounted for qualitatively by assuming an equiIibrium between Tb(II1) and the added metal ion in the protein sites and the quenching capability of the Tb(II1) released from the two sites. Addition of Ca(II) yields the least reduction in emission intensity by Tb(EF) displacement, as it aione is much more weakIy bound than Tb(II1). If non-quenching Gd(III) is bound as strongly as Tb(III) to both the CD and EF sites, then after addition of 2.0 equivalents of Gd(III), the intensity should fall by one-half (to 70 on the scale of Fig. 2) due to replacement of half the Tb(EF) and an additional 43% due to the I:0 eqrrivalent of released Tb(III) which may serve as a‘quencher.

STUDY OF CARP MUSCLE BINDING PARVALBUMIN

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Thus a value of about 30 on the relative intensity scale of Fig. 2 is expected after addition of 2.0 equivalents of Gd(DI). This value conforms closely to that reported in Fig. 2 and could be adjusted exactly by choice of relative equilibrium constants for binding. The lesser intensities upon addition of Eu(II1) and Er(II1) to the Tb(III)protein may be due either to stronger binding than Gd(II1) or some quenching capabilities_ The greater mtensity increase for Eu(II1) and ErfJII) upon subsequent dialyses suggests that they quench fluorescence in the same manner as Tb(IIi). The magnitude of the results for Gd(III) suggests that the first mechanism mentioned in the previous paragraph is unlikely so that the reduction in emission intensity occurs by association of and quenching by solution Tb(HI) at the water molecule bound to Tb(EF). The characteristic terbium emission observed in this study depends upon a dipolequadrupole interaction between the irradiated phenylalanine side chain of residue 57 and the emitting Tb(II1) in the EF site. The probability of such an intramolecular energy transfer exhibits an r-a dependence upon the distance between the donor and acceptor sites. Thus the near identity of the emission curves of Fig. 1 for native and fluorine labeled protein and the similar shapes of the circularly polarized emission spectra of the two proteins provide strong evidence that the conformation about the metal binding sites is the same in both the native and labeled proteins. Closely similar circularly polarized emission spectra are also obtained upon substitution of a single Ca(I1) by Tb(III) in rabbit skeletal muscle troponin-C [16] and in bovine cardiac troponin-C [ 181. The circular dichroism of the four proteins, native parvalbumin, Tb(III)-substituted parvalbumin, F-labeled parvalbumin (with Ca(II)), and Tb(III)-substituted F-labeled parvalbumin, are all similar in the 222 nm region indicating similar conformations for all four protein species. Thus we extend the conclusion of the previous paragraph to suggest that the conformations are insignificantly different in the native and F-labeled proteins. The authors thank Dr_ H. G. Brittain and ProjI F_ S. Richardson for obtaining circularly polanied em&ion spectra on native and fluorine-labeled parvalbumin. This reseatch was supported by NZH Postdoctoral FelIowship No. GM 55692 ro D_ J. N. and NSF Grant No. G5 43286 to R. 3. M.

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(1972). D. L. Enfield, L. H. Ericsson, H. E. Blum, E. H. Fischer, and H. Neurath, Proc. Nat. Acad. Sci. 72,1309-1313 (1975). R. H. Kretsinger, and C. E. NockoIds,J. Biol. Chem 248,3323-3326 (1973). W. A. Hendrickson and J. Karle,J. BioL Chem. 248,3327-3334 (1973). P. C. hfoews, and R. H. Kretsinger,J. Mol. Biol. 91,201-228 (1975).

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H- Donato Jr_ and R. B. Martin, Biochemistry. 13,45X-4579 (1974). P_ C_ Moews and R. H. Kretsinger.J. &foL Biol. 91,229-232 (1975). C- Goss&n-Rey, N. Bernard and C. Gerday, Biochim, Biophys. Acta 303, 90-104 (1973).

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S. J. Opella. D. J. NeIson and 0. Jardetzky, J. Amer. Chem. SW_ 76, 7157-7159 (1974)N. G. B&stain, F. S. Richardson, and R. B. Martin, J. Amer- Chem Sot. 98,825s 8260 (1976). J-F_ Pechere, J. Demaille and J-P.Capony, Biochim. Biophys, Acfa 236, 391-408 (1971).

W_ H. Hues& and M. A. Raftery, Biochemistry IO, 1181-X 186 (1971). 15. F. S. Richardson and C. K. Luk,J. Amer. Chem Sot. 97,6666d675 (1975). 16. T- L- MZlIer, D_ J. Nelson, H. G. Brittain, F. S. Richardson, R. B. Martin and C_ M. Kay. FEBS Letters 58,262-264 (1975)_ 17. G- Stein and E. Wurzberg,J. Chem Phys. 62,208-213 (1975)_ 18. H. G. Brittain, F. S. Richardson, R- B. Martin, L. D. Burtnick and C. M. Kay, Bio&em. Biophys. Res Commun 68,1013-1019 (1976). 14.

Received I8 November

1976; Revised 28 December