J. Phys. Chem. 1990, 94, 7923-7927
7923
Differential Scanning Calorimetric Studies on Calmoduiin in the Presence of Monocations, Dications, and Ethanol Harry P. Hopkins, Jr.,* and Richard H. Gayden Department of Chemistry, Georgia State University, Atlanta, Georgia 30303 (Received: January 29, 1990)
Changing the monocations in solution with calmodulin (0.225-0.450 mM at pH = 7.5) alters the broad reversible denaturization observed with differential scanning calorimetry (maxima near 50 "C), and an overall destabilization of this protein's tertiary and secondary structure is found in solutions containing ethanol (1.5-3.0 M). With tetraethylammonium ions present (12 mM), the excess heat capacity curve, corrected for intrinsic heat capacity variations, can be fit to a single two-state transition with a maximum near 49 OC. With Na+ or K+ present at the same concentration, the maximum is in the 52-54 O C range, and with only Li+ (1 2 mM) present the maximum is near 59 OC. Two transitions are needed to fit adequately the corrected excess heat capacity curves with only alkali-metal ions present. With 5.0 mM Mg2+and 12.5 mM K+ present, the melting region has a maximum near 80 OC and can be represented by two transitions with enthalpy changes similar to those observed with only alkali-metal monocations present; similar effects are seen for Sr2+and Mn2+. Up to a mole ratio of two Ca2+ ions per calmodulin, the excess heat capacity curves are similar in shape and have nearly the same maxima as those found in the presence of monocations, implying that any structural change linked to Ca2+binding and/or preferential binding of Ca2+to a native state is associated with substantial occupation of the second, third, and fourth sites. The unusually large slope in the excess heat capacity curves, seen before the transition region with only monocations present, is maintained up to 100 "C when calmoldulin's four metal ion binding sites are nearly filled with Ca2+, Cd2+,or Co2+ions. These results are adequately represented by ( I ) a sequential melting model involving three states, two being highly structured (native) and one a random coil (denatured), and (2) preferential stabilization of the first two states by metal ion binding to the protein.
Introduction Calmodulin is a small molecular weight protein (17 kDa) which is thought to function in all eukaryotic cells by activating other proteins to which it binds hydrophobically when bound to Ca2+ ions.' This ubiquitous protein's three-dimensional structure2 (3-A-resolution X-ray study) resembles a dumbbell (nearly symmetric) with two metal binding sites in each end composed of carboxylate groups located on peptide loops (12 amino acids). Each metal binding peptide loop is flanked by segments of ahelices oriented at near right angles (EF hand structure), and in each end of the dumbbell these regions are held antiparallel to each other by hydrogen bonding via amino acid linker groups. An a-helix, nearly 6 times as long as the a-helices in the binding regions (six in all), forms the middle region of the dumbbell separating the two binding regions. Each metal binding region has a large affinity3-" for Ca2+ ions (dissocation constants, Kd, between lo-' and but each site has been shown recently to bind Mg2+ions at considerably lower affnitiesl2 (Kd between and lo"). Many other metal cations, e.g., Cd*+, also have been shown to have affinities for ~almodulin,~-"~'~ and even alkali-metal ions are thought to bind to these sites. A conformational transition is thought to occur after the binding of two Ca2+ ions because of changesi7-I9seen in the C D and N M R spectra and the fact that calmodulin's affinity' for other proteins is not very large until at least two calcium ions are bound to it. Obviously, the thermal stability (secondary and tertiary structure) of calmodulin could be profoundly affected by the binding of metal ions to the individual sites, and such effects easily can be investigated by differential scanning calorimetry (DSC). Calorimetric investigations on other small molecular weight proteins2*26 (e.g., ribonuclease A and lysozyme) have demonstrated that such proteins denature along a simple two-state pathway, Le., from a compact form with a definite structure to a random coil. Many larger p r o t e i n ~ ~ appear ~ - ~ l to have distinct domains, which may melt separately depending on affinities27*28*3i-33 of the domains for ligands or ions. Calmodulin's thermal melting curve might be affected by the concentration and type of cations present, and because the three-dimensional structure is known, one should be able to relate these effects to the a-helical regions in the protein. We have investigated with DSC the thermodynamics of denaturization of calmodulin in the To whom correspondence should be addressed
0022-3654/90/2094-7923$02.50/0
presence (moles of cations/calmodulin between 40 and 1 IO) of the monocations Li+, Na+, K+, and tetraethylammonium (N(1) Klee, C. B.; Vanaman, T. C. Advances in Protein Chemistry; Plenun Press: New York, 1982; Vol. 35, p 213. (2) Babu, Y. S.; Sack, J. S.; Greenhough, T. J.; Bugg, C. E.; Means, A. R.;Cook, J. Nature 1985, 315, 37. (3) Cox, J . A.; Mahoe, A.; Stein, E. A. J . Biol. Chem. 1981, 256, 3218. (4) Dedman, H. R.;Potter, J. D.; Jackson, R. L.; Means, A. R.J. Biol. Chem. 1977, 252, 8415. (5) Walsh, M.; Stevens, F. C.; Oikawa, K.; Kay, C. M. Can. J . Biochem. 1979, 57, 261. (6) Wolf, H. U.; Dieckvoss, G . ;Lichtner, R.Acta Biol. Med. Ger. 1977, 36, 847. (7) Crouch, T. H.; Klee, C. B. Biochemistry 1980, 19, 3692. (8) Watterson, D. M.; Harrelson, W. G., Jr.; Keller, P. M.; Sharief, F.; Vanaman, T. C. J . Biol. Chem. 1976, 251, 4501. (9) Ogawa, Y.; Tanokura, M. J . Biochem. 1984, 95, 19. Brostrom, M. A. J . Biol. (10) Wolff, D. J.; Pokier, P. G.; Brostrom, C. 0.; Chem. 1977, 252, 4108. (1 1) Haiech, J.; Klee, C. B.; Demaille, J. G. Biochemistry 1981, 20, 3890. (12) Hopkins, H. P., Jr.; Gayden, R.H. J . Solution Chem. 1989,18,743. (13) Thulin, E.; Andersson, A.; Drakenberg, T.; Forsen, S.; Vogel, H. J. Biochemistry 1984, 23, 1862. (14) Deleted in proof. (1 5 ) Deleted in proof. (16) Deleted in proof. (17) Klevit, R.E.; Dalgarno, D. C.; Levine, B. A,; Williams, R.J. P. FEES Lett. 1984, 138, 281. (18) Ikura, M.; Hiraoki, T.; Hikichi, K.; Minowa, 0.;Yamaguchi, H.; Yazawa, M. Biochemistry 1984, 23, 3124. (19) Hincke, M. T.; Sykes, B. D.; Kay, C . M. Biochemistry 1981,20,3286. (20) Sturtevant, J. M. Annu. Reo. Phys. Chem. 1987, 38, 463. (21) Jackson, W. M.; Brandts, J . F. Biochemisrry 1970, 9, 2294. (22) Privalov, P. L.; Poleklin, T. In Methods of Enzymology; Hirs, C . H. W., Timaskeff, S. N., Eds.; Academic Press: New York, 1986; Vol. 131, p 2. (23) Privalov, P.; Khechinashvili, N. J . Mol. Biol. 1974, 86, 665. (24) Tsong, T.; Hearn, R.;Wrathall, D.; Sturtevant, J. Biochemistry 1970, 9, 2666. (25) Privalov, P. L.; Khechinashvili, N. N.; Atansov, B. P. Biopolymers 1971, 10, 1865. (26) Jocobson, A. L.; Devin, G.; Braun, H. Biochemistry 1981, 20, 1694. (27) Donovan, J . W. Trends Biochem. Sci. 1984, 9, 340. (28) Vickers, L. P.; Donovan, J. W.; Schachman, H. K. J . Biol. Chem. 1978, 253, 8493. (29) Privalov, P. L. Adu. Protein Chem. 1982, 35, I . (30) Privalov, P.; Filimonov, V.; Venkstern, T.; Bayev, A. J . Mol.Biol. 1975, 97, 279. (31) Donovan, J. W.; Ross, K. D. J . Biol. Chem. 1975, 250, 6022. (32) Filimonov, V. V.; Pfeil, W.; Tsalkova, T. N.; Privalov, P. L. Biophys. Chem. 1978, 8 , 1 1 7. (33) Fukada, H.; Sturtevant, J . M.; Quiocho, F. A. J . Biol. Chem. 1983, 258. 13193.
0 1990 American Chemical Society
7924
The Journal of Physical Chemistry, Vol. 94, No. 20, 1990
(Et)4+)and after adding to these solutions Ca2+and Mg2+ions, for which the enthalpy and entropy changes12 for binding at each site are known at 25 OC. For comparison, Ba2+,Sr2+,and Co2+, which also are known to bind to calmodulin. were included in our study. Cadmium was also included because two ~ i g n a l sare ' ~ seen in the cadmium NMR spectra, implying that there are two classes of binding sites, a conclusion consistent with the variations found for the binding constants for Ca2+and Mg2+. Several years before the crystal structure appeared, Tsalkova and P r i v a l o proposed ~~~~~~ a domain-melting model for calmodulin based on their limited studies in the presence of ethylenediaminetetraacetic acid (EDTA) and Ca2+and Mg2+ at different mole ratios. Their model has four domains (one for each metal site), each of which could melt separately or cooperatively, depending on the occupation by metal ions. Obviously, this model is not entirely consistent with the dumbbell structure found in the X-ray studies, i.e., two binding sites being intimately associated in each end and connected by a long a-helix. The DSC curves presented here, in our opinion, are consistent with the crystal structure and a model for melting of calmodulin involving no more than three macromolecular states (allosteric states as defined by Gill and c o - ~ o r k e r s ~but ~ ) ,not individual domains. Our excess heat capacity curves are also free from effects caused by the changing affinity3' of EDTA for Ca2+ as temperaturc increases.
Experimental Section Calmodulin was extracted and purified by a procedure similar to that described by Gopalakrishna and Anderson.38 Bovine brain and/or testes obtained from Walker Meats slaughter house of Carrollton, GA, were homogenized with two volumes of buffer A (50 mM Tris-HCI pH = 7.5, 1 mM EDTA, 1 mM 2mercaptoethanol. 0.5 mM phenylmethylsulfonyl fluoride) and centrifuged at 15 000 g for 30 min. The resultant supernatant was passed through two layers of cheese cloth to remove lipids, and the pH was adjusted to 4 to precipitate the calmodulin. After homogenization in a minimal volume of buffer A, adjustment to pH 7 . 5 , and separation from insoluble proteins by centrifugation at 22 OOOg, the supernatant was adjusted to 5 mM in CaC12 and applied at 4-5 mL/min to a phenylsepharose (Sigma) column (2.5 cm by 8 cm; 40-mL bed volume) at room temperature. The column was washed with buffer B (50 mM Tris-HCI, pH = 7.5, 1 mM 2-mercaptoethanol, 0.1 mM CaCI,) until the absorbance of the eluant reached base line and then washed with buffer C (buffer B + 0.5 M NaCI) to remove S-100 proteins ionically bound to the calmodulin. Eluting with buffer D (buffer B without CaCI, but containing 1 mM EGTA, ethylene glycol bis(aminoethy1 ether)-N,N,N',N'-tetraacetic acid) removed Ca2+ from the Ca2+-calmodulin complex bound to the column and produced high-purity calmodulin which was concentrated by ultrafiltration with a 10000 molecular weight cutoff membrane. A micro-Lowry protein assay was39combined with absorbance measurements to yield an extinction coefficient at 277 nm comparable to that obtained by Haiech et al.," and the molecular weight was found to be between 16000 and 17000 Da by electrophore~is.~~ All calorimetric experiments were performed in 25 mM HEPES which had been adjusted to pH = 7.5 with the hydroxide of a particular monocation. Before adding a dication to the apoprotein with the particular monocation present, all divalent metal ions were removed by placing the calmodulin solution in an Amicon ultrafiltration cell (molecular weight exclusion of 10000) and flushing repeatedly with the 25 mM HEPES buffer containing 0.05 M EDTA. The EDTA was removed by repeated flushing with the 25 mM HEPES containing a particular monocation. The final solutions, concentrated in the ultrafiltration cell, were passed (34) Tsalkova. T. N.; Privalov, P. L. J . Mol. B i d . 1985, 181, 533. (35) Tsalkova, T. N.; Privalov, P. L. Mol. Biol. (Moscow) 1983, 17, 251. (36) Robert, C. H.; Colosimo, A.; Gill, S.J. Biopolymers 1989, 28, 1705. (37) Hovey, J. K.; Hepler, L. G. Inorg. Chem. 1988, 27, 3442. (38) Gopalakrishna. R.; Anderson. W. B. Biochem. Biophys. Res. Commun. 1982, 104. 830. (39) Peterson, G. L. Anal. Eiochem. 1977. 811. 346. (40) Deleted i n proof
Hopkins and Gayden I
1
I
1
Colmodulin
I
I
6
2
0 0 Temperature
20
40
60
80
OC
2
EtOH/K+
T e m p e r a t u r e OC
Figure 1. Excess heat capacity curves for calmodulin in the presence of K', Li', N(Et),+ (1 2 mM for each monocation), and ethanol (1.5 M) compared to ribonuclease. The buffer was 25 mM HEPES at pH = 7.5, and the protein concentrations were in the 0.3-0.6 mM range.
through a Chelex-X-100 ion-exchange column which was previously equilibrated in the buffer being used. If the calorimetric curves for the solutions were identical with those found earlier, then divalent cations dissolved in a chelexed buffer were titrated into the solution to a particular molar ratio of dication to calmodulin. Differential scanning calorimetry experiments were performed and analyzed with a MC-2 scanning calorimeter and associated computer programs purchased from Microcal, Inc. Power meas u r e m e n t ~ ,recorded ~ ~ , ~ ~ in mcal/min, were divided by the scan rate ( 5 5 deg/h), corrected for instrumental effects by subtracting a base line determined with buffer in each cell, and divided by the millimoles of the sample present to yield heat capacities in cal/(deg mol). High-frequency noise was removed by numerical smoothing with a polynomial fit over short temperature ranges. The sensitivity and accuracy of the instrument were determined periodically by passing current through a heater mounted on the sample cell, and the heat capacity measurements were found to have a precision in the 1-2% range. At the end of each scan the solution was removed from the cell and the concentration of calmodulin determined from absorbances at 277 nm and the previously determined extinction coefficient.
Results Excess heat capacities versus temperature for calmodulin in the presence of different monocations are shown in panels a-c in Figure 1 along with a similar plot for ribonuclease A, a small globular p r ~ t e i with n ~ ~a well-characterized ~~~ two-state thermal transition. A thermal transition region is seen for calmodulin in the 30-70 OC range, but it is much smaller in magnitude, and the slope of the excess heat capacity curve below the transition region is about twice as large as that for ribonuclease. With tetraethylammonium present (12 mM), a maximum in the transition region is observed near 49 OC, and replacement of this ion by alkali-metal monocations at the same concentration shifts the maximum upward: near 59 OC for Li+ ions and near 53 "C for Na+ and K+ ions. Adding ethanol (1.5 M) to the solution with K+ ions present lowers the maximum below 45 OC; at 3.1 M ethanol the maximum is near 40 OC and the curve is much broader. Before attempting interpretation of these curves, one must separate any intrinsic temperature d e p e n d e n ~ e ~of~ ,the ~ ' heat capacity changes from those due to transitions between distinct states. When the intrinsic changes are small and/or the slopes are nearly the same before and after the transition region, such
__ (41) Lysko, K . A.; Carlson, R.; Taverna, R.; Snow, J.; Brandts, J. F. B~ochentisrry 1981, 20, 5570.
DSC Studies on Calmodulin
The Journal of Physical Chemistry, Vol. 94, No. 20, 1990 7925 c
OI-JL'1 ."J Q O
0
20
40
60
C
80
-
Temperature
3
2C
AC
60
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20
40
60
80
OC
0
20
40
66
0
-
20
40
o
60 80
SO
T e m p e t a t u r s OC
Figure 2. Excess heat capacity curves for 340 pM calmodulin in the presence of 12 m M N(Et),+ and 593 pM calmodulin in the presence of 12 mM K+ appear in panels on the left. Base lines, generated by connecting the regions before and after the transition region with the method of splines, are shown as the dotted curves. Subtraction of these base lines from the excess heat capacity curves yielded the curves shown in the panels on the right.
20
40
60 a0
Temperature
0
20 40 60
BO
OC
Temperature OC
Figure 3. Corrected excess heat capacity curves, generated in the manner indicated in Figure 2, are shown (solid lines) in the presence of different monocations (1 2 mM) and ethanol when a 25 mM HEPES buffer at pH = 7.5 was used and the protein concentrations were in the 0.3-0.6mM range. A sequential model (NI N 2 D2) or a sum (2)of two-state
- -
transitions generated virtually identical theoretical curves (double-dotted). Curves for the individual transitions are shown as single-dotted curves.
TABLE I: Calorimetric ParameteP for Melting of Calmodulin in the Presence of Monocations and Mg*+ conditions p, M T,, 'C 1.5 M ethanol 274 44.4 12 m M Kt 240 49.2 I2 mM N(Et),+ 12 mM Kt 593 52.8
AH-,
56.1 36.7 53.0
12 mM Na'
225
54.2
63.2
12 mM Lit
420
58.9
68.4
5 mm Mg2+ 12 mM Kt
440
82.4
67.4
T,,b "C 33.8 45.0 49.2 33.6 51.7 35.6 54.0 44.1 59.5 65.7 81.7
AH:
21 38 43 18 41 21 43 29 42 26 46
a Tm= peak temperature of melting region; AHa,= enthalpy change for entire melting region in kcal/mol, and the estimated relative uncertainties are in the 3 4 % range (see Results section); Tm= temperature at peak of the transition; AH, = enthalpy changes for the individual theoretical transitions. From deconvolution analysis.
as those for ribonuclease, a simple extrapolation is adequate. Unfortunately, such a procedure does not suffice for calmodulin with monocations present, and one must perform a somewhat arbitrary c o n n e c t i ~ n ~of~ the * ~ regions ~ * ~ ~ before and after the transition region. We chose to use the least arbitrary method, which in our opinion is the connection of the curves above and below the transition regions by the method of splines. Examples of the application of this method to generate the excess heat capacity curves for the transition region are shown in Figure 2. The panels on the right contain the experimental curves with the splines-generated curves shown as the dashed lines connecting the upper and lower temperature regions. Subtraction of the splines curves from the experimental curves produced the base line subtracted excess heat capacity curves for the transition regions (panels on right, Figure 2). Curves generated in this manner are shown in Figure 3 and compared to the curves generated from models discussed later. The parameters, temperature maxima, and enthalpy changes (AH,) for the transitions used to produce the curves in Figure 3 are listed in Table I along with the enthalpy changes (AHca,)for the entire transition region. Variations in AHH,,are related to errors in the protein concentration, subtraction of the intrinsic heat capacity curve, and estimation of the areas under the corrected curves (see Figure 2), and we estimate that the relative precision associated with these parameters is in the 3-5% range. In all cases the curves generated from the models are very close to those derived from the data, but the sum of the
%!L+ ou ! ou 4 0 0
20 40 60 80
0
20 40 BO BO
Temperature
--
0
20
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80
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BO
a0
C'
1
/(dl
:2u 2
Q o
0
20
40 BO
0
80
o
20
40 BO a0
o
Temperature OC
Figure 4. Excess heat capacity curves for calmodulin (concentration in the 0.3-0.6 mM range) at different Ca2+concentrations in 12 mM Kt and 25 mM HEPES at pH = 7.5. Curves for solutions with very small Ca2' concentrations are shown in the far left panels, curves with two ratios of EDTA to Ca2' are shown in panels b and c, and curves with 4:l and 20:l ratios of Ca2' to calmodulin are shown in panels e and f.
AH, values for the individual transitions is always greater than AHcalfor the entire region because of difficulties in fitting the edges of the transition region. Errors associated with the temperatures in Table I are in the 0.1-0.3OC range. Minimal changes in the excess heat capacity curves were observed up to a 2:1 ratio of Ca2+to calmodulin in solutions with monocations present; Le., the curves were virtually identical with panel d in Figure 4 and those in Figure 1, Similar curves were found with excess EDTA present (panel a, Figure 4),a condition (25 "C) at which very little calmodulin is bound to Ca2+and also when the EDTA to Ca2+ ratio was 1.2 (panel c, Figure 4), a condition at which the first site is nearly filled with Caz+, the second is partially filled Ca2+, and Ca2+occupies very few of the other sites. At a ratio of 4:l for Ca2+ to calmodulin and at an EDTA to Ca2+ratio of 1.0, nearly all the first, second, and third sites are filled with Ca2+,and the excess heat capacity curves are virtually a straight line in the 0-100 OC region (panels c and e in Figure 4). Increasing the occupation (25 "C) of sites past this point does not appear to affect the curves (panel f, Figure 4). A small peak may exist near 40 OC in panels c and e and may also be present in panel f for which the CaZ+to calmodulin ratio is
7926 The Journal of Physical Chemistry, Vol. 94, No. 20, 1990
-
r----
I
E-
Mg2+
APO
T e m p e r a t u r e :O
----6.
Temperature
OC
Figure 5. Excess heat capacity curves for calmodulin (concentrationsin the 0.3-0.6mM range) are shown for metal ion to calmodulin ratios of 1O:l for Mg2+, 3:1 for Mn2+, 2:l for Sr2+,4:l for Cd2+,and 3:l for Co2+.
20:I . Adding ethanol to these solution up to I .5 M does not change appreciably the shape of the excess heat capacity curves shown in Figure 4. Excess heat capacity curves with 12 mM K+ ions present and several other dications present are shown in panels in Figure 5 and compared with the curve for calmodulin with only K + ions present. Addition of Mg2+, Mn2+,and Sr2+,all of which are known to bind to calmodulin, shift the maximum for the transition region above 80 OC at metal ion to calmodulin ratios of l O : l , 2:1, and 2: I , respectively. At Cd2+and Coz+ ion to calmodulin ratios of 4: I , the curves (Figure 5) are similar to those observed with Ca2+ present at the same ratio.
Discussion When alkali-metal monocations are present (12 mM), subtracting the intrinsic heat capacity variations by the method of splines produced asymmetrical curves that could not be fit to a simple two-state model.zo*22*41 Application of a sequential mode137*42,44 with three macroscopic states reproduced the experimental curves; however, a sum of two-state transitions also produced excellent fits. The transition profiles derived from the two-state analyses and shown in the panels in Figure 3 are identical with those produced from the sequential analyses, and the parameters used to generate these theoretical curves are given in Table I. With tetraethylammonium ions present (1 2 mM), the corrected excess heat capacity curve for calmodulin could be reproduced extremely well by a single two-state transition (Figure 3). Within experimental error, AH (40 kcal/mol) is nearly the same as AH for the main transition for the asymmetrical curves found at the same concentration of alkali-metal monocations and when Mg2+(5 mM) is present with K+ (12 mM). The first transition, which is not seen in the presence of tetraethylammonium ions, has an enthalpy change in the 20-25 kcal/mol range, even when both K+ (1 2 mM) and Mg2+ (5.0 mM) ions are present. Adding either Sr2+or Mn2+ affects the excess heat capacity curves in a manner similar to Mg2+, but an analysis based on the model for solutions with alkali-metal monocations could not adequately reproduce the curves. The excess heat capacity curves shown here in the presence of alkali-metal ions are similar in shape to those found by Tsaklova and P r i ~ a l o vin~ ~a 0.010 . ~ ~ M cacodylate buffer (pH = 7.28) with 0.002 M EDTA, either before or after correcting for the intrinsic variations. Our total enthalpy changes (AH,,) for the transition region with alkali-metal cations present (sum of the two transitions) are 20-25% lower than the corresponding values reported by Tsalkova and P r i v a l o ~probably , ~ ~ ~ ~ due ~ to errors i n concen(42) Biltonen, R. L.; Freire, E. CRC Crir. Rev. Biochem. 1978. 5. 8 5 (43) Velicelebi, G . ; Sturtevant, J. M. Biochemisrry 1979, 18, 1180 (44) Freire, E . ; Biltonen, R. L Biopolymers 1978, 17. 463
Hopkins and Gayden tration for the protein and problems associated with compensating for the large heat capacity variation found in this work and by Tsalkova and P r i v a l o ~before ~ ~ , ~the ~ transition region. Our enthalpy parameters in the presence of Mg2+agree well with those reported by Tsalkova and P r i ~ i l o v ~(29 ~ *and ' ~ 47 kcal/mol compared to 26 and 46 kcal/mol, Table I), but the corresponding T,,, values are nearly 4 deg higher. Differences in the occupation of sites by Mg2+ in the two experiments will cause the maxima to vary somewhat if the affinities of the sites change significantly upon denaturization. (See recent analyses by Sturtevant20 and et al.) The binding of Mg2+ to calmodulin is nearly enthalpically neutral,12 and the enthalpy changes for the transitions should not be affected much by the presence of this ion at any concentration, just as observed. The addition of Ca2+ ions (affinities for sites on calmodulin at least 100 times greateri2than Mg2+) up to a mole ratio of 2:1 causes very little change in the shape of the excess heat capacity curves. From this observation we conclude that the affinities of the first two sites for Ca2+ions do not change much upon melting of calmodulin, and major conformational changes do not accompany the filling of the first site and substantial filling of the second site. Past the ratio of two CaZ+ions per calmodulin, the melting of calmodulin moves toward temperatures above 100 OC; thus, significantly increasing the occupation of the second, third, and fourth sites by Ca2+ions has a profound effect on the heat capacity curves. Apparently, the second, third, and fourth sites have far greater affinities for Ca2+in the native state than in the denatured state, and this could be due to a conformational change, postulated from spectral and enzyme activation studies, that accompanys the filling of these site^."-]^,^^ When all four sites are nearly filled with Ca2+ ions (ratio of CaZ+to calmodulin greater than four, panels e and f, Figure 4), our curves up to 100 OC are remarkably ~~~~ similar to those of Tsalkova and P r i v a l o ~at~ comparable conditions; i.e., the excess heat capacity curves increase nearly linearly with increasing temperature from 5 to 100 OC. Clearly, the thermal melting of calmodulin bound at all four sites with Ca2+must be above 100 "C, and even at 110 OC we do not see evidence of a decrease in heat capacity with all four sites bound, just as found by Tsalkova and P r i ~ a l o at v ~similar ~ ~ ~ conditions. ~ Decreases in the excess heat capacity curves seen by them and by us (panels b and c, Figure 4), with EDTA and Ca2+present at ratios where a substantial portion of sites are unfilled, could be due to changes in the affinity of EDTA for Ca2+ a t higher temperatures. On the basis of AH for binding46 Ca2+ to the tetraanion of EDTA being nearly -6 kcal/mol and the known thermodynamics4' for ionization of EDTA, we estimate that the apparent binding constant of Ca2+for EDTA could increase by nearly a factor of IO2 when the temperature is raised from 25 to 100 OC; thus, heat could be produced upon transfer of some CaZ+ ions from calmodulin ( I .3 kcal/mol of Ca2+ions boundI2 at 25 "C) to EDTA. A simple sequential melting model, or allosteric framework in the terms used by Gill et al.,36appears to us to be sufficient to explain all of our results, and one does not need to postulate, as Tsalkova and P r i v a l o ~did, ~ ~ .four ~ ~ melting domains, each of which can be affected differently by cations and each of which can interact with each other. Furthermore, the dumbbell shape of calmodulin with two Ca2+sites at each end does not seem consistent with their domain model, and we propose three macromolecular (allosteric) states for calmodulin, each of which can bind cations. With excess tetraethylammonium ions present, the fraction of calmodulin molecules in the least stable state (native, N I ) is very small, and calmodulin proceeds from the intermediate state (native, N2) to the high-temperature state (denatured, D) via a simple two-state transition. Replacing this cation with alkali-metal monocations stabilizes the N 1 state via binding of (45) Watterson, D. M.; Vincenzi, F. F., Eds. Calmodulin and Cell Functions. Ann. N.Y. Acad. Sci. 1980, 356.
(46) Christensen, J. J.; Eatough, D. J.; Izatt, R. M. Handbook oJMetal Ligand Hears, 2nd ed.; Marcel Dekker: New York, 1975. (47) Christensen. J. J.; Hansen, L. D.; Izatt, R. M. Handbook oJProron Ionization Heats: Wiley: New York, 1976.
J. Phys. Chem. 1990, 94, 7927-7935
7927
these cations to the metal binding sites, causing the excess heat capacity curves to be representative of sequential melting involving states N1, N2, and D. The order of stabilization of the N1 state by binding to cations is Li+ > Na+ > K+ >> tetraethylammonium (see Table 1 for transition temperatures at 12 mM), but the same order of binding affinities exists for the N 2 state because the transition from state N 2 to D depends on which cation is present, e.g., 49.2 “ C for tetraethylammonium and 59.5 O C for Li+, both at 12 mM. Breaking calmodulin in the middle or clipping off the fourth metal binding region produces proteins that melt, according to Tsalkova and P r i v a l ~ v via , ~ ~an apparent two-state process, which is consistent with the entire calmodulin molecule being involved in the final transition. Addition of ethanol to calmodulin solutions (1 2 mM K+) moves the entire transition region to lower temperatures, but the change is about 6 times what would be expected from predictions based on studies by Sturtevant and V e l i ~ e l e bon i ~ ~lysozyme (1 29 amino acid residues and a single two-state transition). All calmodulin’s a-helices have a net h y d r ~ p h o b i c i t y ,in~ ~spite of a regular in-
terspersion of polar and hydrophobic amino acid residues, which is unusual in a small protein. Thus, the large shift in ethanol solutions is probably due to calmodulin’s hydrophobicity; Le., each a-helix will be less stable in the ethanol-water environment because the denatured state is favored via solvation of the hydrophobic residues by ethanol. The very large slope seen in the excess heat capacity curves for the native protein (also found by Tsalkova and P r i v a l ~ v ~ ~ * ~ ~ ) appears to be characteristic of calmodulin’s dumbbell shape, which would be expected to have many low-frequency vibrations that would contribute substantial changes to the excess heat capacity curve. Calmodulin has a large number of hydrophobic residues, but the excess heat capacity actually decreases with increasing t e m p e r a t ~ r efor ~ ~small apolar solutes which might be models for these residues. Thus, the unusual slope seen for calmodulin is unlikely related to its hydrophobicity. Registry No. Et, 64-17-5; N(Et),+, 66-40-0; Li, 7439-93-2; N a , 7440-23-5;K, 7440-09-7;Ca, 7440-70-2;Mg, 7439-95-4; Mn, 7439-96-5; Sr, 7440-24-6; Cd, 7440-43-9;Co, 7440-48-4.
(48) Cox, J. A,; Comte, M.; MalnGe, A. In Metal Ions in Biological Sysfems; Segiel, H., Ed.; Marcel Dckker: New York, 1984; Vol. 17.
(49) Gill, S.J.; Dec, S.F.; Olofsson, G.; Wadso, 1. J . Phys. Chem. 1985, 89, 3758.
Characterization of Ground and Electronically Excited States of o-Hydroxybenzaldehyde and Its Non-Hydrogen-Bonded Photorotamer in 12 K Rare Gas Matrices Meredith A. Morgan,+ Edward Orton,* and George C. Pimentelt Laboratory of Chemical Biodynamics, University of California. Berkeley, Berkeley, California 94720 (Received: January 30, 1990: In Final Form: May 3, 1990)
Intramolecularly hydrogen bonded o-hydroxybenzaldehyde (OHBA-C) isblated in 12 K rare gas matrices photolyzes to a non-hydrogen-bonded rotamer (OHBA-F). IR spectra of OHBA-C, OHBA-F, and several model and isotopically substituted compounds are consistent with identification of the OHBA-F conformer as that formed by 180-deg rotation of both the hydroxy and aldehyde groups. For the two rotamers, electronic absorption, excitation, and emission spectra are presented together with time-resolved emission measurements and estimates of a ground-state reaction enthalpy. From these data, it is proposed that the S, state of OHBA-C is an n,r* hydrogen atom transfer state, and Sz is a r,r* proton-transfer state with a large (- 18 kcal) barrier to reaction. Rotamerization is reversed by SI or S2excitation of OHBA-F. The conversion of OHBA-C to OHBA-F is - 5 times as efficient as the reverse process upon excitation at the respective SI 0-0 energies. An increase in photolysis quantum yield of OHBA-C is measured at energies well above the 0-0 energy and may correspond to reaction over the proposed -8 kcal SI barrier.
Introduction The spectroscopy of o-hydroxybenzaldehyde (OHBA-C in Figure 1) has been actively investigated for many years. Structurally, it is the simplest of the intramolecularly hydrogen bonded salicylic acid derivatives which are thought to undergo proton or hydrogen-atom transfer in the S, state. It remains an area of debate which of these transfer processes takes place in the SI state, and whether SIis n , r * or r,r*.’s2OHBA-C forms a non intramolecularly hydrogen bonded rotamer upon UV photolysis in cryogenic mat rice^.^ The precise structure of the non-hydrogen-bonded conformer has not been resolved.’ Previous studies have been hampered by reliance on either emission or infrared absorption spectroscopy alone, as well as by complications from intermolecularly hydrogen bonding solvents or impurities. Proton-transfer systems are of interest for four-level lasers, and photochemically reversible isomerizations are of interest as prototypes for molecular information or energy storage systems. ‘Author to whom correspondence should be addressed. ‘Current address: Rohm and Haas Co., Architectural Coatings Research, Spring House, PA 19477. *Deceased June 18, 1989.
0022-3654/90/2094-7927$02.50/0
In this study of OHBA in cryogenic rare gas matrices, we identify the conformation of the non intramolecularly hydrogen bonded rotamer from high-resolution infrared spectra. We also present high-resolution steady-state and time-resolved emission spectra of OHBA-C and its photorotamer in argon and xenon matrices. From these spectra we derive information on the excited states of both conformers: 0-0 energies; vibrational modes active in the chromophores; and the electronic configurations of SI and S2. Combining these data with the vibrational structure in the rotamer’s excitation spectrum, and with ground-state enthalpiese6 and barriers to reaction4*’measured and estimated by others, we ( 1 ) Nagaoka, S.;Hirota, N.; Sumitani, M.; Yoshihara, K. J. A m . Chem. SOC.1983, 105, 4220. (2) Nagaoka, S.; Nagashima, U.; Ohta, N.; Fujita, M.; Takemura, T. J . Phys. Chem. 1988, 92, 166. (3) Gebicki, J.; Krantz, A. J . Chem. Soc., Perkin Trans. 2 1984, 1617. (4) Tabei. M.: Tezuka. T.: Hirota. M. Tetrahedron 1971. 27. 301. ( 5 ) Rajogopal, E.; Sivakumar, K. V.; Subrahmanyam, S.V. J . Chem. SOC., Faraday Trans. I 1987, 77, 2149. (6) Schaefer, T.; Sebastian, R.; Laatikainen, R.; Salman, S. Can. J. Chem. 1984, 62, 326. (7) Anet, F. A. L.; Ahmad, M. J . A m . Chem. SOC.1964, 86, 119.
0 1990 American Chemical Society
