- Email: [email protected]
Release of Two Peripheral Proteins from Chloroplast Thylakoid Membranes in the Presence of a Hofmeister Series of Chaotropic Anions
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 358, No. 2, October 15, pp. 385–390, 1998 Article No. BB980866
Release of Two Peripheral Proteins from Chloroplast Thylakoid Membranes in the Presence of a Hofmeister Series of Chaotropic Anions1 Dirk K. Hincha2 Institut fu¨r Pflanzenphysiologie und Mikrobiologie, Freie Universita¨t, Ko¨nigin Luise Strasse 12-16, D-14195 Berlin, Germany
Received June 30, 1998
The dissociation of two peripheral spinach (Spinacia oleracea L.) thylakoid membrane proteins, coupling factor CF1 and ferredoxin-NADP1-oxidoreductase, in the presence of chaotropic sodium salts has been investigated, using monospecific antibodies against the proteins as probes. Release of both proteins followed the Hofmeister series of anions (Cl2 < NO32 < Br2 < I2 < SCN2). In mixtures, the different salts had an additive effect. In addition, there were also qualitative differences in the action of the anions, such that NaI and NaSCN led to a different concentration and time dependence of the dissociation of the peripheral proteins from thylakoids. An analysis of the temperature dependence of protein release showed that the more chaotropic ions reduced the activation energy required for the dissociation of the proteins from their binding sites on the membrane. The addition of sugars (glucose, sucrose, or trehalose) reduced the amount of protein released from the membranes in the presence of NaI or NaSCN. © 1998 Academic Press
Key Words: coupling factor CF1; ferredoxin-NADP1oxidoreductase; lyotropy; protein solubility; salt effects; Spinacia oleracea.
The stability and solubility of proteins in aqueous solutions has received widespread interest, as it is important both from a theoretical and from a practical point of view (see, e.g., 1 for a review). It has been recognized for a long time that the solubility of proteins can be greatly influenced by cosolutes, which can lead to reversible precipitation or to reversible or irrevers1 Financial support was provided by the Deutsche Forschungsgemeinschaft through the Heisenberg program. 2 Fax: 149 30 838 4313. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
ible denaturation. The addition of other compounds, such as sugars, can counteract the effects of denaturants. In general, substances that destabilize proteins in solution and increase the solubility of proteins are called chaotropes, while cosolutes that stabilize protein structure are called kosmotropes. The stability of soluble proteins is usually measured as the temperature of denaturation, and the effects of cosolutes on the denaturation temperature are explained in terms of the “preferential exclusion theory” (see 2 for a review). Briefly, this theory states that kosmotropic cosolutes are preferentially excluded from the hydration shell of proteins, leading to a preferential hydration of the protein surface. This makes the exposure of hydrophobic amino acids from the interior of the protein to the solution energetically more unfavorable. This effect is based on an increase in the surface tension of water and consequently the interfacial tension between water and the protein. Chaotropic salts, on the other hand, destabilize proteins, because they have the opposite effect (3). The same phenomenon can also be interpreted in terms of water structure. The cluster structure of water (4, 5) is stabilized by kosmotropic solutes (structure makers, salting-out effect on proteins) and destabilized by chaotropic solutes (structure breakers, salting-in effect on proteins) (6, 7). Of course, in reality there is a continuum between the two extremes described above. This has first been recognized by Hofmeister in his classical paper, where he described the effects of a wide variety of salts on the solubility of egg white proteins (8). These effects of salts on protein solubility have later been quantified by Voet, who assigned a lyotropic number (N) to each ion (9). It has been shown that, among other physical characteristics of aqueous solutions, also the change in surface tension upon addition of different salts is a linear function of N (10). In the present investigation, 385
386
DIRK K. HINCHA
N values have been used as a convenient means to quantitatively compare the effects of a Hofmeister series of sodium salts on two peripheral thylakoid membrane proteins. Most of the literature on the effects of solutes on proteins deals with soluble proteins. It seems interesting to extend this research to include peripheral membrane proteins, which are normally membrane bound but are soluble in the absence of detergent once they are dissociated from their binding sites. The two proteins that were studied for this purpose are the peripheral part of the chloroplast thylakoid ATPase (chloroplast coupling factor CF1) and the ferredoxin-NADP1-oxidoreductase (FNR; EC 1.18.1.2).3 Both proteins are localized on the outer surface of the chloroplast thylakoid membrane, protruding into the chloroplast stroma in vivo and into the incubation medium after isolation of the thylakoid membranes. Treatment of thylakoids with dilute EDTA solutions released a large part of both proteins, while rebinding only took place in the presence of divalent cations (11, 12). This indicates that tight binding occurs by hydrophobic interaction. Negative charges on proteins and membrane have to be screened by cations to reduce electrostatic repulsion (13). CF1 is a large complex of five different subunits with a stoichiometry of a3b3gde, bound hydrophobically to the CF0 transmembrane proton channel (13). FNR is a monomeric protein (14) that is bound hydrophobically to the PSI-E subunit of photosystem I (15). The possibility of using chaotropic salts to dissociate peripheral proteins from a variety of membranes has been described before (16, 17) and this technique has been used to remove CF1 from thylakoids (18), as well as the peripheral part of other ATPases from their binding sites (19 –22). These were, however, no systematic, quantitative studies, similar to those on soluble proteins, but, rather, chaotropic salts were used as a tool to study the respective proteins or membranes. In the present report, the effects of a Hofmeister series of chaotropic sodium salts on the dissociation of CF1 and FNR from their hydrophobic binding sites on thylakoid membranes were analyzed as a contribution to our understanding of the stability properties of proteins. MATERIALS AND METHODS Plant material. Spinach plants (Spinacia oleracea L. cv. Monnopa) were grown in a growth chamber under controled conditions as described recently (23, 24). Proteins and antibodies. FNR and CF1 were isolated from spinach thylakoid membranes as described by Wolter et al. (12), and monospecific, polyclonal antibodies were raised against the native proteins in rabbits as described by Hincha et al. (25). Isolation and incubation of thylakoid membranes. Thylakoids were isolated from spinach leaves as described previously (26) and
3
Abbreviation used: FNR, ferredoxin-NADP1-oxidoreductase.
FIG. 1. Release of the peripheral proteins CF1 and FNR from thylakoid membranes. The membranes were incubated for 2 h at 0°C in the presence of 0.5 M of a Hofmeister series of sodium salts. The chaotropic properties of the salts were ranked according to their lyotropic number N (see text for details; N 5 10.0, NaCl; 11.3, NaBr; 11.6, NaNO3; 12.5, NaI; 13.3, NaSCN). In the case of NaI and NaSCN, the appropriate data from all experiments in this study were used to calculate the means and standard deviations from 21 samples each, measured over the course of several months, to give an indication of the overall variability of the data.
washed three times by centrifugation and resuspension in 10 mM MgCl2. The final membrane suspension was adjusted to 1 mg chlorophyll mL21. Chlorophyll was determined according to Arnon (27). Samples (200 mL) were incubated for 2 h at 0°C, unless otherwise specified in the figure legends, and contained 5 mM MgCl2, 25 mM Hepes (pH 7.8), and 500 mM of NaCl, NaNO3, NaBr, NaI, or NaSCN. Control samples were kept under the same conditions in the presence of 5 mM MgCl2, 25 mM Hepes (pH 7.8). Quantitation of protein release. After the incubation, the membranes were sedimented by centrifugation for 15 min at 16,000g and 4°C. CF1 and FNR were determined in the supernatants by quatitative single radial immunodiffusion in agarose gels (28) as described in detail before (25, 29). As a reference, an aliquot of the original thylakoid suspension was lysed in 2% Triton X-100 and a dilution series was applied to the same gels. From this calibration, values for 100% CF1 and FNR content of each thylakoid preparation were determined and release of the proteins was calculated relative to this total content. The amount of the proteins released from the membranes in control samples containing only MgCl2 and Hepes were set as 0% protein release, to calculate the percentage of the proteins released during incubation, due to the presence of the chaotropic salts. The data shown in the figures are means 6 S.D. from three parallel samples, unless otherwise specified in the figure legends. Where no error bars are visible, S.D. was smaller than the symbols. Where correlation coefficients (r) are indicated in the figures, the lines were fitted to the data by linear or exponential correlation analysis using the Macintosh Cricket Graph software.
RESULTS
Figure 1 shows that during an incubation for 2 h at 0°C both peripheral thylakoid proteins investigated can be dissociated from the membranes by chaotropic salts. The effectiveness of the different anions used in this study clearly follows the well-known Hofmeister series (8). In order to facilitate a quantitative comparison between the anions, they have been plotted ac-
HOFMEISTER EFFECTS ON PERIPHERAL MEMBRANE PROTEINS
FIG. 2. Release of CF1 and FNR from thylakoid membranes as a function of the lyotropic number N of the salts in the suspending medium (see the legend to Fig. 1 and the text for details). The different lyotropic numbers were achieved by mixing either NaCl and NaI or NaNO3 and NaSCN in different proportions. The final salt concentration during incubation was always 0.5 M.
cording to their lyotropic numbers N. These numbers have been determined from solubility studies with soluble proteins, i.e., proteins that are not normally associated with a membrane. For such proteins, solubility is a linear function of N (9). It is obvious from Fig. 1 that this linearity is not maintained when chaotropic ions are used to dissociate proteins from their binding sites on a membrane. Nevertheless, for both FNR and CF1 the effectiveness of the five different sodium salts investigated shows the same ranking as that for soluble proteins. The effects of the different anions are additive (Fig. 2), as has also been shown for soluble proteins (9). The whole range of N values was achieved by mixing different fractions of either NaCl (N 5 10) and NaI (N 5 12.5), or NaNO3 (N 5 11.6) and NaSCN (N 5 13.3) to a final total concentration of 0.5 M. It can be seen that the release of FNR and CF1 showed the same dependence on N, regardless of whether the pure salts (Fig. 1) or mixtures of different salts (Fig. 2) were used. However, the effects of the investigated anions on the dissociation of peripheral membrane proteins were also qualitatively different and not only quantitatively. Figure 3 shows the dependence of the solubilization of CF1 and FNR on the concentration of either NaI or NaSCN during incubation. The release of both proteins from thylakoids showed a linear dependence on the concentration of NaSCN over the whole concentration range investigated (Fig. 3B). In the presence of NaI, on the other hand, such a linear dependence was only found at concentrations above approximately 100 mM (Fig. 3A). An increase in the concentration of NaI below 100 mM caused a disproportionately large increase in the dissociation of both proteins, perhaps indicating a more specific interaction at low concentrations. The two anions also led to a different time dependence of protein release (Fig. 4). The amount of both
387
proteins that was released from the membranes increased as a linear function of time in the presence of NaSCN (Fig. 4B). In the presence of NaI, protein release showed kinetics that could be fitted more accurately with exponential curves (Fig. 4A) than with linear curves (r 5 0.99 for both proteins vs r 5 0.95 for FNR and 0.97 for CF1). The curves for both CF1 and FNR could be linearized in a double-reciprocal plot (Fig. 4A, inset). From this plot the maximum release at infinite incubation time in the presence of 0.5 M NaI was calculated as 18.9% for CF1 and 20.4% for FNR. This is similar to the values found after a 2-h incubation (Fig. 1), indicating that saturation kinetics are consistent with the data obtained in other experiments. To gain more insight into the physical basis of the action of chaotropic ions, the temperature dependence of the dissociation of FNR and CF1 was determined between 0 and 30°C. In order to minimize the effects of the different kinetics of protein release in the presence of different salts (Fig. 4), the incubation time was reduced to 1 h. The resulting data were analyzed in the form of Arrhenius plots (Fig. 5). It can be seen that linear curves were obtained in the presence of all five salts for both FNR (Fig. 5A) and CF1 (Fig. 5B). The slopes of these regression lines, however, varied between the different salts. Figure 6 shows that the slopes of all Arrhenius plots shown in Figure 5 were a linear function of the lyotropic number N of the salts. Since the slope of an Arrhenius plot is a direct measure of the activation energy of the observed process, it can be concluded from these data that the presence of chaotropic salts reduces the energy barrier for the dissociation of the proteins from their binding sites on the membranes. It has been shown before that the presence of sugars can ameliorate the effects of chaotropic ions and other denaturants, such as urea or guanidinium hydrochloride, on the stability of soluble proteins. Figure 7 shows that the same is also true for peripheral membrane
FIG. 3. Dependence of the release of CF1 and FNR from thylakoid membranes on the concentration of NaI or NaSCN in the incubation solution. The data are means 6 S.D. of six samples from two independent experiments.
388
DIRK K. HINCHA
DISCUSSION
The data presented in this paper show some interesting similarities and differences between soluble proteins, or protein complexes, and peripherally bound membrane proteins. First of all, it is noteworthy that the simple monomeric protein FNR and the multisubunit protein complex CF1 showed a very similar behavior under all conditions investigated in this study. This can be explained by the similar binding mechanism. Both CF1 and FNR are bound hydrophobically to other membrane proteins (11, 13, 15). Earlier work on CF1 has shown that in the presence of chaotropic agents, all subunits are released from the membranes (30, 31).
FIG. 4. Time dependence of the release of CF1 and FNR from thylakoids in the presence of 0.5 M NaI (A) or NaSCN (B) at 0°C. The inset shows the fit of the data in (A) to linear curves in a double reciprocal plot.
proteins. Loss of CF1 and FNR from thylakoids was greatly reduced when a sugar was present in the incubation solution in addition to either NaI or NaSCN. There were no systematic differences between the effects of the three sugars that were investigated, glucose, sucrose, and trehalose. In all cases, loss of the proteins from the membranes decreased as a linear function of the sugar concentration. While the presence of sugars in the solutions clearly stabilized the proteins on the membranes, the addition of a soluble protein (bovine serum albumin up to 10 mg mL-1) had no influence on protein release in the presence of 500 mM NaI and NaSCN (data not shown). Likewise, the substrates NAD1 or NADP1 (up to 5 mM) had no effect on the release of FNR, and ADP or ATP (up to 5 mM) had no effect on the release of CF1, in the presence of NaI or NaSCN (data not shown).
FIG. 5. Arrhenius plots of the temperature dependence of the release of FNR (A) and CF1 (B) from thylakoids. Samples were incubated for 1 h in the presence of 0.5 M of the different Hofmeister salts at temperatures between 0 and 30°C. The logarithm of the percentage of the different proteins released from the membranes during incubation is plotted as a function of the reciprocal incubation temperature in Kelvin.
HOFMEISTER EFFECTS ON PERIPHERAL MEMBRANE PROTEINS
FIG. 6. Correlation between the slopes of the Arrhenius plots shown in Fig. 5 with the lyotropic numbers of the different salts (compare Fig. 1) present during incubation (see the legend to Fig. 5 for details). The solid symbols represent data obtained from FNR measurements and the open symbols data from CF1. The slopes of the Arrhenius plots are a direct measure for the activation energy required to release the proteins from their binding sites on the membranes.
Once released from the membrane, CF1 is very coldlabile and dissociates rapidly. This dissociation is largely prevented in the presence of micromolar concentrations of ATP or ADP (32). In the presence of the chaotropic salts NaI or NaSCN, on the other hand, none of the substrates for FNR or CF1 provided any stabilization against dissociation of the proteins from the membranes. This is different from the situation during freezing of thylakoids in the presence of NaCl, where ATP, but not ADP, was able to stabilize CF1 on the membranes (33, 34). Under these conditions FNR was not released from the membranes (25). It can therefore be assumed that the conditions during freezing in the presence of NaCl were considerably milder than during incubation at 0°C in the presence of either NaI or NaSCN. Therefore, under those milder conditions a certain degree of substrate-induced stabilization of CF1 on the membranes occurs. This is in accordance with the fact that Santarius (35) found some protection of CF1 from membrane dissociation in the presence of NaCl under nonfreezing conditions. In general, the dissociation of proteins from thylakoids in the presence of chaotropic anions follows the Hofmeister series, both during freezing (36, 37) and in unfrozen solutions. This is similar to the effects of such salts on protein solubility in general. However, a quatitative analysis of a series of sodium salts revealed that, contrary to the situation with soluble proteins, the effect of the salts on the binding of peripheral membrane proteins was not a linear function of the lyotropic number N, assigned to the different anions (Fig. 1; compare 9). Nevertheless, the effects of the salts in
389
mixtures were strictly additive (Fig. 2) when analyzed on the basis of their lyotropic numbers. From Fig. 4 it is evident that the samples were not in equilibrium after 1 h of incubation. This, however, in no way invalidates the conclusions drawn above, as longer incubation times, which might result in an equilibrium situation, would necessarily lead to even stronger deviations from linearity. An analysis of the temperature dependence of the dissociation of CF1 and FNR from thylakoids in the presence of the different salts (Fig. 5) revealed that the activation energy for the release of both proteins was a linear function of N (Fig. 6). This reduction in activation energy in the presence of chaotropic anions is most likely related to the reduction in the interfacial energy between water and protein, affected by the salts (3), as the surface tension of water is again a linear function of N (10). The crucial role of surface tension for the stability of the binding of peripheral membrane proteins is emphasized by the fact that sugars reduce the dissociation of CF1 and FNR in a concentration-dependent manner (Fig. 7). It has been shown for soluble proteins that sucrose and glucose increase protein stability in solution by increasing the surface-tension of water and that kosmotropes can compensate the effects of chaotropes in solution (see 2 and 38 for reviews). Also, the stabilizing effect of sugars (Fig. 7) shows that the release of peripheral proteins in the presence of salts is
FIG. 7. Sugars protect FNR and CF1 from the effects of the chaotropes NaI and NaSCN. Thylakoids were incubated for 2 h at 0°C in the presence of 0.5 M of the indicated salts and increasing concentrations of glucose, sucrose, or trehalose. The correlation coefficients of the linear curves fitted to the data were between 0.99 and 0.95.
390
DIRK K. HINCHA
not releated to the effects of osmotic dehydration, as this is further increase by the addition of sugars. Still, changes in the surface tension of water are probably not the only mechanism through which chaotropic ions destabilize the binding of peripheral membrane proteins. This is indicated by the fact that surface tension is a linear function of N (10), while the dissociation of CF1 and FNR is not (Figs. 1 and 2). More detailed investigations with NaI and NaSCN support the idea of more specific effects of the different anions. Both the concentration and the time dependence of the release of CF1 and FNR show marked differences between the two anions (Figs. 3 and 4). The fact that FNR and CF1 behaved very similar in these experiments seems to exclude a specific interaction between one of the anions and either protein. Since the stability of soluble proteins is a linear function of N, this may point to an effect of the anions on the membranes that is unrelated to the effects on the surface tension of water. This may involve competition for charged sites on the protein and/or the membrane that are normally involved in electrostatic binding to the membrane in addition to hydrophobic binding (39). Alternatively, the ions could have direct effects on membrane lipid structure, as shown for pure phosphatidylethanolamine membranes (40). It could be easily conceived that such effects would indirectly influence the binding strength of peripheral membrane proteins. Further investigations will be necessary to clarify this point. REFERENCES 1. Carpenter, J. F., Crowe, J. H., and Arakawa, T. (1990) J. Dairy Sci. 73, 3627–3636. 2. Timasheff, S. N. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 67–97. 3. Bull, H. B., and Breese, K. (1980) Arch. Biochem. Biophys. 202, 116 –120. 4. Kusalik, P. G., and Svishchev, I. M. (1994) Science 265, 1219 – 1221. 5. Liu, K., Cruzan, J. D., and Saykally, R. J. (1996) Science 271, 929 –933. 6. Leberman, R., and Soper, A. K. (1995) Nature 378, 364 –366. 7. Parsegian, V. A. (1995) Nature 378, 335–336. 8. Hofmeister, F. (1888) Arch. Exp. Pathol. 24, 247–260. 9. Voet, A. (1937) Chem. Rev. 20, 169 –179. 10. Melander, W., and Horvath, C. (1977) Arch. Biochem. Biophys. 183, 200 –215. 11. Matthiijs, H. C. P., Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 12154 –12158. 12. Wolter, F. P., Schmitt, J. M., Bohnert, H. J., and Tsugita, A. (1984) Plant Sci. Lett. 34, 323–334.
13. Strotmann, H., and Bickel-Sandko¨tter, S. (1984) Annu. Rev. Plant Physiol. 35, 97–120. 14. Karplus, P. A., Daniels, M. J., and Herriott, J. R. (1991) Science 251, 60 – 66. 15. Andersen, B., Scheller, H. V., and Moller, B. L. (1992) FEBS Lett. 311, 169 –173. 16. Hatefi, Y., and Hanstein, W. G. (1969) Proc. Natl. Acad. Sci. USA 62, 1129 –1136. 17. Hatefi, Y., and Hanstein, W. G. (1974) Methods Enzymol. 31, 770 –790. 18. Kamienietzky, A., and Nelson, N. (1975) Plant Physiol. 55, 282– 287. 19. Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967–973. 20. Gra¨f, R., Lepier, A., Harvey, W. R., and Wieczorek, H. (1994) J. Biol. Chem. 269, 3767–3774. 21. Kaiser, G., Martinoia, E., Schmitt, J. M., Hincha, D. K., and Heber, U. (1986) Planta 169, 345–355. 22. Lepier, A., Gra¨f, R., Azuma, M., Merzendorfer, H., Harvey, W. R., and Wieczorek, H. (1996) J. Biol. Chem. 271, 8502– 8508. 23. Hincha, D. K., Sonnewald, U., Willmitzer, L., and Schmitt, J. M. (1996) J. Plant Physiol. 147, 604 – 610. 24. Sieg, F., Schro¨der, W., Schmitt, J. M., and Hincha, D. K. (1996) Plant Physiol. 111, 215–221. 25. Hincha, D. K., Heber, U., and Schmitt, J. M. (1985) Biochim. Biophys. Acta 809, 337–344. 26. Hincha, D. K., and Schmitt, J. M. (1992) J. Plant Physiol. 140, 236 –240. 27. Arnon, D. J. (1949) Plant Physiol. 24, 1–15. 28. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965) Immunochemistry 2, 235–254. 29. Hincha, D. K., and Schmitt, J. M. (1995) in Methods in Molecular Biology, Vol. 38, Cryopreservation and Freeze-Drying Protocols (Day, J. G., and McLellan, M. R., Eds.), pp. 71– 80, Humana, New Jersey. 30. Schmitt, J. M., Schramm, M. J., Pfanz, H., Coughlan, S., and Heber, U. (1985) Cryobiology 22, 99 –104. 31. Volger, H., Heber, U., and Berzborn, R. J. (1978) Biochim. Biophys. Acta 511, 455– 469. 32. Hightower, K. E., and McCarty, R. E. (1996) Biochemistry 35, 10051–10057. 33. Santarius, K. A. (1984) Planta 161, 555–561. 34. Hincha, D. K., and Schmitt, J. M. (1988) Plant Cell Environ. 11, 41– 46. 35. Santarius, K. A. (1984) Physiol. Plant. 61, 591–598. 36. Mollenhauer, A., Schmitt, J. M., Coughlan, S., and Heber, U. (1983) Biochim. Biophys. Acta 728, 331–338. 37. Hincha, D. K., and Schmitt, J. M. (1985) Biochim. Biophys. Acta 812, 173–180. 38. Somero, G. N. (1992) in Water and Life (Somero, G. N., Osmond, C. B., and Bolis, C. L., Eds.), pp. 3–18, Springer, Berlin. 39. Telfer, A., Barber, J., and Jagendorf, A. T. (1980) Biochim. Biophys. Acta 591, 331–345. 40. Sanderson, P. W., Lis, L. J., Quinn, P. J., and Williams, W. P. (1991). Biochim. Biophys. Acta 1067, 43–50.
Vol. 358, No. 2, October 15, pp. 385–390, 1998 Article No. BB980866
Release of Two Peripheral Proteins from Chloroplast Thylakoid Membranes in the Presence of a Hofmeister Series of Chaotropic Anions1 Dirk K. Hincha2 Institut fu¨r Pflanzenphysiologie und Mikrobiologie, Freie Universita¨t, Ko¨nigin Luise Strasse 12-16, D-14195 Berlin, Germany
Received June 30, 1998
The dissociation of two peripheral spinach (Spinacia oleracea L.) thylakoid membrane proteins, coupling factor CF1 and ferredoxin-NADP1-oxidoreductase, in the presence of chaotropic sodium salts has been investigated, using monospecific antibodies against the proteins as probes. Release of both proteins followed the Hofmeister series of anions (Cl2 < NO32 < Br2 < I2 < SCN2). In mixtures, the different salts had an additive effect. In addition, there were also qualitative differences in the action of the anions, such that NaI and NaSCN led to a different concentration and time dependence of the dissociation of the peripheral proteins from thylakoids. An analysis of the temperature dependence of protein release showed that the more chaotropic ions reduced the activation energy required for the dissociation of the proteins from their binding sites on the membrane. The addition of sugars (glucose, sucrose, or trehalose) reduced the amount of protein released from the membranes in the presence of NaI or NaSCN. © 1998 Academic Press
Key Words: coupling factor CF1; ferredoxin-NADP1oxidoreductase; lyotropy; protein solubility; salt effects; Spinacia oleracea.
The stability and solubility of proteins in aqueous solutions has received widespread interest, as it is important both from a theoretical and from a practical point of view (see, e.g., 1 for a review). It has been recognized for a long time that the solubility of proteins can be greatly influenced by cosolutes, which can lead to reversible precipitation or to reversible or irrevers1 Financial support was provided by the Deutsche Forschungsgemeinschaft through the Heisenberg program. 2 Fax: 149 30 838 4313. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
ible denaturation. The addition of other compounds, such as sugars, can counteract the effects of denaturants. In general, substances that destabilize proteins in solution and increase the solubility of proteins are called chaotropes, while cosolutes that stabilize protein structure are called kosmotropes. The stability of soluble proteins is usually measured as the temperature of denaturation, and the effects of cosolutes on the denaturation temperature are explained in terms of the “preferential exclusion theory” (see 2 for a review). Briefly, this theory states that kosmotropic cosolutes are preferentially excluded from the hydration shell of proteins, leading to a preferential hydration of the protein surface. This makes the exposure of hydrophobic amino acids from the interior of the protein to the solution energetically more unfavorable. This effect is based on an increase in the surface tension of water and consequently the interfacial tension between water and the protein. Chaotropic salts, on the other hand, destabilize proteins, because they have the opposite effect (3). The same phenomenon can also be interpreted in terms of water structure. The cluster structure of water (4, 5) is stabilized by kosmotropic solutes (structure makers, salting-out effect on proteins) and destabilized by chaotropic solutes (structure breakers, salting-in effect on proteins) (6, 7). Of course, in reality there is a continuum between the two extremes described above. This has first been recognized by Hofmeister in his classical paper, where he described the effects of a wide variety of salts on the solubility of egg white proteins (8). These effects of salts on protein solubility have later been quantified by Voet, who assigned a lyotropic number (N) to each ion (9). It has been shown that, among other physical characteristics of aqueous solutions, also the change in surface tension upon addition of different salts is a linear function of N (10). In the present investigation, 385
386
DIRK K. HINCHA
N values have been used as a convenient means to quantitatively compare the effects of a Hofmeister series of sodium salts on two peripheral thylakoid membrane proteins. Most of the literature on the effects of solutes on proteins deals with soluble proteins. It seems interesting to extend this research to include peripheral membrane proteins, which are normally membrane bound but are soluble in the absence of detergent once they are dissociated from their binding sites. The two proteins that were studied for this purpose are the peripheral part of the chloroplast thylakoid ATPase (chloroplast coupling factor CF1) and the ferredoxin-NADP1-oxidoreductase (FNR; EC 1.18.1.2).3 Both proteins are localized on the outer surface of the chloroplast thylakoid membrane, protruding into the chloroplast stroma in vivo and into the incubation medium after isolation of the thylakoid membranes. Treatment of thylakoids with dilute EDTA solutions released a large part of both proteins, while rebinding only took place in the presence of divalent cations (11, 12). This indicates that tight binding occurs by hydrophobic interaction. Negative charges on proteins and membrane have to be screened by cations to reduce electrostatic repulsion (13). CF1 is a large complex of five different subunits with a stoichiometry of a3b3gde, bound hydrophobically to the CF0 transmembrane proton channel (13). FNR is a monomeric protein (14) that is bound hydrophobically to the PSI-E subunit of photosystem I (15). The possibility of using chaotropic salts to dissociate peripheral proteins from a variety of membranes has been described before (16, 17) and this technique has been used to remove CF1 from thylakoids (18), as well as the peripheral part of other ATPases from their binding sites (19 –22). These were, however, no systematic, quantitative studies, similar to those on soluble proteins, but, rather, chaotropic salts were used as a tool to study the respective proteins or membranes. In the present report, the effects of a Hofmeister series of chaotropic sodium salts on the dissociation of CF1 and FNR from their hydrophobic binding sites on thylakoid membranes were analyzed as a contribution to our understanding of the stability properties of proteins. MATERIALS AND METHODS Plant material. Spinach plants (Spinacia oleracea L. cv. Monnopa) were grown in a growth chamber under controled conditions as described recently (23, 24). Proteins and antibodies. FNR and CF1 were isolated from spinach thylakoid membranes as described by Wolter et al. (12), and monospecific, polyclonal antibodies were raised against the native proteins in rabbits as described by Hincha et al. (25). Isolation and incubation of thylakoid membranes. Thylakoids were isolated from spinach leaves as described previously (26) and
3
Abbreviation used: FNR, ferredoxin-NADP1-oxidoreductase.
FIG. 1. Release of the peripheral proteins CF1 and FNR from thylakoid membranes. The membranes were incubated for 2 h at 0°C in the presence of 0.5 M of a Hofmeister series of sodium salts. The chaotropic properties of the salts were ranked according to their lyotropic number N (see text for details; N 5 10.0, NaCl; 11.3, NaBr; 11.6, NaNO3; 12.5, NaI; 13.3, NaSCN). In the case of NaI and NaSCN, the appropriate data from all experiments in this study were used to calculate the means and standard deviations from 21 samples each, measured over the course of several months, to give an indication of the overall variability of the data.
washed three times by centrifugation and resuspension in 10 mM MgCl2. The final membrane suspension was adjusted to 1 mg chlorophyll mL21. Chlorophyll was determined according to Arnon (27). Samples (200 mL) were incubated for 2 h at 0°C, unless otherwise specified in the figure legends, and contained 5 mM MgCl2, 25 mM Hepes (pH 7.8), and 500 mM of NaCl, NaNO3, NaBr, NaI, or NaSCN. Control samples were kept under the same conditions in the presence of 5 mM MgCl2, 25 mM Hepes (pH 7.8). Quantitation of protein release. After the incubation, the membranes were sedimented by centrifugation for 15 min at 16,000g and 4°C. CF1 and FNR were determined in the supernatants by quatitative single radial immunodiffusion in agarose gels (28) as described in detail before (25, 29). As a reference, an aliquot of the original thylakoid suspension was lysed in 2% Triton X-100 and a dilution series was applied to the same gels. From this calibration, values for 100% CF1 and FNR content of each thylakoid preparation were determined and release of the proteins was calculated relative to this total content. The amount of the proteins released from the membranes in control samples containing only MgCl2 and Hepes were set as 0% protein release, to calculate the percentage of the proteins released during incubation, due to the presence of the chaotropic salts. The data shown in the figures are means 6 S.D. from three parallel samples, unless otherwise specified in the figure legends. Where no error bars are visible, S.D. was smaller than the symbols. Where correlation coefficients (r) are indicated in the figures, the lines were fitted to the data by linear or exponential correlation analysis using the Macintosh Cricket Graph software.
RESULTS
Figure 1 shows that during an incubation for 2 h at 0°C both peripheral thylakoid proteins investigated can be dissociated from the membranes by chaotropic salts. The effectiveness of the different anions used in this study clearly follows the well-known Hofmeister series (8). In order to facilitate a quantitative comparison between the anions, they have been plotted ac-
HOFMEISTER EFFECTS ON PERIPHERAL MEMBRANE PROTEINS
FIG. 2. Release of CF1 and FNR from thylakoid membranes as a function of the lyotropic number N of the salts in the suspending medium (see the legend to Fig. 1 and the text for details). The different lyotropic numbers were achieved by mixing either NaCl and NaI or NaNO3 and NaSCN in different proportions. The final salt concentration during incubation was always 0.5 M.
cording to their lyotropic numbers N. These numbers have been determined from solubility studies with soluble proteins, i.e., proteins that are not normally associated with a membrane. For such proteins, solubility is a linear function of N (9). It is obvious from Fig. 1 that this linearity is not maintained when chaotropic ions are used to dissociate proteins from their binding sites on a membrane. Nevertheless, for both FNR and CF1 the effectiveness of the five different sodium salts investigated shows the same ranking as that for soluble proteins. The effects of the different anions are additive (Fig. 2), as has also been shown for soluble proteins (9). The whole range of N values was achieved by mixing different fractions of either NaCl (N 5 10) and NaI (N 5 12.5), or NaNO3 (N 5 11.6) and NaSCN (N 5 13.3) to a final total concentration of 0.5 M. It can be seen that the release of FNR and CF1 showed the same dependence on N, regardless of whether the pure salts (Fig. 1) or mixtures of different salts (Fig. 2) were used. However, the effects of the investigated anions on the dissociation of peripheral membrane proteins were also qualitatively different and not only quantitatively. Figure 3 shows the dependence of the solubilization of CF1 and FNR on the concentration of either NaI or NaSCN during incubation. The release of both proteins from thylakoids showed a linear dependence on the concentration of NaSCN over the whole concentration range investigated (Fig. 3B). In the presence of NaI, on the other hand, such a linear dependence was only found at concentrations above approximately 100 mM (Fig. 3A). An increase in the concentration of NaI below 100 mM caused a disproportionately large increase in the dissociation of both proteins, perhaps indicating a more specific interaction at low concentrations. The two anions also led to a different time dependence of protein release (Fig. 4). The amount of both
387
proteins that was released from the membranes increased as a linear function of time in the presence of NaSCN (Fig. 4B). In the presence of NaI, protein release showed kinetics that could be fitted more accurately with exponential curves (Fig. 4A) than with linear curves (r 5 0.99 for both proteins vs r 5 0.95 for FNR and 0.97 for CF1). The curves for both CF1 and FNR could be linearized in a double-reciprocal plot (Fig. 4A, inset). From this plot the maximum release at infinite incubation time in the presence of 0.5 M NaI was calculated as 18.9% for CF1 and 20.4% for FNR. This is similar to the values found after a 2-h incubation (Fig. 1), indicating that saturation kinetics are consistent with the data obtained in other experiments. To gain more insight into the physical basis of the action of chaotropic ions, the temperature dependence of the dissociation of FNR and CF1 was determined between 0 and 30°C. In order to minimize the effects of the different kinetics of protein release in the presence of different salts (Fig. 4), the incubation time was reduced to 1 h. The resulting data were analyzed in the form of Arrhenius plots (Fig. 5). It can be seen that linear curves were obtained in the presence of all five salts for both FNR (Fig. 5A) and CF1 (Fig. 5B). The slopes of these regression lines, however, varied between the different salts. Figure 6 shows that the slopes of all Arrhenius plots shown in Figure 5 were a linear function of the lyotropic number N of the salts. Since the slope of an Arrhenius plot is a direct measure of the activation energy of the observed process, it can be concluded from these data that the presence of chaotropic salts reduces the energy barrier for the dissociation of the proteins from their binding sites on the membranes. It has been shown before that the presence of sugars can ameliorate the effects of chaotropic ions and other denaturants, such as urea or guanidinium hydrochloride, on the stability of soluble proteins. Figure 7 shows that the same is also true for peripheral membrane
FIG. 3. Dependence of the release of CF1 and FNR from thylakoid membranes on the concentration of NaI or NaSCN in the incubation solution. The data are means 6 S.D. of six samples from two independent experiments.
388
DIRK K. HINCHA
DISCUSSION
The data presented in this paper show some interesting similarities and differences between soluble proteins, or protein complexes, and peripherally bound membrane proteins. First of all, it is noteworthy that the simple monomeric protein FNR and the multisubunit protein complex CF1 showed a very similar behavior under all conditions investigated in this study. This can be explained by the similar binding mechanism. Both CF1 and FNR are bound hydrophobically to other membrane proteins (11, 13, 15). Earlier work on CF1 has shown that in the presence of chaotropic agents, all subunits are released from the membranes (30, 31).
FIG. 4. Time dependence of the release of CF1 and FNR from thylakoids in the presence of 0.5 M NaI (A) or NaSCN (B) at 0°C. The inset shows the fit of the data in (A) to linear curves in a double reciprocal plot.
proteins. Loss of CF1 and FNR from thylakoids was greatly reduced when a sugar was present in the incubation solution in addition to either NaI or NaSCN. There were no systematic differences between the effects of the three sugars that were investigated, glucose, sucrose, and trehalose. In all cases, loss of the proteins from the membranes decreased as a linear function of the sugar concentration. While the presence of sugars in the solutions clearly stabilized the proteins on the membranes, the addition of a soluble protein (bovine serum albumin up to 10 mg mL-1) had no influence on protein release in the presence of 500 mM NaI and NaSCN (data not shown). Likewise, the substrates NAD1 or NADP1 (up to 5 mM) had no effect on the release of FNR, and ADP or ATP (up to 5 mM) had no effect on the release of CF1, in the presence of NaI or NaSCN (data not shown).
FIG. 5. Arrhenius plots of the temperature dependence of the release of FNR (A) and CF1 (B) from thylakoids. Samples were incubated for 1 h in the presence of 0.5 M of the different Hofmeister salts at temperatures between 0 and 30°C. The logarithm of the percentage of the different proteins released from the membranes during incubation is plotted as a function of the reciprocal incubation temperature in Kelvin.
HOFMEISTER EFFECTS ON PERIPHERAL MEMBRANE PROTEINS
FIG. 6. Correlation between the slopes of the Arrhenius plots shown in Fig. 5 with the lyotropic numbers of the different salts (compare Fig. 1) present during incubation (see the legend to Fig. 5 for details). The solid symbols represent data obtained from FNR measurements and the open symbols data from CF1. The slopes of the Arrhenius plots are a direct measure for the activation energy required to release the proteins from their binding sites on the membranes.
Once released from the membrane, CF1 is very coldlabile and dissociates rapidly. This dissociation is largely prevented in the presence of micromolar concentrations of ATP or ADP (32). In the presence of the chaotropic salts NaI or NaSCN, on the other hand, none of the substrates for FNR or CF1 provided any stabilization against dissociation of the proteins from the membranes. This is different from the situation during freezing of thylakoids in the presence of NaCl, where ATP, but not ADP, was able to stabilize CF1 on the membranes (33, 34). Under these conditions FNR was not released from the membranes (25). It can therefore be assumed that the conditions during freezing in the presence of NaCl were considerably milder than during incubation at 0°C in the presence of either NaI or NaSCN. Therefore, under those milder conditions a certain degree of substrate-induced stabilization of CF1 on the membranes occurs. This is in accordance with the fact that Santarius (35) found some protection of CF1 from membrane dissociation in the presence of NaCl under nonfreezing conditions. In general, the dissociation of proteins from thylakoids in the presence of chaotropic anions follows the Hofmeister series, both during freezing (36, 37) and in unfrozen solutions. This is similar to the effects of such salts on protein solubility in general. However, a quatitative analysis of a series of sodium salts revealed that, contrary to the situation with soluble proteins, the effect of the salts on the binding of peripheral membrane proteins was not a linear function of the lyotropic number N, assigned to the different anions (Fig. 1; compare 9). Nevertheless, the effects of the salts in
389
mixtures were strictly additive (Fig. 2) when analyzed on the basis of their lyotropic numbers. From Fig. 4 it is evident that the samples were not in equilibrium after 1 h of incubation. This, however, in no way invalidates the conclusions drawn above, as longer incubation times, which might result in an equilibrium situation, would necessarily lead to even stronger deviations from linearity. An analysis of the temperature dependence of the dissociation of CF1 and FNR from thylakoids in the presence of the different salts (Fig. 5) revealed that the activation energy for the release of both proteins was a linear function of N (Fig. 6). This reduction in activation energy in the presence of chaotropic anions is most likely related to the reduction in the interfacial energy between water and protein, affected by the salts (3), as the surface tension of water is again a linear function of N (10). The crucial role of surface tension for the stability of the binding of peripheral membrane proteins is emphasized by the fact that sugars reduce the dissociation of CF1 and FNR in a concentration-dependent manner (Fig. 7). It has been shown for soluble proteins that sucrose and glucose increase protein stability in solution by increasing the surface-tension of water and that kosmotropes can compensate the effects of chaotropes in solution (see 2 and 38 for reviews). Also, the stabilizing effect of sugars (Fig. 7) shows that the release of peripheral proteins in the presence of salts is
FIG. 7. Sugars protect FNR and CF1 from the effects of the chaotropes NaI and NaSCN. Thylakoids were incubated for 2 h at 0°C in the presence of 0.5 M of the indicated salts and increasing concentrations of glucose, sucrose, or trehalose. The correlation coefficients of the linear curves fitted to the data were between 0.99 and 0.95.
390
DIRK K. HINCHA
not releated to the effects of osmotic dehydration, as this is further increase by the addition of sugars. Still, changes in the surface tension of water are probably not the only mechanism through which chaotropic ions destabilize the binding of peripheral membrane proteins. This is indicated by the fact that surface tension is a linear function of N (10), while the dissociation of CF1 and FNR is not (Figs. 1 and 2). More detailed investigations with NaI and NaSCN support the idea of more specific effects of the different anions. Both the concentration and the time dependence of the release of CF1 and FNR show marked differences between the two anions (Figs. 3 and 4). The fact that FNR and CF1 behaved very similar in these experiments seems to exclude a specific interaction between one of the anions and either protein. Since the stability of soluble proteins is a linear function of N, this may point to an effect of the anions on the membranes that is unrelated to the effects on the surface tension of water. This may involve competition for charged sites on the protein and/or the membrane that are normally involved in electrostatic binding to the membrane in addition to hydrophobic binding (39). Alternatively, the ions could have direct effects on membrane lipid structure, as shown for pure phosphatidylethanolamine membranes (40). It could be easily conceived that such effects would indirectly influence the binding strength of peripheral membrane proteins. Further investigations will be necessary to clarify this point. REFERENCES 1. Carpenter, J. F., Crowe, J. H., and Arakawa, T. (1990) J. Dairy Sci. 73, 3627–3636. 2. Timasheff, S. N. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 67–97. 3. Bull, H. B., and Breese, K. (1980) Arch. Biochem. Biophys. 202, 116 –120. 4. Kusalik, P. G., and Svishchev, I. M. (1994) Science 265, 1219 – 1221. 5. Liu, K., Cruzan, J. D., and Saykally, R. J. (1996) Science 271, 929 –933. 6. Leberman, R., and Soper, A. K. (1995) Nature 378, 364 –366. 7. Parsegian, V. A. (1995) Nature 378, 335–336. 8. Hofmeister, F. (1888) Arch. Exp. Pathol. 24, 247–260. 9. Voet, A. (1937) Chem. Rev. 20, 169 –179. 10. Melander, W., and Horvath, C. (1977) Arch. Biochem. Biophys. 183, 200 –215. 11. Matthiijs, H. C. P., Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 12154 –12158. 12. Wolter, F. P., Schmitt, J. M., Bohnert, H. J., and Tsugita, A. (1984) Plant Sci. Lett. 34, 323–334.
13. Strotmann, H., and Bickel-Sandko¨tter, S. (1984) Annu. Rev. Plant Physiol. 35, 97–120. 14. Karplus, P. A., Daniels, M. J., and Herriott, J. R. (1991) Science 251, 60 – 66. 15. Andersen, B., Scheller, H. V., and Moller, B. L. (1992) FEBS Lett. 311, 169 –173. 16. Hatefi, Y., and Hanstein, W. G. (1969) Proc. Natl. Acad. Sci. USA 62, 1129 –1136. 17. Hatefi, Y., and Hanstein, W. G. (1974) Methods Enzymol. 31, 770 –790. 18. Kamienietzky, A., and Nelson, N. (1975) Plant Physiol. 55, 282– 287. 19. Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967–973. 20. Gra¨f, R., Lepier, A., Harvey, W. R., and Wieczorek, H. (1994) J. Biol. Chem. 269, 3767–3774. 21. Kaiser, G., Martinoia, E., Schmitt, J. M., Hincha, D. K., and Heber, U. (1986) Planta 169, 345–355. 22. Lepier, A., Gra¨f, R., Azuma, M., Merzendorfer, H., Harvey, W. R., and Wieczorek, H. (1996) J. Biol. Chem. 271, 8502– 8508. 23. Hincha, D. K., Sonnewald, U., Willmitzer, L., and Schmitt, J. M. (1996) J. Plant Physiol. 147, 604 – 610. 24. Sieg, F., Schro¨der, W., Schmitt, J. M., and Hincha, D. K. (1996) Plant Physiol. 111, 215–221. 25. Hincha, D. K., Heber, U., and Schmitt, J. M. (1985) Biochim. Biophys. Acta 809, 337–344. 26. Hincha, D. K., and Schmitt, J. M. (1992) J. Plant Physiol. 140, 236 –240. 27. Arnon, D. J. (1949) Plant Physiol. 24, 1–15. 28. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965) Immunochemistry 2, 235–254. 29. Hincha, D. K., and Schmitt, J. M. (1995) in Methods in Molecular Biology, Vol. 38, Cryopreservation and Freeze-Drying Protocols (Day, J. G., and McLellan, M. R., Eds.), pp. 71– 80, Humana, New Jersey. 30. Schmitt, J. M., Schramm, M. J., Pfanz, H., Coughlan, S., and Heber, U. (1985) Cryobiology 22, 99 –104. 31. Volger, H., Heber, U., and Berzborn, R. J. (1978) Biochim. Biophys. Acta 511, 455– 469. 32. Hightower, K. E., and McCarty, R. E. (1996) Biochemistry 35, 10051–10057. 33. Santarius, K. A. (1984) Planta 161, 555–561. 34. Hincha, D. K., and Schmitt, J. M. (1988) Plant Cell Environ. 11, 41– 46. 35. Santarius, K. A. (1984) Physiol. Plant. 61, 591–598. 36. Mollenhauer, A., Schmitt, J. M., Coughlan, S., and Heber, U. (1983) Biochim. Biophys. Acta 728, 331–338. 37. Hincha, D. K., and Schmitt, J. M. (1985) Biochim. Biophys. Acta 812, 173–180. 38. Somero, G. N. (1992) in Water and Life (Somero, G. N., Osmond, C. B., and Bolis, C. L., Eds.), pp. 3–18, Springer, Berlin. 39. Telfer, A., Barber, J., and Jagendorf, A. T. (1980) Biochim. Biophys. Acta 591, 331–345. 40. Sanderson, P. W., Lis, L. J., Quinn, P. J., and Williams, W. P. (1991). Biochim. Biophys. Acta 1067, 43–50.