Interaction of bovine S100 protein with detergents and phosphatidylcholine vesicles

Btochimica et Biophysica Acta. 703 (1982) 241-246

241

Elsevier Biomedical Press BBA 31121

INTERACTION OF BOVINE SI00 PROTEIN W I T H D E T E R G E N T S AND P H O S P H A T I D Y L C H O L I N E VESICLES R O B E R T O D. M O R E R O * and G R E G O R I O WEBER

Department of Biochemistry. School of Chemical Sctences. Unit;erst(v of lllmois. Urbana. IL 61801 ( U.S.A. ) (Received September 29th, 1981 )

Key word~: Protein binding," Fluorescence polartcation; SIO0 protein; Phosphattd)'h'holine veswle

The interaction of bovine SI00 protein with sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), octaethyleneglycol dodecyl ether (OEGDE), and egg phosphatidylcholine vesicles was followed by fluorescence methods. The monomeric forms of both anionic (SDS) and cationic (CTAB) detergents interact with the protein, producing changes in the tryptophan and tyrosine fluore~ence similar to those seen when the protein is exposed to low or high pH. No additional interaction was detected between the SI00 protein and the micelles for the charged detergents. No interactions were detected with either the monomeric form or the micelles of the non-ionic detergent OEGDE. Fluore~ence polarization studies of SI00 protein photo-labelled with azidonaphthalene sulfonate show that the protein binds to egg phosphatidylcholine vesicles, even at neutral pH and in the absence of bivalent or monovalent cations and that the strength of the binding of the protein to the vesicles increases at acid pH. At neutral pH binding of the intact protein results in a 90% increase in tryptophan fluorescence and 60% increase of tyrosine fluorescence. The set of observations supports the ty.pe of interaction between SI00 and lipid vesicles postulated by Calissano and coworkers (Calissano, P., Alema, S. and Fasella, P. (1974) Biochemistry 13, 4553-4560) to explain the increased leakage of rubidium induced by SI00. Superficial interactions of this type may be at the basis of the actions of many peripheral membrane proteins.

Many proteins are immersed deeply into the lipid bilayer with direct contact between the non-polar amino acids of the protein and hydrocarbon interior of the membrane, but other proteins are known to be associated with the membrane primarily through ionic interactions, e.g., the ATPase of Streptococcus fecalis [1], and the basic protein of myelin [2]. * International Postdoctoral Fellow from Department of Health, Education, and Welfare, Public Health Service. Present address: lnstituto de Quimica Biol6gica, Facultad de Bioquimica, Quimica y Farmacia, Universidad Nacional de Tucumb.n, San Miguel de Tucuman, Argentina. Abbreviations: SDS, sodium dodecyl sulfate: CTAB, cetyltrim e t h y l a m m o n i u m bromide; O E G D E , octaethyleneglycol dodecyl ether. 0167-4838/82/0000-0000502.75 ~ 1982 El.~vier Biomedical Press

Non-ionic detergents have been proposed as probes for hydrophobic binding sites on proteins [3,4], as these compounds combine poorly with typical water-soluble proteins, but interact strongly with membrane proteins which can act as a nucleus for formation of micelles [5]. On the other hand. ionic detergents such as SDS and CTAB drastically alter the conformation of the proteins with either hydrophilic or hydrophobic surfaces. In most cases, binding of the detergents occurs below the critical micelle concentration, and no compelling evidence for the binding of micelles by hydrophilic proteins has been presented. The brain protein, SI00, has been found in the soluble fraction, but also in very significant proportion in the microsomal fraction bound to the

242 mcmhranes [6,7]. It has also been show,'n that the mentioned protein interacts with lipid membranes. liposon3es [8] and with synaptosc, mal particles [9]. In this paper we report a study of the protein intrinsic fluorescence, and also of its conjugate with I-azidonaphthalene-5-sulfonate, during interaction of the purified bovine SIO0 with selected detergents and phosphalidylcholine vesicles.

Experimen|al procedures MateriaLs Sodium dodecyl sulfate (SDS) and cetyltrimethyl ammoniunl bromide (('TAB) were purchased from Sigma Chemical Co. Octaethyleneglycol dodecyl ether (OEGDE) was purchased from Nikkol Co. C/'okio. Japan). LPhosphatidylcholine from egg yolk type IX-E was obtained from Sigma Chemical (_'o. and purified bv the procedure of Singleton et al. [10]. Tris buffer, reagent grade, was obtained from Sigma Chemical Co. All other reagents were analytical grade. These were used without further purification.

Metho~Ls S100 was prepared and labelled with Iazidonaphthalene-5-sulfonate by the procedure described in the preceding paper [18]. Protein concentration was measured using the molar absorption coefficient E 8.260 mol i cm t [11]. or bv the method of Lowry et al. [12]. Phospholipid concentration was determined by' measuring inorganic phosphorus by' the method of Bartlett [13]. Fluorescence spectra, polarization (P), lifetime (r), and rotational relaxation times (p). were obtained by using equipment and methods described in the preceding paper. All experiments were done in 20 mM Tris-HCl buffer, pH 7.3, or in the same buffer to which different amounts of detergents or egg phosphatidylcholine vesicles had been added. Other pl! values were obtained by the addition of small amounts of 2 M HCI from a Hamilton syringe.

Preparation of phosphatid.vk'holine vesicles Vesicles were prepared by ultrasonic irradiation of phosphatidylcholine suspension in 20 mM TrisH('I buffer, pH 3.0. A 20 KHz Branson sonifier

was used at 30 W output on 5 ml solution under a nitrogen atmosphere for 40 rain while the temperature was maintained between 0 5°C by the use of an ice bath. The sonicated phospholipids suspension was filtered on a Sepharose 4B column (22 × 1.6 cm), equilibrated and eluted with 20 mM Tris-HCI buffer, pH 7.3, to remove large unfractionated particles [14]. Critical micelle concentration was measured using the fluorescence enhancement of anilinonaphthalene sulfonate, excited at 360 nm, which accompanies its association with CTAB or O E G D E micelles. The critical micelle concentration of SDS was measured by the same procedure using perylene instead of anilinonaphthalene sulfonate. The value of 1.0 mM was obtained for SDS. similar to that reported by Emerson and Holtzer [15].

Resulls Effect of detergents on the intrinsic .fluorescence and fluorescence polari:ation of the S I O0 protein Fig. I shows typical intrinsic fluorescence spectra of bovine SI00 protein at neutral pH (pl-! 7.3) in the absence of any detergent, and in the presence of an anionic (SDS). a cationic (CTAB), and a no,a-ionic ( O E G D E ) detergent, respectively, at concentrations higher than the critical mieelle concentration. As can be seen in Fig. 2, SDS increased 2-fold the tryptophan fluorescence and 5-fold the tyrosine fluorescence. The presence of CTAB at concentrations higher than the critical micelle concentration produced an increase of almost 7-fold in tryptophan fluorescence, but tvrosine fluorescence was not changed much. The non-ionic detergent, OEGDE. did not affect the intrinsic fluorescence of SI00 protein and both tryptophan and tvrosine contributions resemble those of the fluorescence of the native protein. The changes in the tryptophan and tyrosine fluorescence of S100 protein, as a function of the detergent concentration, are shown in Fig. 2. As the concentration of SI)S was increased, both tyrosine and tryptophan fluorescence increased, giving a typical sigmoidal curve (Fig. 2A). At the ionic strength employed in these experiments the critical micelle concentration of SDS is about I • 10 ~ M, so that the fluorescence changes measured were in the range where the detergent concentra-

243 OEGDE

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Fig 2. Plots of the total intrinsic fluorescence ('--' "~-). tryptophan fluore.,,cence ( 0 C)). and tvrosine fluorescence (@ @) of SI(X) protein as a function of detergent concentration. The arrows indicate the critical micellc concentration of each amphiphile. Buffer: 20 mM Tris-||('l, ptl 7.4.

I

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ta.I u Fig. 3. O E G D E did not c h a n g e the p o l a r i z a t i o n v a l u e at a n y c o n c e n t r a t i o n tested (Fig. 3B): b o t h ionic d e t e r g e n t s , S D S a n d C T A B , i n c r e a s e d the

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DS3 mM

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350 WAVELENGTH

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Fig. I. Intrinsic fluorescence spectra of bovine SI00 protein in the absence of detergents ( . . . . . ), and in the presence of: 2.0 mM SDS ( ), 2.5 mM ('TAB ( . . . . . . ) or 0.05~. OEGI)E ( . . . . . ). Buffer: 20 mM Tris-HCI, pll 7.3. Excitation vqp.,elength: 275 nm. Protein concentration: 0.6 mg/ml.

0.12

0.18 0.16

tion j u s t r e a c h e d the critical micelle c o n c e n t r a t i o n . T h e large i n c r e a s e in t r y p t o p h a n f l u o r e s c e n c e , on a d d i t i o n of C T A B , was also a typical s i g m o i d a l c u r v e with m i d p o i n t in the z o n e of the critical m i c e l l e c o n c e n t r a t i o n (Fig. 2C). T h e f l u o r e s c e n c e o f t y r o s i n e r e m a i n e d a l m o s t c o n s t a n t d u r i n g the e x p e r i m e n t . T h e n o n - i o n i c d e t e r g e n t ( O E G D E ) did n o t p r o d u c e any effect on the intrinsic fluor e s c e n c e o f S I 0 0 p r o t e i n e v e n at a c o n c e n t r a t i o n 2 - f o l d h i g h e r t h a n the critical micelle c o n c e n t r a t i o n (Fig. 2B). In o r d e r to establish w h e t h e r or not s o m e intera c t i o n b e t w e e n the p r o t e i n a n d the micelles o f the d e t e r g e n t s m e n t i o n e d a b o v e occurs, we s t u d i e d the d e p e n d e n c e o f the p o l a r i z a t i o n v a l u e s of the a z i d o c o m p o u n d - p r o t e i n c o n j u g a t e as a f u n c t i o n of the d e t e r g e n t c o n c e n t r a t i o n . T h e results are s h o w n in

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Fig. 3. The influence of detergent concentration on the fluorescence polarization values of I-azidonaphthalene-5-sulfonateSI00 conjugate. Protein concentration: 20 mg/ml. Buffer: 20 mM Tris-HC1, pl[ 7.3. Temperature: 25°C. Excitation wa'.elength: 340 nm The fluorescence was passed through a 2 M NaNO, filter.

244

polarization values parallel to the intrinsic fluorescence (compare Figs. 3A and 3C with 2A and 2C, respectively), when the detergent concentration was below the critical micelle concentration. The polarization values were maximum at a detergent concentration below the critical micelle concentration, and at a concentration larger than the critical micelle concentration the polarization valeus decreased, reaching even lower values than those obtained in the absence of detergents. The possible dissociation of the protein into subunits, when subjected to high concentration of detergents, was investigated by electrophoresis on cellulose acetate. The electrophoretic patterns of the protein ahme, and in the presence of SDS. CTABr or O E G D E at concentrations at least 2-fold higher than its corresponding critical micelle concentration, were determined. After exposure of the protein to the effect of 2.0 mM CTABr two bands were obtaiend. The non-ionic detergent, OEGDE, was unable to change the electrophoretic pattern, even at a concentration of 0.15% (not shown).

lTw interaction of SIO0 protein with phosphatidvhholine cesicles The interaction of SI00 protein with egg phosphatidylcholine was followed by measuring the polarization values of the protein labelled with l-azidonaphthalene-5-sulfonate. Fig. 4 shows the variation of the polarization value of the azido compound-Sl00 conjugate as a function of pH, in the presence and in the absence of phospholipid vesicles. Higher polarization values were obtained in the presence of vesicles than in their absence. Inset in Fig. 4, the difference between the curve in the presence of the vesicles and the control is plotted as a function of pH. The difference ( A P ) increased abruptly at pH values lower than 5.0. These results can be reasonably interpreted as the result of interaction between the SI00 protein and the vesicles that already occurs at neutral pH and becomes stronger as the pH is lowered. Table l shows the pH dependence of the lifetime and the rotational relaxation time of azidoSI00 conjugate in the presence of phosphatidylcholine vesicles and its respective controls. The lifetime of the protein fluorescence alone did not change significantly with pH, and the same was seen for the protein with phosphatidylcholine. Acid

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}Zig. 4. The influence of pH on the polarization fluorescence of I-5-azido compound-SI00 conjugate in the absence (O O). and in the presence ( O O ) of egg phosp h a t i d y l c h o l i n e vesicles. Protein concentration: 10 ,uM (200 / x g / m l ) . Phospholipid concentration: 2.8 m M (2.7 m g / m l ) . Buffer: 20 m M Tris-HCI. The pH was adjusted with 2.0 M IICI. Temperature: 25°C. Excitation wavelenghth: 340 nm. The fluorescence was passed through a 2 M N a N O 2 filter and 3-73 C o r n i n g filter. Inset: the difference of both curves l A p ) as a function of p i t .

TABLE I LIFETIME AND ROTATIONAL RELAXATION TIME OF AZIDO NAPIITHALENE-SI00 CONJUGATE AT DIFFERE N T pH V A L U E S IN T I l E P R E S E N C E O R IN T H E ABSENCE OF PHOSPHATIDYLCItOLINE VESICLES pH

7.3 6.(1 5.0 4.0 3.5 2.8

-- Vesicles

- Vesicles

r(ns)

o(ns)

r(ns)

1902 "- 0.08 19.16 ' 0.18 19.23 ~0.15 19.52-'.0.13 18.93 " 0.13 20. I I ." 0.0l

43 54 63 95 15(1 54

17.90 IR.59 18.77 17.59 17.42 19.36

o(ns) * 0.04 -0.03 :.0.04 • 0.07 ' 0.15 ' 0.28

6I 113 165 275

Lifetime ( r ) was measured by' m o d u l a t i o n at 3{1 MHz. Rotational relaxation time was calculated by the e q u a t i o n

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(I/PI'

1/'3)

(t/p

~/3)

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'~,ith p~ o b t a i n e d by e x t r a p o l a t i n g the l}errin plots.

245 T A B L E II E F F E C T OF P H O S P H A T I D Y L ( H O I . I N E VESICI.ES ON T H F INTRINSIC F L U O R E S C E N C E OF SI00 PROTEIN

6£ t"luorcscence {arbitra U units)

~ 5.0 z w Tryptophan Tyrosinc T~'ptophan + tyrosine

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- Vesicles

4 Vesicles

91Increase

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94 63 87

a

~2.(1

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zlo

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pH Fig. 5. The fluorescence of I-azidonaphthalene-5-sulfonatc-S100 protein conjugate in the absence ( 0 O), and in the presence ( O O I of egg phosphatidylcholine vesicles as a function of pH. Buffer: 20 m M Tris-HC1. The pH was adjusted by adding small volumes of 2.0 M HCI. Temperature: 25°C. The protein/phospholipid concentration ratio was as described in Fig. 4. Excitation wavelength: 340 n m

titration of the protein-lipid complex revealed an increasing difference in the rotational relaxation time as the pH decreased. At neutral pH a decrease of almost 50% in the rotational rate could be detected, while at pH 3.0 when the rotational relaxation time reached the maximum value, the difference between the control and the SI00-plus vesicles was a factor of five. To investigate further the nature of the SI00 protein phosphatidylcholine interaction, the fluorescence emission spectra of the azido-conjugate of SI00 protein and the tryptophan and tyrosine fluorescence of the intact protein in the presence of an excess of vesicles, were studied. The relative fluorescence emission of azido-protein conjugate in the presence and in the absence of phosphatidylcholine vesicles, as a function of pH, is shown in Fig. 5. There were no changes in the fluorescence emission from pH 7.5 to 4.5. Below pH 4.5 the fluorescence decreased significantly, and this decrease was greater in the presence of vesicles. Results shown in Figs. 4 and 5 indicate that two possible interactions may occur: one at neutral pH

with no change in the fluoresence emission of the azido-protein conjugate, and another at acid pH. It is interesting to remark that no shift in the maximum emission could be detected over the whole pH range tested (not shown). The integrated intrinsic fluorescence of SI00 in the presence and in the absence of vesicles at neutral pH, excited at 275 nm and 295 nm, is shown in Table 1I. The presence of phospholipid vesicles did not change the maxima of emission of tryptophan fluorescence, but the fluorescence intensity was enhanced; the percentage increase ranging from 80 to 100% in different experiments. Tyrosine fluorescence was also enhanced to the extent of about 70%.

Discussion The data presented in this paper indicate that the positive and the negative charged detergents produce the same effects as the large shifts in pH, both as regards the fluorescence changes and the dissociation into subunits; taken together with the absence of effects seen upon addition of the neutral detergent they permit the conclusion that the electrostatic repulsion among the subunits is necessary for the appearance of the effects. The association of the protein with vesicles of phosphatidylcholine is demonstrated by the increase in the rotational relaxation times (Table I) and in the fluorescence efficiency of tyrosine and tryptophan (Table ill. It is worth recalling that transferring the protein into a non-polar medium produces a very dramatic decrease in the rotational relaxation time (Oh= 16.5 ns in isobutanol as opposed to Ph = 185 ns at the same pH in water). This large

246

decrease, which must result from a disorganization of the structure to the extent of permitting large partial rotations on the nanosecond timescale, is replaced here by an increase in Ph- and is readily interpreted as resulting from assc, iation or complex formation with the vesicles without appreciable penetration of the protein into the non-polar medium. However. one must realize that this association could still have a profound effect on the time-dependent properties of the bilaver that occur on the timescale larger than the fluorescence emission. Without constituting direct proof, the present studies lend strong support to the hypothesis of Calissano et al. [8] that superficial association of the SI(X) protein with the lipid vesicles, rather than penetration into the lipid bilayer, is the cause of the increased leakage of Rb" or Ca:" from the vesicles upon addition of SIO0. Changes in the permeability of the lipid bilayer following this type of superficial association may indeed be at the basis of the properties of many peripheral proteins. In these associations the structural properties of the protein deduced from its behavior in water solutions may be irrelevant to their functionalism, and even those properties deduced from the average structure of the lipid-protein complex may be unable to explain the effect of the protein upon associated vesicles, like the leakage of cytochrome h 5 observed by Robinson and Tanford [16] or rohdopsin observed by Dufourcq and Faucon [I 7]. For this purpose one requires also to know the possible fluctuations of the structure over the time range in which the observed experimental effects (e.g., leakage) take place. The fluorescence methods permit us to explore only the time range of 10 m to 10 vs, and the development of methods able to follow fluctuations in the microseconds to milliseconds timescale are greatly

needed for understanding of the effective interaction of proteins with membranes.

Acknowledgements This investigation was supported in part by a Public Health Service International Research Fellowship (1-F05-tw-2224-01) and by a Grant G M 11223 from The National Institute of Health.

References 1 Abrans A. and Baron C. (1968) Bitx:hemistry 7, 501-506 2 I:,ylar t-.H.. Salk J., Beveridge G.C. and Brown LV. (1969) Arch. Bit~:hem. Biophys 132, 34-49 3 Helenius A. and Simons K. (1972) J. Biol. 247, 3656-3661 4 Makino S., Reynolds J.A. and Tanford C. (1973) J. Biol. ('hem. 248. 4926-4932 5 Sardet ('., Tardieu A. and I,uzzati V. ~1976) J. Mol. Biol. 105. 393-407 6 Moore B.W. (1965) Biochem. Biophys. Res. ('ommun. 19, 739-744 7 Rusca G., Calissano P. and Alema A. (1973) Brain Res. 49, 383 407 8 ('alissano P., Alema S. and Fassella P. (1974) Bicv,:hemistr,, 13, 4553 .4560 9 Donato R. ( 19761J. Neurochern. 27. 439 447 IO Singleton, W.S., Gra',. M.S., Brown, M.I,. and White, J.l,. (1965} J. Am. Oil ('hem. Soc. 42, 53-56 II Calissano P., Moore B.W. and Friesen A. (1969) Bit~,:hemistrv :g, 4318-4326 12 I,owry O.['1., Ro,,,ebrough N.J.. Farr A.I,. and Randall R.J. (1951) J. Biol. ('hem. 193, 265-275 13 Bartletl G.R. (1959) J. Biol. ('hem. 234. 466-468 14 lluang C'. (1969} Bituehemistry 8, 344-352 15 Emerson M.F. and Hohzer A. (19761 J. Ph'~'s. Chem. 69, 3718 3729 16 Robinson N.C. and "lanford C. (1975) J. Biol. Chem. 14. 360-378 17 I)ufourcq J. and Faucon J.[:. (1977) Biochim. Biophys. Acta 467. I I 1 I g Mc, rero. R.O. and Weber. (i, (1982) Biochim. Biophys. Acta 704, 231 240