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A model for ionic and hydrophobic interactions and hydrogen-bonding in sodium dodecyl sulfate-protein complexes
20
Bioehimi¢a et Biophvsica Acta 873 (1986) 20 26 Elsevier
BBA 32604
A model for ionic and hydrophobic interactions and hydrogen-bonding in s o d i u m d o d e c y i s u l f a t e - p r o t e i n c o m p l e x e s Per Lundahl, Eva Greijcr, Maria Sandberg, Susanna Cardell and Kjell-Ove Eriksson Institute of Biochemistry. Biomedical Center, Unit~ersi(v of Uppsala, P.O. Box 5 76, S-751 23 Uppsala (Sweden)
(Received April 23rd, 1986)
Key words: Sodium dodecyl sulfate protein complex: Membrane protein; Dodecyl sulfate-polypeptide hydrogen-bonding; Detergent protein interaction; Structural model
We propose a new hypothetical model for the structure of complexes between sodium dodecyl sulfate and proteins as described below: the detergent forms a flexible capped cylindrical miceile around which the hydrophilic segments of the polypeptide are helically wound. These polypeptide segments are attached by hydrogen bonds from the nitrogens of the peptide bonds to the sulfate oxygens of the detergent molecules. Cationic amino acid side-chains can also form ionic bonds with the sulfate groups. When the latter occurs the polypeptide assumes an a-helical conformation at the surface of the detergent cylinder in accordance with interpretations of circular dichroism measurements (Mattice, W.L., Riser, J.M. and Clark, D.S. (1976) BiochemistD' 15, 4264-4272). An essentially regular arrangement of one hydrogen bond per peptide bond and two per dodecyl sulfate monomer is consistent with the known binding ratio of approximately one detergent molecule per two amino acid residues and with the proportionality between the polypeptide molecular mass or the number of residues and the length of the complex (Reynolds, J.A. and Tanford, C. (1970) J. Biol. Chem. 245, 5161-5165). The hydrogen-bonded structure of our model also agrees with the finding that dodecyl sulfate associates much more readily with proteins than does tetradecyltrimethylammonium chloride (Nozaki, Y., Reynolds, J.A. and Tanford, C. (1974) J. Biol. Chem. 249, 4452-4459). The axial length of the structure we propose can be estimated at approximately 0.6 A per amino acid residue. Hydrophobic polypeptide segments, including membrane-spanning a-helices of integral membrane proteins, can be accommodated in the interior of the elongated dodecyi sulfate micelle. Therefore, not only water-soluble proteins but also some integral membrane proteins may form the proposed type of complex, in the case of glycosylated membrane proteins the sugar moieties will protrude from the surface of the rod-shaped complex. We have named our model 'the flexible helix ~.
Introduction
Sodium dodecyl sulfate (SDS) is used for solubilization and fractionation of amphiphilic proteins, for dissociation of protein complexes and quaternary protein structures and for molecular mass estimations by electrophoresis or gel chromatography [1 8], but no suitable model for the structure of an SDS-protein complex has been 0167-4838/86/$03.50
proposed. The detergent may interact with polypeptides both 'hydrophobically' and by ionic or hydrogen-bonding interactions with the head group. Non-hydrophobic interactions are suggested by the fact that hydrophilic proteins that lack disulfide bonds, either naturally or after reduction, bind SDS approximately in proportion to their molecular masses [9-12] although hydrophilic polypeptide segments cannot interact hy-
1986 Elsevier Science Publishers B.V. (Biomedical Division)
21
drophobically with the detergent molecules and do not bind non-ionic detergents [1,2,13-15]. Furthermore, the cationic detergent tetradecyltrimethylammonium chloride binds much less efficiently to polypeptides than does SDS, although the alkyl chain of the former detergent is longer than that of SDS. This was shown by Nozaki and coworkers [16], who stated: " W e are thus forced to conclude that the observed over-all advantage of the anionic detergent (SDS) ... must be taken to indicate that the head group plays a significant role in the mechanism of binding". In the present work we propose a new and detailed model for the structure of SDS-protein complexes. Our model seems plausible in the light of earlier experimental results. It includes hydrogen-bonding of non-charged and anionic amino acid residues and ionic bonding of cationic amino acid side-groups to sulfate groups on the surface of a cylindrical SDS micelle.
K°I
0.7
0.6
0
2 L 6 8 10 Dodecyt sutfate (raM)
Fig. 1, K d values for molecular-sieve chromatography of ovalbumin as a function of the SDS concentration in the eluent (gel filtration calibration protein, Pharmacia). Column: Superose 6 TM, 28x1 cm (Pharmacia). Eluent: 0.1 M sodium phosphate, pH 7.4, 1 mM EDTA, 1 mM dithioerythritol and the given concentration of SDS. Temperature: 21-24°C. For chromatography in the absence of SDS the proteins were solubilized in the eluent alone. Otherwise, 0.1 M SDS was included and the proteins were reduced and S-carboxymethylated. The data for 0.6-10 mM SDS are from Fig. 2 in Ref. 8. Major changes in K d value take place between 0 and 0.6 mM SDS and between 1.2 and 2.5 mM SDS, in agreement with data in Refs. 10 and 12.
Model for the structure of SDS-protein complexes
Hydrogen-bonding. Reduced water-soluble proteins bind SDS in approximate proportion to their molecular masses. Reported values are 1.4 [9,10], 1.1-2.2 [11] and 1.2-1.5 [12] g SDS per g protein for SDS concentrations above approx. 1.5 mM. The latter range corresponds to 0.96-1.2 SDS molecules per two amino acid residues for an average M, of 115 per residue. Binding or release takes place in two major steps as SDS is added or removed (Refs. 8, 10, 12 and Fig. 1). This binding ratio may reflect a stoichiometric binding of one detergent molecule per two amino acid residues. A hydrogen-bonding structure that gives the latter stoichiometry is illustrated in Fig. 2. Two hydrogen bonds attach a single sulfate head group of an SDS molecule to the nitrogens of peptide bonds Nos. n and n + 2. Another head group is attached to peptide bonds Nos. n + 1 and n + 3. The repetitive unit comprises four amino acid residues and two SDS molecules (Fig. 3A). Proline residues break the structure (Fig. 3B), but the ratio between the number of bound SDS molecules and the number of non-imino acid residues may be preserved (see legend to Fig. 3). The three-dimensional structure of the repetitive unit is illustrated in Fig. 4, in which the hydrogen bond angles are
a p p r o x . 160 °. I n t h i s w a y m o s t o f t h e p e p t i d e g r o u p n i t r o g e n s m a y b e h y d r o g e n - b o n d e d in a cooperative manner to the detergent molecules, w h i c h i n t e r a c t h y d r o p h o b i c a l l y as d e s c r i b e d b e low.
Hydrophobic interaction between detergent alkyl chains. W e a s s u m e t h a t t h e a l k y l c h a i n s o f t h e d e t e r g e n t i n t e r a c t h y d r o p h o b i c a l l y , e s s e n t i a l l y as i n a s p h e r i c a l S D S m i c e l l e [18]. H o w e v e r , m e a surements of intrinsic viscosity indicate that SDSprotein complexes are rod-shaped with an axial l e n g t h of a b o u t 0.74 ,~ p e r a m i n o a c i d r e s i d u e [19]. T h i s v a l u e w a s o b t a i n e d b y u s e o f t h e S i m h a
~) R 3 0 ...H-N-
,,s:,
O "°"
H
C-
C-
c:o
o
®
R4
N-
? ......
..
O,, I ~ ~O" " ~" 0'.-C I -R2 ~ S "O
I ...... C-N-C-C ii ® i O R~
I
.v
- N-H" ©
'.,.
Fig. 2. Proposed hydrogen-bonding between oxygens in the SDS head groups and nitrogens of peptide bonds. The figure shows a repetitive unit with two sulfate groups and four amino acid residues.
22
R~
~3
R,~
,,A_~ i ~ t n •
n "
l""t"
Rr
N i .
R6
R~
Rt
~,,_I-N L.~ L v , _ _ [;
.
"i""'I"
_
Fig. 3. Two consecutive units of the proposed hydrogen-bonded SDS-protein complex (cf. Fig. 2). (A) Regular structure with s - a m i n o acids. The filled circles represent the sulfate groups• (B) Structure interrupted by proline or hydroxyproline. A large proportion of this structure may be formed for instance by collagen. This protein contains approx. 10 mol% each of proline and hydroxyproline and binds approx• 1.4 g SDS per g protein [171. The number of residues per unit molecular mass is 26% higher for collagen than for average water-soluble proteins [17[. Therefore, the number of non-imino acid residues in collagen is nearly the same per unit molecular mass as in water-soluble proteins, and the normal weight per weight SDS binding [171 is consistent with the structure in panel B.
equation [20], which applies to rigid ellipsoids. Measurements of transient electric birefringence show an extended shape with a length of 0.6 _+ 0.2 ,~ (n = 6) per residue, as calculated from data in Ref. 21, Table III. Electro-optical data are con-
Fig. 4. The repetitive unit of Figs. 2 and 3. The polypeptide is white with the nitrogens of the peptide bonds numbered as in Fig. 2. Of the SDS molecules only the sulfur atoms are shown (black), The connections between the nitrogens and the sulfur atoms represent the hydrogen atoms as well as the bonded oxygen atoms. The hydrogen bond angles are approx. 160 ° .
sistent with a flexible structure of the complex [21,22]. Hydrophobic contact between the dodecyl chains in a flexible, rod-shaped complex of a length proportional to the number of amino acid residues is obtained by coiling the SDS-polypeptide complex (Fig. 3), thereby arranging the detergent into a cylindrical micelle (Fig. 5). This is only a description of the final structure, and does not imply any mechanism for the complex formation. Dense packing of the SDS molecules corresponds to a rod length of 0.32 A per residue (Fig. 5A). This is a lower limit, since the hydrogenbonded structure (Figs. 2-4) requires more space per detergent molecule. The rod length 0.74 A per amino acid residue [19] corresponds to the pitch of the polypeptide helices illustrated in Figs. 5B and
J
I
"
I
1 Fig. 5. Flexible helix models for the complex between SDS and a polypeptide. The polypeptide with SDS attached by hydrogen bonds (Fig. 3) is helically wound, forming a flexible, cylindrical structure with the polypeptide and the sulfate groups on the outside and the dodecyl chains in the interior. The radius of the detergent cylinder is approx. 16 ,~ (cf. Ref. 1,8), the outer radius 24 A as estimated from a detailed model, which shows about 40 amino acid residues per polypeptide turn (Fig. 6, below). (A) Complex with a polypeptide of the size of serum albumin, M r 66000. This model shows the lower limit of cylinder length and helix pitch: 13 A per turn or 0.32 A, per amino acid residue, as calculated by use of the cross-sectional area, 59 ~ 2 of SDS [18] and the assumption that the detergent molecules are densely packed. However, this maximal degree of packing is not consistent with the hydrogenbonded structure of Figs. 2-4. (B) Less dense packing with a helix pitch of 0.74 ,~ per amino acid residue as estimated by Reynolds and Tanford [19]. Due to the flexibility of this structure this value of the helix pitch may be too high, (C) Structure as in (B), except that a segment composed of 20-30 hydrophobic amino acid residues traverses the core of the detergent cylinder.
23 C. Since the c o m p l e x m a y be flexible, this estimate of the rod length is a p p r o x i m a t e . F u r t h e r details are given in the legend to Fig. 5.
Hydrophobic interaction between detergent alkyl chains and hydrophobic amino acid side-groups. F o r solubility reasons, h y d r o p h o b i c p o l y p e p t i d e segm e n t s should traverse the interior of the detergent micelle (Fig. 5C). M a n y water-soluble p r o t e i n s c o n t a i n only short h y d r o p h o b i c sequences, whereas some m e m b r a n e proteins c o n t a i n h y d r o p h o b i c m e m b r a n e - s p a n n i n g a-helices that include 2 0 - 3 0 a m i n o acid residues [23 25], a n u m b e r which corr e s p o n d s to 0.5-0.75 turn of the p o l y p e p t i d e helix (Fig. 5). These a-helices are 3 0 - 4 5 ,~ long, whereas the d i a m e t e r of the h y d r o p h o b i c core of the cylindrical SDS micelle is a b o u t 25 A [18]. The helices can be a c c o m m o d a t e d within this core p r o v i d e d that they are tilted at an angle of 40 60 ° from the long axis of the detergent cylinder. A given number of SDS molecules will s u r r o u n d each c~-helix a n d the a m o u n t of SDS that interacts with the helices will be a p p r o x i m a t e l y p r o p o r t i o n a l to the n u m b e r of m e m b r a n e - s p a n n i n g helices (cf. Fig. 7 below). Ionic bonds. The interactions between SDS and p o l y p e p t i d e s naturally include ionic b o n d s between cationic a m i n o acid side-groups a n d the sulfate group of the detergent. M a t t i c e and coworkers [26] have shown b y m e a s u r e m e n t s of circular d i c h r o i s m that the content of arginine and lysine residues and the p r o p o r t i o n of e~-helical
TABLE I TORSION ANGLES FOR THE a-CARBONS OF THE REPETITIVE UNIT OF THE FLEXIBLE HELIX MODEL FOR A SDS-POLYPEPTIDE COMPLEX The a-carbon numbers refer to Figs. 2 and 4. The angles are average estimates from four repetitive units of the model in Fig. 6, with an inaccuracy of about +_10°, and are given according to the system in Ref. 27. a-carbon No.
~
1 2 3 4
-130 ° - 160 ° 40 ° - 180 °
-70 ° 50 ° 40 o 160 °
structure in SDS p r o t e i n complexes are positively correlated. Accordingly, we p r o p o s e that hydrophilic p o l y p e p t i d e segments that contain cationic side-groups m a y form short a-helices at the surface of the detergent micelle, a-helices that are posit i o n e d tangentially so that the cationic groups face the micelle. Energetically this is plausible, since h y d r o g e n b o n d s to the SDS molecules (Figs. 2 4) w o u l d be replaced by ionic b o n d s a n d h y d r o g e n b o n d s within the c~-helices. Models. Half a turn of a h y d r o g e n - b o n d e d p o l y p e p t i d e - S D S c o m p l e x such as those in Figs. 2 - 5 is shown in the m o d e l d e p i c t e d in Fig. 6. The m o d e l contains 40 a m i n o acid residues and 20 S D S molecules per full turn. T h e pitch of the helix is approx. 0.6 A per a m i n o acid residue or 24
Fig. 6. Model of half a turn of the helix in Fig. 5B, except that the axial length is about 0.6 ,~ per amino acid residue. The polypeptide is white, the sulfurs black and the dodecyl chains grey. The detergent molecules are arranged as the steps of a winding stair-case of low pitch and large diameter. (A) View at an angle of approx. 60 ° toward the cylinder axis: (B) view in the direction of the axis.
24 p e r turn. The torsion angles for the a - c a r b o n s in the p o l y p e p t i d e are allowed (Table I). The model is sterically possible. In Fig. 7 a section of the r o d - s h a p e d complex is schematically illustrated. In a d d i t i o n to h y d r o g e n - b o n d e d p o l y p e p t i d e segments this figure shows h y d r o p h o b i c c~-helices traversing the interior of the micelle and hydrophilic, cationic a-helices at the surface of the micelle. H y d r o g e n - b o n d e d SDS molecules are shown for only two turns of the large-radius p o l y p e p t i d e coil. See also the legend to Fig. 7. In reality most water-soluble proteins would form complexes with the structure shown in the lower half of Fig. 7, whereas m a n y integral m e m b r a n e proteins m a y form complexes as shown in the u p p e r half of the figure. W e have shown both types in the same d r a w i n g to emphasize the basic similarities that are possible.
Discussion
Fig. 7. The flexible-helix model for SDS-protein complexes. This drawing by M. Sandberg illustrates six turns of a complex, corresponding to a polypeptide segment with Mr approx. 28000. Hydrogen-bonded SDS molecules of two central turns are drawn in grey. The dodecyl chains are shown wedge-shaped and shorter than in scale for clarity of the figure (cf. Fig. 6). Two five-turn hydrophobic a-helices traverse the structure. a-Helices of this type are found in some membrane proteins [23-25]. Only the hydrogen-bonded SDS molecules in hydrophobic contact with the helices are shown. Opposite these SDS molecules more detergent molecules may be bound by the hydrophobic effect and van der Waals forces alone. There may be space for one SDS molecule per amino acid residue, twice as many as in the hydrogen-bonded structure. Short hydrophobic segments of water-soluble proteins may also pass through the interior of the cylinder (not illustrated). At the bottom of the figure two short a-helices are positioned at the surface of the cylinder, illustrating the proposed structure in which lysine and arginine side-groups form ionic bonds with the sulfate groups of the detergent (cf. Ref. 26). At high ionic strength, additional non-hydrogen-bonded SDS molecules can be accommodated between the turns of the large-radius helix, whereas at low ionic strength this is prevented by electrostatic repulsion. This is consistent with increased SDS binding at high ionic strength [11].
Earlier models for the structure of S D S - p r o t e i n complexes lack detail. Such a complex was, for instance, described by R e y n o l d s and T a n f o r d as " a rod-like particle" [19] and later by T a n f o r d as " s h o r t rod-like segments with intervening regions possessing some flexibility" [18]. S h i r a h a m a and coworkers [28] i n t r o d u c e d " t h e necklace m o d e l " which they characterized in the following way: " T h e p o l y m e r chain is considered to be mostly flexible, a n d s o d i u m dodecyl sulfate clusters are a r r a n g e d along the p o l y m e r chain". The main distinction between these models is the degree of flexibility and compactness. T h e m o d e l of Rey n o l d s and T a n f o r d is based m a i n l y on measurem e n t s of intrinsic viscosity. The d i m e n s i o n s of the complexes were calculated by use of the Simha equation, which applies to rigid particles [20,29,30]. T h e results are only a p p r o x i m a t e , since evidence for the rigidity of the complex does not exist. M e a s u r e m e n t s of the specific Kerr constants for S D S - p o l y p e p t i d e complexes [22] and the electric birefringence relaxation times [21,22] are not easily interpreted, b u t m a y indicate a flexible structure of the complex. The electro-optical d a t a c a n n o t directly s u p p o r t the S h i r a h a m a model [28], which is based m a i n l y on the observations that the electrophoretic mobilities and the SDS b i n d i n g ratios for p o l y p e p t i d e s , polyvinyl alcohol and
25
polyvinylpyrrolidone are independent of the polymer molecular mass. In our opinion a weakness of the necklace model is that the distribution, sizes, number and structure of the proposed SDS clusters and the interactions with the polypeptide have not been specified. SDS gel electrophoresis affords high resolution: polypeptides that differ less than 1000 in relative molecular mass can be separated, which would mean that the hypothetical clusters could include only 3 6 SDS molecules. Complexes between a few hydrophobic amino acid side-chains and such a small number of SDS molecules do not seem sterically or energetically favourable, since SDS micelles normally comprise 50-130 detergent molecules [18]. Our new model unites the results of earlier measurements and concepts of the previous models. We propose that the SDS molecules are distributed along the polymer chain in hydrogenbonded pairs and also that the complex is rod-shaped and flexible. This is possible if the polypeptide forms essentially a large-radius helix surrounding the detergent molecule pairs, which are arranged as the steps of a winding staircase with a hydrophilic outer structure and a hydrophobic center. Hydrogen-bonding between the peptide bonds and the sulfate groups of the SDS molecules stabilizes the structure, which also allows some ionic as well as hydrophobic interactions between the polypeptide and the detergent. The new model seems qualitatively consistent with earlier data from physical measurements, but additional evidence, possibly by N M R spectroscopy, is needed to verify or disprove the model. Conclusive data should be easier to collect than for any previous model, since we have elaborated our 'flexible helix model' in considerable detail. It can serve, we hope, as a point of departure for further experiments. Some simple predictions can immediately be made from our hypothetical structural model. For instance, tetradecyltrimethylammonium chloride should bind to polypeptides less efficiently than does SDS, since it cannot form hydrogen bonds. As mentioned in the Introduction, this has already been shown by Nozaki and coworkers [16]. Another consequence of the hydrogen bonds in the SDS-polypeptide complexes is that urea should decrease the SDS binding. According to Takagi
and Kubo [31] the binding of SDS to some reduced proteins is actually reduced by almost half by the presence of 8 M urea and the SDS concentration needed to reach saturation is increased. This can partly explain the usefulness of urea in removing SDS from proteins by electrophoresis or ion-exchange chromatography [32,33] or isoelectric focusing [34] although in these contexts urea is usually thought of as an agent for increasing the solubility of proteins. The efficient removal of SDS from proteins by ion-pair formation in organic solvents [35] suggests, on the other hand, that, as expected, hydrogen-bond pairs alone are not sufficient to stabilize an SDS-polypeptide complex, not even at low dielectric constants. Furthermore, we may predict that electrophoretic separation of SDS complexes of small-sized polypeptides, M r 10000 or less, may give low resolution, since according to our model the number of turns of the flexible helix will be only two or less and the complex thus approaches the structure of a single spherical SDS micelle with an M r of
06
0.4E, 0.2
" ~c 00
h 400
~ 800 ~ Mr2/3
~ 12~00 ~ 1600 /
Fig. 8. The relationship between - l o g K d and M 2/3 for SDS complexes with oligopeptides and proteins upon molecularsieve chromatography on a column of 12% agarose gel beads (cf. Ref. 7). The beads were prepared as in Ref. 36. Eluent: 100 m M sodium phosphate, pH 7.0, with 3.5 m M SDS. (M) SDS micelles, (polypeptide M r zero); (A) insulin A-chain, oxidized ( M r 2300); (B) insulin B-chain, oxidized (3300); (C) adrenocorticotropic hormone (ACTH, Serva) (4500); (D) the snake-venom toxin Notechis 111:4 (see Ref. 37) (8050); (E), cytochrome c (12400); (F) myoglobin (17200); (G) immunoglobulin, light chain (23000); (H) h u m a n serum albumin (66400). Proteins ( D ) - ( H ) were reduced and alkylated. The SDS concentration in the samples applied to the column was 170 raM. For an interpretation of this diagram in terms of our flexible-helix model, see Discussion.
26
approx. 25000 [18]. The intrinsic viscosity for SDS-polypeptide complexes will also be essentially independent of Mr below M~ 10000, which agrees with results of Takagi and Kubo (Fig. 4 in Ref. 31). Similarly, resolution in molecular-sieve chromatography deteriorates below Mr 8000. as shown by the diminished slope of - log K d versus M//3 (Fig. 8). The steep slope of this calibration line corresponds to our flexible helix model, whereas the shallow slope at M~ 2000-5000 represents the lower limit of our model where the polypeptides are attached around spherical SDS micelles (compare with the micelle caps in Fig. 5A). This dual-slope calibration diagram is not consistent with the necklace model of Shirahama and coworkers [28]. Acknowledgements We thank Lars Liljas for valuable discussions. The Notechis III:4 toxin was kindly supplied by David Eaker. We are grateful for support from the O.E. and Edla Johansson Science Foundation and the Swedish Natural Science Research Council. References 1 Makino, S. (1979) Adv. Biophys 12, 131-184 2 Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79 3 MaizeL J.V. (197l) in Methods in Virology (Maramorosch, K. and KoprowskL H., eds.), Vol. 5, pp. 179-246, Academic Press, New York 4 Becker, R.~ Helenius, A. and Simons, K. (1975) Biochemistry 14. 1835-1841 5 Dunker, A.K. and Rueckert, R.R. (1969) J. Biol. Chem. 244, 5074-5080 6 Weber, K. and Osborn, M. (1975) in The Proteins, 3rd Edn. (Neurath, H. and Hill, R.L., eds.), Vol. I. pp. 179 223, Academic Press, New York 7 Eriksson, K.-O. (1985) J. Biochem. Biophys. Methods 11, 145 152 8 Mascher~ E. and Lundahl, P. (1986) Biochim, Biophys. Acta 856, 505-514 9 Pitt-Rivers, R. and Impiombato, F.S.A. (1968) Biochem. J. 109, 825-830 10 Reynolds, J.A. and Tanford, C. (1970) Proc. Natl. Acad. Sci. USA 66, 1002-1007
11 Nelson, C.A. (1971) J. Biol. ('hem. 246, 3895-3901 12 Takagi, T., TsujiL K. and Shirahama, K. (1975) J. Biochem 77, 939 947 13 Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656 366l 14 Helenius, A. and Simons, K. (1977) Proc. Natl. Acad. Sci. USA 74, 529 532 15 Robinson. N.C. and Tanford, C. (1975) Biochemistry 14, 369 378 16 Nozaki, Y., Reynolds, J.A. and f'anford. C. (1974) J. Biol. Chem. 249, 4452 4459 17 Freytag, J.W., Noelken, M.E. and Hudson, B.G. (I979) Biochemistry 18, 4761 4768 18 Tanh;rd, C. (1980) The Hydrophobic Effect, pp. 51-59, 79-89, 146 164, 213 214, Wiley, New York 19 Reynolds, J.A. and Tanford, C. (1970) J. Biol. ('hem. 245, 5161 5165 20 Simha, R. (1940) J, Phys. Chem. 44, 25 34 21 Wright, A K . , Thompson, M.R. and Miller, R.L. (1975) Biochemistry 14. 3224-3228 22 Rowe, E.S. and Steinhardt, J. (1976) Biochemistry I5, 2579-2585 23 Kopito, R.R. and Lodish, H.F. (1985) Nature 316, 234-238 24 Mueckler, M., Caruso. C., Baldwin, S.A., Panico. M., Blench, 1., Morris, H.R., Allard, W.J., Lienhard, G.E. and kodish, H.F. (1985) Science 229, 941-945 25 Deisenhofer. J.. Epp, O., Miki, K., Huber, R. and Michel. H. (1985) Nature 318, 618-624 26 Manice, W.L., Riser, J.M. and Clark. D.S. (1976) Biochemistry 15. 4264-4272 27 Venkatachalam, C,M. and Ramachandran, G.N. (1967)in Conformation of Biopolymers, Vol. 1 (Ramachandran, G.N., ed.), pp. 83 97, Academic Press, New York 28 Shirahama, K., Tsujii, K. and TakagL T. (1974)J. Biochem. 75, 309 319 29 Mehl, J.W., Oncley, ,I.E. and Simha, R. (1940) Science 92, 132-133 30 Tanford, C, (1961) Physical Chemistry of Macromolecules, pp. 333-346. Wiley, New York 31 Takagi, T. and Kubo, K. (1979) Biochim. Biophys. Acta 578, 68 75 32 Weber, K. and Kuter, D.J. (1971) J. Biol. Chem. 246. 4504 4509 33 Lenard. J. (1971) Biochem. Biophys. Res. Commun. 45, 662-668 34 Ames, G.F,-L. and Nikaido, K. (1976) Biochemistry 15, 616-623 35 Henderson, L.E., Oroszlar, S. and Konigsberg, W. (1979) Anal. Biochem. 93, 153-157 36 Hjerten, S. and Eriksson, K.-O. (1984) Anal. Biochem. 137, 313-317 37 Halpert, J., Fohlman, J. and Eaker, D. (1979) Biochimie 61, 719-723
Bioehimi¢a et Biophvsica Acta 873 (1986) 20 26 Elsevier
BBA 32604
A model for ionic and hydrophobic interactions and hydrogen-bonding in s o d i u m d o d e c y i s u l f a t e - p r o t e i n c o m p l e x e s Per Lundahl, Eva Greijcr, Maria Sandberg, Susanna Cardell and Kjell-Ove Eriksson Institute of Biochemistry. Biomedical Center, Unit~ersi(v of Uppsala, P.O. Box 5 76, S-751 23 Uppsala (Sweden)
(Received April 23rd, 1986)
Key words: Sodium dodecyl sulfate protein complex: Membrane protein; Dodecyl sulfate-polypeptide hydrogen-bonding; Detergent protein interaction; Structural model
We propose a new hypothetical model for the structure of complexes between sodium dodecyl sulfate and proteins as described below: the detergent forms a flexible capped cylindrical miceile around which the hydrophilic segments of the polypeptide are helically wound. These polypeptide segments are attached by hydrogen bonds from the nitrogens of the peptide bonds to the sulfate oxygens of the detergent molecules. Cationic amino acid side-chains can also form ionic bonds with the sulfate groups. When the latter occurs the polypeptide assumes an a-helical conformation at the surface of the detergent cylinder in accordance with interpretations of circular dichroism measurements (Mattice, W.L., Riser, J.M. and Clark, D.S. (1976) BiochemistD' 15, 4264-4272). An essentially regular arrangement of one hydrogen bond per peptide bond and two per dodecyl sulfate monomer is consistent with the known binding ratio of approximately one detergent molecule per two amino acid residues and with the proportionality between the polypeptide molecular mass or the number of residues and the length of the complex (Reynolds, J.A. and Tanford, C. (1970) J. Biol. Chem. 245, 5161-5165). The hydrogen-bonded structure of our model also agrees with the finding that dodecyl sulfate associates much more readily with proteins than does tetradecyltrimethylammonium chloride (Nozaki, Y., Reynolds, J.A. and Tanford, C. (1974) J. Biol. Chem. 249, 4452-4459). The axial length of the structure we propose can be estimated at approximately 0.6 A per amino acid residue. Hydrophobic polypeptide segments, including membrane-spanning a-helices of integral membrane proteins, can be accommodated in the interior of the elongated dodecyi sulfate micelle. Therefore, not only water-soluble proteins but also some integral membrane proteins may form the proposed type of complex, in the case of glycosylated membrane proteins the sugar moieties will protrude from the surface of the rod-shaped complex. We have named our model 'the flexible helix ~.
Introduction
Sodium dodecyl sulfate (SDS) is used for solubilization and fractionation of amphiphilic proteins, for dissociation of protein complexes and quaternary protein structures and for molecular mass estimations by electrophoresis or gel chromatography [1 8], but no suitable model for the structure of an SDS-protein complex has been 0167-4838/86/$03.50
proposed. The detergent may interact with polypeptides both 'hydrophobically' and by ionic or hydrogen-bonding interactions with the head group. Non-hydrophobic interactions are suggested by the fact that hydrophilic proteins that lack disulfide bonds, either naturally or after reduction, bind SDS approximately in proportion to their molecular masses [9-12] although hydrophilic polypeptide segments cannot interact hy-
1986 Elsevier Science Publishers B.V. (Biomedical Division)
21
drophobically with the detergent molecules and do not bind non-ionic detergents [1,2,13-15]. Furthermore, the cationic detergent tetradecyltrimethylammonium chloride binds much less efficiently to polypeptides than does SDS, although the alkyl chain of the former detergent is longer than that of SDS. This was shown by Nozaki and coworkers [16], who stated: " W e are thus forced to conclude that the observed over-all advantage of the anionic detergent (SDS) ... must be taken to indicate that the head group plays a significant role in the mechanism of binding". In the present work we propose a new and detailed model for the structure of SDS-protein complexes. Our model seems plausible in the light of earlier experimental results. It includes hydrogen-bonding of non-charged and anionic amino acid residues and ionic bonding of cationic amino acid side-groups to sulfate groups on the surface of a cylindrical SDS micelle.
K°I
0.7
0.6
0
2 L 6 8 10 Dodecyt sutfate (raM)
Fig. 1, K d values for molecular-sieve chromatography of ovalbumin as a function of the SDS concentration in the eluent (gel filtration calibration protein, Pharmacia). Column: Superose 6 TM, 28x1 cm (Pharmacia). Eluent: 0.1 M sodium phosphate, pH 7.4, 1 mM EDTA, 1 mM dithioerythritol and the given concentration of SDS. Temperature: 21-24°C. For chromatography in the absence of SDS the proteins were solubilized in the eluent alone. Otherwise, 0.1 M SDS was included and the proteins were reduced and S-carboxymethylated. The data for 0.6-10 mM SDS are from Fig. 2 in Ref. 8. Major changes in K d value take place between 0 and 0.6 mM SDS and between 1.2 and 2.5 mM SDS, in agreement with data in Refs. 10 and 12.
Model for the structure of SDS-protein complexes
Hydrogen-bonding. Reduced water-soluble proteins bind SDS in approximate proportion to their molecular masses. Reported values are 1.4 [9,10], 1.1-2.2 [11] and 1.2-1.5 [12] g SDS per g protein for SDS concentrations above approx. 1.5 mM. The latter range corresponds to 0.96-1.2 SDS molecules per two amino acid residues for an average M, of 115 per residue. Binding or release takes place in two major steps as SDS is added or removed (Refs. 8, 10, 12 and Fig. 1). This binding ratio may reflect a stoichiometric binding of one detergent molecule per two amino acid residues. A hydrogen-bonding structure that gives the latter stoichiometry is illustrated in Fig. 2. Two hydrogen bonds attach a single sulfate head group of an SDS molecule to the nitrogens of peptide bonds Nos. n and n + 2. Another head group is attached to peptide bonds Nos. n + 1 and n + 3. The repetitive unit comprises four amino acid residues and two SDS molecules (Fig. 3A). Proline residues break the structure (Fig. 3B), but the ratio between the number of bound SDS molecules and the number of non-imino acid residues may be preserved (see legend to Fig. 3). The three-dimensional structure of the repetitive unit is illustrated in Fig. 4, in which the hydrogen bond angles are
a p p r o x . 160 °. I n t h i s w a y m o s t o f t h e p e p t i d e g r o u p n i t r o g e n s m a y b e h y d r o g e n - b o n d e d in a cooperative manner to the detergent molecules, w h i c h i n t e r a c t h y d r o p h o b i c a l l y as d e s c r i b e d b e low.
Hydrophobic interaction between detergent alkyl chains. W e a s s u m e t h a t t h e a l k y l c h a i n s o f t h e d e t e r g e n t i n t e r a c t h y d r o p h o b i c a l l y , e s s e n t i a l l y as i n a s p h e r i c a l S D S m i c e l l e [18]. H o w e v e r , m e a surements of intrinsic viscosity indicate that SDSprotein complexes are rod-shaped with an axial l e n g t h of a b o u t 0.74 ,~ p e r a m i n o a c i d r e s i d u e [19]. T h i s v a l u e w a s o b t a i n e d b y u s e o f t h e S i m h a
~) R 3 0 ...H-N-
,,s:,
O "°"
H
C-
C-
c:o
o
®
R4
N-
? ......
..
O,, I ~ ~O" " ~" 0'.-C I -R2 ~ S "O
I ...... C-N-C-C ii ® i O R~
I
.v
- N-H" ©
'.,.
Fig. 2. Proposed hydrogen-bonding between oxygens in the SDS head groups and nitrogens of peptide bonds. The figure shows a repetitive unit with two sulfate groups and four amino acid residues.
22
R~
~3
R,~
,,A_~ i ~ t n •
n "
l""t"
Rr
N i .
R6
R~
Rt
~,,_I-N L.~ L v , _ _ [;
.
"i""'I"
_
Fig. 3. Two consecutive units of the proposed hydrogen-bonded SDS-protein complex (cf. Fig. 2). (A) Regular structure with s - a m i n o acids. The filled circles represent the sulfate groups• (B) Structure interrupted by proline or hydroxyproline. A large proportion of this structure may be formed for instance by collagen. This protein contains approx. 10 mol% each of proline and hydroxyproline and binds approx• 1.4 g SDS per g protein [171. The number of residues per unit molecular mass is 26% higher for collagen than for average water-soluble proteins [17[. Therefore, the number of non-imino acid residues in collagen is nearly the same per unit molecular mass as in water-soluble proteins, and the normal weight per weight SDS binding [171 is consistent with the structure in panel B.
equation [20], which applies to rigid ellipsoids. Measurements of transient electric birefringence show an extended shape with a length of 0.6 _+ 0.2 ,~ (n = 6) per residue, as calculated from data in Ref. 21, Table III. Electro-optical data are con-
Fig. 4. The repetitive unit of Figs. 2 and 3. The polypeptide is white with the nitrogens of the peptide bonds numbered as in Fig. 2. Of the SDS molecules only the sulfur atoms are shown (black), The connections between the nitrogens and the sulfur atoms represent the hydrogen atoms as well as the bonded oxygen atoms. The hydrogen bond angles are approx. 160 ° .
sistent with a flexible structure of the complex [21,22]. Hydrophobic contact between the dodecyl chains in a flexible, rod-shaped complex of a length proportional to the number of amino acid residues is obtained by coiling the SDS-polypeptide complex (Fig. 3), thereby arranging the detergent into a cylindrical micelle (Fig. 5). This is only a description of the final structure, and does not imply any mechanism for the complex formation. Dense packing of the SDS molecules corresponds to a rod length of 0.32 A per residue (Fig. 5A). This is a lower limit, since the hydrogenbonded structure (Figs. 2-4) requires more space per detergent molecule. The rod length 0.74 A per amino acid residue [19] corresponds to the pitch of the polypeptide helices illustrated in Figs. 5B and
J
I
"
I
1 Fig. 5. Flexible helix models for the complex between SDS and a polypeptide. The polypeptide with SDS attached by hydrogen bonds (Fig. 3) is helically wound, forming a flexible, cylindrical structure with the polypeptide and the sulfate groups on the outside and the dodecyl chains in the interior. The radius of the detergent cylinder is approx. 16 ,~ (cf. Ref. 1,8), the outer radius 24 A as estimated from a detailed model, which shows about 40 amino acid residues per polypeptide turn (Fig. 6, below). (A) Complex with a polypeptide of the size of serum albumin, M r 66000. This model shows the lower limit of cylinder length and helix pitch: 13 A per turn or 0.32 A, per amino acid residue, as calculated by use of the cross-sectional area, 59 ~ 2 of SDS [18] and the assumption that the detergent molecules are densely packed. However, this maximal degree of packing is not consistent with the hydrogenbonded structure of Figs. 2-4. (B) Less dense packing with a helix pitch of 0.74 ,~ per amino acid residue as estimated by Reynolds and Tanford [19]. Due to the flexibility of this structure this value of the helix pitch may be too high, (C) Structure as in (B), except that a segment composed of 20-30 hydrophobic amino acid residues traverses the core of the detergent cylinder.
23 C. Since the c o m p l e x m a y be flexible, this estimate of the rod length is a p p r o x i m a t e . F u r t h e r details are given in the legend to Fig. 5.
Hydrophobic interaction between detergent alkyl chains and hydrophobic amino acid side-groups. F o r solubility reasons, h y d r o p h o b i c p o l y p e p t i d e segm e n t s should traverse the interior of the detergent micelle (Fig. 5C). M a n y water-soluble p r o t e i n s c o n t a i n only short h y d r o p h o b i c sequences, whereas some m e m b r a n e proteins c o n t a i n h y d r o p h o b i c m e m b r a n e - s p a n n i n g a-helices that include 2 0 - 3 0 a m i n o acid residues [23 25], a n u m b e r which corr e s p o n d s to 0.5-0.75 turn of the p o l y p e p t i d e helix (Fig. 5). These a-helices are 3 0 - 4 5 ,~ long, whereas the d i a m e t e r of the h y d r o p h o b i c core of the cylindrical SDS micelle is a b o u t 25 A [18]. The helices can be a c c o m m o d a t e d within this core p r o v i d e d that they are tilted at an angle of 40 60 ° from the long axis of the detergent cylinder. A given number of SDS molecules will s u r r o u n d each c~-helix a n d the a m o u n t of SDS that interacts with the helices will be a p p r o x i m a t e l y p r o p o r t i o n a l to the n u m b e r of m e m b r a n e - s p a n n i n g helices (cf. Fig. 7 below). Ionic bonds. The interactions between SDS and p o l y p e p t i d e s naturally include ionic b o n d s between cationic a m i n o acid side-groups a n d the sulfate group of the detergent. M a t t i c e and coworkers [26] have shown b y m e a s u r e m e n t s of circular d i c h r o i s m that the content of arginine and lysine residues and the p r o p o r t i o n of e~-helical
TABLE I TORSION ANGLES FOR THE a-CARBONS OF THE REPETITIVE UNIT OF THE FLEXIBLE HELIX MODEL FOR A SDS-POLYPEPTIDE COMPLEX The a-carbon numbers refer to Figs. 2 and 4. The angles are average estimates from four repetitive units of the model in Fig. 6, with an inaccuracy of about +_10°, and are given according to the system in Ref. 27. a-carbon No.
~
1 2 3 4
-130 ° - 160 ° 40 ° - 180 °
-70 ° 50 ° 40 o 160 °
structure in SDS p r o t e i n complexes are positively correlated. Accordingly, we p r o p o s e that hydrophilic p o l y p e p t i d e segments that contain cationic side-groups m a y form short a-helices at the surface of the detergent micelle, a-helices that are posit i o n e d tangentially so that the cationic groups face the micelle. Energetically this is plausible, since h y d r o g e n b o n d s to the SDS molecules (Figs. 2 4) w o u l d be replaced by ionic b o n d s a n d h y d r o g e n b o n d s within the c~-helices. Models. Half a turn of a h y d r o g e n - b o n d e d p o l y p e p t i d e - S D S c o m p l e x such as those in Figs. 2 - 5 is shown in the m o d e l d e p i c t e d in Fig. 6. The m o d e l contains 40 a m i n o acid residues and 20 S D S molecules per full turn. T h e pitch of the helix is approx. 0.6 A per a m i n o acid residue or 24
Fig. 6. Model of half a turn of the helix in Fig. 5B, except that the axial length is about 0.6 ,~ per amino acid residue. The polypeptide is white, the sulfurs black and the dodecyl chains grey. The detergent molecules are arranged as the steps of a winding stair-case of low pitch and large diameter. (A) View at an angle of approx. 60 ° toward the cylinder axis: (B) view in the direction of the axis.
24 p e r turn. The torsion angles for the a - c a r b o n s in the p o l y p e p t i d e are allowed (Table I). The model is sterically possible. In Fig. 7 a section of the r o d - s h a p e d complex is schematically illustrated. In a d d i t i o n to h y d r o g e n - b o n d e d p o l y p e p t i d e segments this figure shows h y d r o p h o b i c c~-helices traversing the interior of the micelle and hydrophilic, cationic a-helices at the surface of the micelle. H y d r o g e n - b o n d e d SDS molecules are shown for only two turns of the large-radius p o l y p e p t i d e coil. See also the legend to Fig. 7. In reality most water-soluble proteins would form complexes with the structure shown in the lower half of Fig. 7, whereas m a n y integral m e m b r a n e proteins m a y form complexes as shown in the u p p e r half of the figure. W e have shown both types in the same d r a w i n g to emphasize the basic similarities that are possible.
Discussion
Fig. 7. The flexible-helix model for SDS-protein complexes. This drawing by M. Sandberg illustrates six turns of a complex, corresponding to a polypeptide segment with Mr approx. 28000. Hydrogen-bonded SDS molecules of two central turns are drawn in grey. The dodecyl chains are shown wedge-shaped and shorter than in scale for clarity of the figure (cf. Fig. 6). Two five-turn hydrophobic a-helices traverse the structure. a-Helices of this type are found in some membrane proteins [23-25]. Only the hydrogen-bonded SDS molecules in hydrophobic contact with the helices are shown. Opposite these SDS molecules more detergent molecules may be bound by the hydrophobic effect and van der Waals forces alone. There may be space for one SDS molecule per amino acid residue, twice as many as in the hydrogen-bonded structure. Short hydrophobic segments of water-soluble proteins may also pass through the interior of the cylinder (not illustrated). At the bottom of the figure two short a-helices are positioned at the surface of the cylinder, illustrating the proposed structure in which lysine and arginine side-groups form ionic bonds with the sulfate groups of the detergent (cf. Ref. 26). At high ionic strength, additional non-hydrogen-bonded SDS molecules can be accommodated between the turns of the large-radius helix, whereas at low ionic strength this is prevented by electrostatic repulsion. This is consistent with increased SDS binding at high ionic strength [11].
Earlier models for the structure of S D S - p r o t e i n complexes lack detail. Such a complex was, for instance, described by R e y n o l d s and T a n f o r d as " a rod-like particle" [19] and later by T a n f o r d as " s h o r t rod-like segments with intervening regions possessing some flexibility" [18]. S h i r a h a m a and coworkers [28] i n t r o d u c e d " t h e necklace m o d e l " which they characterized in the following way: " T h e p o l y m e r chain is considered to be mostly flexible, a n d s o d i u m dodecyl sulfate clusters are a r r a n g e d along the p o l y m e r chain". The main distinction between these models is the degree of flexibility and compactness. T h e m o d e l of Rey n o l d s and T a n f o r d is based m a i n l y on measurem e n t s of intrinsic viscosity. The d i m e n s i o n s of the complexes were calculated by use of the Simha equation, which applies to rigid particles [20,29,30]. T h e results are only a p p r o x i m a t e , since evidence for the rigidity of the complex does not exist. M e a s u r e m e n t s of the specific Kerr constants for S D S - p o l y p e p t i d e complexes [22] and the electric birefringence relaxation times [21,22] are not easily interpreted, b u t m a y indicate a flexible structure of the complex. The electro-optical d a t a c a n n o t directly s u p p o r t the S h i r a h a m a model [28], which is based m a i n l y on the observations that the electrophoretic mobilities and the SDS b i n d i n g ratios for p o l y p e p t i d e s , polyvinyl alcohol and
25
polyvinylpyrrolidone are independent of the polymer molecular mass. In our opinion a weakness of the necklace model is that the distribution, sizes, number and structure of the proposed SDS clusters and the interactions with the polypeptide have not been specified. SDS gel electrophoresis affords high resolution: polypeptides that differ less than 1000 in relative molecular mass can be separated, which would mean that the hypothetical clusters could include only 3 6 SDS molecules. Complexes between a few hydrophobic amino acid side-chains and such a small number of SDS molecules do not seem sterically or energetically favourable, since SDS micelles normally comprise 50-130 detergent molecules [18]. Our new model unites the results of earlier measurements and concepts of the previous models. We propose that the SDS molecules are distributed along the polymer chain in hydrogenbonded pairs and also that the complex is rod-shaped and flexible. This is possible if the polypeptide forms essentially a large-radius helix surrounding the detergent molecule pairs, which are arranged as the steps of a winding staircase with a hydrophilic outer structure and a hydrophobic center. Hydrogen-bonding between the peptide bonds and the sulfate groups of the SDS molecules stabilizes the structure, which also allows some ionic as well as hydrophobic interactions between the polypeptide and the detergent. The new model seems qualitatively consistent with earlier data from physical measurements, but additional evidence, possibly by N M R spectroscopy, is needed to verify or disprove the model. Conclusive data should be easier to collect than for any previous model, since we have elaborated our 'flexible helix model' in considerable detail. It can serve, we hope, as a point of departure for further experiments. Some simple predictions can immediately be made from our hypothetical structural model. For instance, tetradecyltrimethylammonium chloride should bind to polypeptides less efficiently than does SDS, since it cannot form hydrogen bonds. As mentioned in the Introduction, this has already been shown by Nozaki and coworkers [16]. Another consequence of the hydrogen bonds in the SDS-polypeptide complexes is that urea should decrease the SDS binding. According to Takagi
and Kubo [31] the binding of SDS to some reduced proteins is actually reduced by almost half by the presence of 8 M urea and the SDS concentration needed to reach saturation is increased. This can partly explain the usefulness of urea in removing SDS from proteins by electrophoresis or ion-exchange chromatography [32,33] or isoelectric focusing [34] although in these contexts urea is usually thought of as an agent for increasing the solubility of proteins. The efficient removal of SDS from proteins by ion-pair formation in organic solvents [35] suggests, on the other hand, that, as expected, hydrogen-bond pairs alone are not sufficient to stabilize an SDS-polypeptide complex, not even at low dielectric constants. Furthermore, we may predict that electrophoretic separation of SDS complexes of small-sized polypeptides, M r 10000 or less, may give low resolution, since according to our model the number of turns of the flexible helix will be only two or less and the complex thus approaches the structure of a single spherical SDS micelle with an M r of
06
0.4E, 0.2
" ~c 00
h 400
~ 800 ~ Mr2/3
~ 12~00 ~ 1600 /
Fig. 8. The relationship between - l o g K d and M 2/3 for SDS complexes with oligopeptides and proteins upon molecularsieve chromatography on a column of 12% agarose gel beads (cf. Ref. 7). The beads were prepared as in Ref. 36. Eluent: 100 m M sodium phosphate, pH 7.0, with 3.5 m M SDS. (M) SDS micelles, (polypeptide M r zero); (A) insulin A-chain, oxidized ( M r 2300); (B) insulin B-chain, oxidized (3300); (C) adrenocorticotropic hormone (ACTH, Serva) (4500); (D) the snake-venom toxin Notechis 111:4 (see Ref. 37) (8050); (E), cytochrome c (12400); (F) myoglobin (17200); (G) immunoglobulin, light chain (23000); (H) h u m a n serum albumin (66400). Proteins ( D ) - ( H ) were reduced and alkylated. The SDS concentration in the samples applied to the column was 170 raM. For an interpretation of this diagram in terms of our flexible-helix model, see Discussion.
26
approx. 25000 [18]. The intrinsic viscosity for SDS-polypeptide complexes will also be essentially independent of Mr below M~ 10000, which agrees with results of Takagi and Kubo (Fig. 4 in Ref. 31). Similarly, resolution in molecular-sieve chromatography deteriorates below Mr 8000. as shown by the diminished slope of - log K d versus M//3 (Fig. 8). The steep slope of this calibration line corresponds to our flexible helix model, whereas the shallow slope at M~ 2000-5000 represents the lower limit of our model where the polypeptides are attached around spherical SDS micelles (compare with the micelle caps in Fig. 5A). This dual-slope calibration diagram is not consistent with the necklace model of Shirahama and coworkers [28]. Acknowledgements We thank Lars Liljas for valuable discussions. The Notechis III:4 toxin was kindly supplied by David Eaker. We are grateful for support from the O.E. and Edla Johansson Science Foundation and the Swedish Natural Science Research Council. References 1 Makino, S. (1979) Adv. Biophys 12, 131-184 2 Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79 3 MaizeL J.V. (197l) in Methods in Virology (Maramorosch, K. and KoprowskL H., eds.), Vol. 5, pp. 179-246, Academic Press, New York 4 Becker, R.~ Helenius, A. and Simons, K. (1975) Biochemistry 14. 1835-1841 5 Dunker, A.K. and Rueckert, R.R. (1969) J. Biol. Chem. 244, 5074-5080 6 Weber, K. and Osborn, M. (1975) in The Proteins, 3rd Edn. (Neurath, H. and Hill, R.L., eds.), Vol. I. pp. 179 223, Academic Press, New York 7 Eriksson, K.-O. (1985) J. Biochem. Biophys. Methods 11, 145 152 8 Mascher~ E. and Lundahl, P. (1986) Biochim, Biophys. Acta 856, 505-514 9 Pitt-Rivers, R. and Impiombato, F.S.A. (1968) Biochem. J. 109, 825-830 10 Reynolds, J.A. and Tanford, C. (1970) Proc. Natl. Acad. Sci. USA 66, 1002-1007
11 Nelson, C.A. (1971) J. Biol. ('hem. 246, 3895-3901 12 Takagi, T., TsujiL K. and Shirahama, K. (1975) J. Biochem 77, 939 947 13 Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656 366l 14 Helenius, A. and Simons, K. (1977) Proc. Natl. Acad. Sci. USA 74, 529 532 15 Robinson. N.C. and Tanford, C. (1975) Biochemistry 14, 369 378 16 Nozaki, Y., Reynolds, J.A. and f'anford. C. (1974) J. Biol. Chem. 249, 4452 4459 17 Freytag, J.W., Noelken, M.E. and Hudson, B.G. (I979) Biochemistry 18, 4761 4768 18 Tanh;rd, C. (1980) The Hydrophobic Effect, pp. 51-59, 79-89, 146 164, 213 214, Wiley, New York 19 Reynolds, J.A. and Tanford, C. (1970) J. Biol. ('hem. 245, 5161 5165 20 Simha, R. (1940) J, Phys. Chem. 44, 25 34 21 Wright, A K . , Thompson, M.R. and Miller, R.L. (1975) Biochemistry 14. 3224-3228 22 Rowe, E.S. and Steinhardt, J. (1976) Biochemistry I5, 2579-2585 23 Kopito, R.R. and Lodish, H.F. (1985) Nature 316, 234-238 24 Mueckler, M., Caruso. C., Baldwin, S.A., Panico. M., Blench, 1., Morris, H.R., Allard, W.J., Lienhard, G.E. and kodish, H.F. (1985) Science 229, 941-945 25 Deisenhofer. J.. Epp, O., Miki, K., Huber, R. and Michel. H. (1985) Nature 318, 618-624 26 Manice, W.L., Riser, J.M. and Clark. D.S. (1976) Biochemistry 15. 4264-4272 27 Venkatachalam, C,M. and Ramachandran, G.N. (1967)in Conformation of Biopolymers, Vol. 1 (Ramachandran, G.N., ed.), pp. 83 97, Academic Press, New York 28 Shirahama, K., Tsujii, K. and TakagL T. (1974)J. Biochem. 75, 309 319 29 Mehl, J.W., Oncley, ,I.E. and Simha, R. (1940) Science 92, 132-133 30 Tanford, C, (1961) Physical Chemistry of Macromolecules, pp. 333-346. Wiley, New York 31 Takagi, T. and Kubo, K. (1979) Biochim. Biophys. Acta 578, 68 75 32 Weber, K. and Kuter, D.J. (1971) J. Biol. Chem. 246. 4504 4509 33 Lenard. J. (1971) Biochem. Biophys. Res. Commun. 45, 662-668 34 Ames, G.F,-L. and Nikaido, K. (1976) Biochemistry 15, 616-623 35 Henderson, L.E., Oroszlar, S. and Konigsberg, W. (1979) Anal. Biochem. 93, 153-157 36 Hjerten, S. and Eriksson, K.-O. (1984) Anal. Biochem. 137, 313-317 37 Halpert, J., Fohlman, J. and Eaker, D. (1979) Biochimie 61, 719-723