(soluble proteins/polar amino acids/hydrophobicity/polarity index) ... their surfaces are likely to be water soluble, whereas proteins .... most soluble pro- r%-1arc!
Proc. Nat. Acad. Sci. USA Vol. 69, No. 4, pp. 930-932, April 1972
/
The Low Polarity of Many Membrane Proteins (soluble proteins/polar amino acids/hydrophobicity/polarity index)
RODERICK A. CAPALDI AND GARRET VANDERKOOI Institute for Enzyme Research, University of Wisconsin, Madison, Wis. 53706
Communicated by David E. Green, February 7, 1972 ABSTRACT The polarities of a large number of soluble and membrane proteins have been calculated by summing the mole fractions of polar amino acids. It was found that 85% of the 205 soluble proteins considered in this study had polarities of 47 1 6%. Only 2% of the soluble proteins had polarities below 40%, whereas 47% of the 19 membrane proteins had polarities below 40%. The membrane proteins with polarities below 40% could be separated from their respective membranes only by detergents or organic solvents, indicating the importance of hydrophobic forces in their interaction with other membrane components. It is concluded that the. majority of "intrinsic" membrane proteins have low polarity, and that the polarity index is therefore a useful parameter for characterization of membrane proteins.
We have tested this hypothesis by comparing the polarities of those membrane proteins whose amino-acid composition is known, with the polarities of a large number of soluble proteins. Similar studies have been done before (10, 11), but the results obtained were unclear since much of the amino-acid composition data used pertained to whole membrane preparations, rather than to purified membrane proteins. METHODS The 208 proteins for which amino-acid compositions are listed in the Chemical Rubber Co. Handbook of Biochemistry (12) were used as the sample of soluble proteins, but with the omission of visual pigment, cytochrome c, and structural protein, which are clearly of membrane origin. The amino-acid compositions of membrane proteins were obtained by a search of the literature. Only data from preparations that we considered to be fairly homogeneous were used. Thus spectrin, for example (13), which has recently been shown to be a mixture of proteins (14), was not included. Similarly, proteins that appeared to be fragments of much larger polypeptide chains were not included, e.g., the sialoprotein reported by Winzler (15) has a molecular weight of 36,000 and is likely to be a tryptic fragment of a larger
An analysis of the x-ray diffraction data of several globular proteins has shown that, as a rule, all the polar amino-acid residues are situated on the surface of these molecules (1). It is the spatial distribution and proportion of polar and nonpolar groups on the surface of proteins that determines their solubility. Proteins with a high proportion of polar groups on their surfaces are likely to be water soluble, whereas proteins with a major proportion of their surface covered with nonpolar groups are likely to be water insoluble, unless solubilizing agents such as detergents are added. There is evidence from circular dichroism (2-4), infrared spectroscopy (5), and x-ray diffraction studies (6) to indicate that the majority of membrane proteins are fundamentally similar to soluble proteins, in the sense that they are globular with considerable a-helix content and very little 0-structure. One would expect that they will also have all their polar residues on the surface. There is abundant evidence to indicate that some membrane proteins are located at a hydrocarbon/water interface, partly exposed to the aqueous medium and partly buried in the hydrophobic interior of the lipid bilayer (7). These proteins have been called "intrinsic" (8), or "integral" (9), membrane proteins, to differentiate them from "extrinsic" (8), or "peripheral" (9), membrane proteins, which by definition do not penetrate into the lipid bilayer. Intrinsic membrane proteins might be expected to have a nonrandom spatial distribution of polar and nonpolar groups in order to give them the required surface active property. Thus, the majority of the polar residues are likely to be on the region of protein surface that is exposed to water, while the surface area of the molecule that is in contact with the nonpolar environment will predominantly consist of nonpolar residues. One might therefore expect intrinsic proteins to have a reduced number of polar amino-acid residues, in comparison with proteins that are readily soluble in an aqueous environment.
87,000-dalton glycoprotein (16). However, protein complexes such as cytochrome oxidase and sarcoplasmic reticulum ATPase, which probably exist as complexes in the membrane, were included (17, 18). For the present work, the polarity of a protein was defined as the sum of the residue mole percentages of polar amino acids. We have classified Asp, Asn, Glu, Gln, Lys, Ser, Arg, Thr, and His as polar residues and the remaining amino acids as nonpolar. This classification closely follows that proposed by Hatch and Bruce (10), and is similar to the one proposed by Fisher (19), with the exception of Tyr, which Fisher included in the polar group. However, readjustment of our classifications to include Tyr in the polar class would not significantly alter the results. In a preliminary presentation of this work (20), we divided the amino acids into three classes: polar (Asp, Asn, Glu, Gin, Lys, and Arg), intermediate (Ser, Thr, Tyr, His, and Gly) and nonpolar (Ala, Val, Leu, Ile, Cys, Met, Pro, Phe, and Trp), summing the polar groups + half the total of the intermediate group to give a polarity index. Since the distribution of protein polarities obtained with this division into three classes was essentially the same as that found with the division into two classes, there is evidently no need to introduce a third class. 930
Proc. Nat. Acad. Sci. USA 69
Polarity of Membrane Proteins
(1972)
RESULTS AND DISCUSS]ION The distribution of polarities of the soluble> proteins is shown in histogram form in Fig. 1, lower portion. I'he remarkable result was obtained that 85% of these solublLe proteins had polarities within the narrow range of 47 i 6% The polarity distribution of soluble proteins displays a fairly abrupt lowpolarity cutoff; considerably more tailing ()f the distribution is found at the high polarity end. Only 13 c f the 205 proteins (6%) had polarities less than 40% and only two had polarities less than 37%; these were al-collagen (25.91S%) and proinsulin (36.9%). Both of these proteins are highly ESpecialized, al-collagen being a fibrous structural element, and proinsulin a precursor of a hormone that is active at tihe membrane and
931
z
155-
20
25
30
35
40
45
50
55
PERCENT POLAR AMINO ACIDS
FIG. 1. (Lower part) Histogram showing the polarity disthat is characteristically hydrophobic. tribution of the 205 soluble proteins. The ordinate scale gives the The distribution of polarities of the 19 miembrane proteins number of proteins. (Upper part) Distribution of polarities of listed in Table 1 is shown in the upper port ion of Fig. 1. It is membrane proteins. Each square represents one protein; the apparent that there is a very wide distributiion of polarities of open squares are in group A, while the cross-hatched squares are in group B (see Table 1). membrane proteins, but a large proportion of them have polarities that are much lower than those of most soluble proA(17arc! +h n IJInZuL as iess teins. Nine out of 19 (47%) have polaritiEr%-1 Two of the proteins in group A, rhodopsin and the purple and five out of 19 (26%) have polarities le,ss than 37%O.TwoftepoesngruAroosnadheupl have been conmembrane protein of Halobacterium ..in the halobium, .. It is difficult at the present time to decidle unambiguously on the basis of x-ray to be partially buried sidered lipid and other data (21-24), while the available structural evidence how many of the membrane proteins in I 'ablepener ate deeply into the hydrocarbon interior of the for membranous cytochrome oxidase has been explained in However, the the should be called "intrinsic" membrane prote ,ins. However, terms of the penetration of the protein completely through the bilayer (8, 25). Each of these three proteins has a polarity 13 proteins listed as group A all require deteZrgents or organic of less than 40%. solvents to liberate them from the membr ane, indicating a In contrast, several proteins can be removed from their restrong hydrophobic interacting with lipid or possibly with . other proteins that are themselves hydropholbically associated as aqueous exmildbeprocedures such by not spective membrane .asited the lipid which would expected to disrupt have polarities traction, with lipid. All of the membrane proteins theatbicalvy bilayer. These proteins are listed in group B of Table 1. Their below 40% are in this group (69% of the tot;al in group A). solubility properties would indicate that their interaction with the membrane is not primarily of a hydrophobic nature, but TABLE 1. Polarities of membrane proteins rather that electrostatic forces play a dominant role in their attachment. None of the proteins with low polarity fall into PolaryRef. Polart this group. These should probably all be called extrinsic membrane proteins. Group A. Proteins that require detergents or organic We do not mean to suggest that all proteins that are buried solvents to extract them from the me:Mbrane. in the lipid will necessarily have low polarity, but only that Folch-Lees proteolipid protein, sciatic myelin 27 28.6 proteins that do have a low polarity are likely to be intrinsic C55 isoprenoid alcohol phosphokinase 31.2 28 to their respective membranes. An asymmetric distribution Purple membrane protein of Halobacterium of polar groups between the hydrocarbon and aqueous phases 34.3 29 halobium can be achieved even in proteins with high polarity. Thus Folch-Lees proteolipid protein, brain myelin 35.2 27 cytochrome b5 has a concentration of nonpolar groups at one Rhodopsin, bovine 36.2 30 end of its polypeptide chain that may interact hydrophobiChlorophyll complex II 37.2 31 Chlorophyll complex I 38.3 cally with the lipid (26), even though the molecule as a whole 31 Carotenoid glycoprotein, Sarcina flava 38.4 32 has a high polarity. Cytochrome oxidase 39.7 33 In summary, we have demonstrated that a considerable Sarcoplasmic reticulum ATPase 42.9 34 proportion of known membrane proteins has polarities that Oligomycin-sensitivity-conferring-protein, are significantly lower than those of the majority of soluble mitochondria proteins. This finding is consistent with the other lines of evi2 Cytochrome b5, microsomal dence that indicate that these proteins are most likely parCalsequestrin, sarcoplasmic reticulum 53.6 36 53.6 36 tially buried in the hydrophobic interior of the membrane. Group B. Proteins that can be solubilized from their Thus, the polarity index appears to be a useful parameter for respective membranes by aqueous media. the characterization of membrane proteins. "
se uhanambiguouy lins.
43.48
Mitochondrial ATPase Monoamine oxidase, mitochondrial Cytochrome c, human ATPase from Streptococcus faecalis Basic protein, sciatic myelin Basic protein, brain myelin
45.6 46.1 48.1
37 38 12
48.3
39 27 27
50.0 52.0
This work was supported by grant GM-12847 from the National Institute of General Medical Studies, United States Public Health Service. One of us (R.A.C.) is grateful to the Wellcome Trustees for the award of a Wellcome Research Travel Grant. 1. Klotz, I. M. (1970) Arch. Biochem. Biophys. 138, 704-706. 2. Lenard, J. & Singer, S. J. (1966) Proc. Nat. Acad. Sci. USA
56, 1828-1835.
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Biochemistry: Capaldi and Vanderkooi
3. Wigglesworth, J. M. & Packer, L. (1968) Arch. Biochem. Biophys. 128, 790-801. 4. Urry, D. W. & Ji, T. H. (1968) Arch. Biochem. Biophys. 128, 802-807. 5. Gordon, A. S., D. F. H. Wallach & Straus, J. H. (1969) Biochim. Biophys. Acta 193, 225-227. 6. Mathews, F. S., Levine, M. & Argus, P. (1971) Nature New Biol. 233, 15-17. 7. Vanderkooi, G. & Green, D. E. (1971) BioScience ?1, 409415. 8. Vanderkooi, G. (1972) "Conference on Membrane Structure and Its Biological Implications," eds. Green, D. E. & Danielli, J. F., Ann. N.Y. Acad. Sci., in press. 9. Singer, S. J. & Nicolson, G. L. (1972) Science 175, 720-731. 10. Hatch, F. T. & Bruce, A. L. (1968) Nature 218, 1166-1168. 11. Wallach, D. F. H. & Gordon, A. (1968) Fed. Proc. 27, 12631268. 12. Sober, H. A., ed. (1970) Handbook of Biochemistry (The Chemical Rubber Co., Cleveland), 2nd ed., pp. C281-287. 13. Marchesi, S. L., Steers, E., Marchesi, V. T. & Tillack, T. W. (1970) Biochemistry 9, 50-56. 14. Trayer, H. R., Nozaki, Y., Reynolds, J. A. & Tanford, C. (1971) J. Biol. Chem. 246, 4485-4488. 15. Winzler, R. J. (1969) in Red Cell Membrane Structure and Function, eds. Jamieson, F. A. & Greenwalt, T. H. (J. B. Lippincott, Philadelphia, Pa.), pp. 159-165. 16. Bretscher, M. S. (1971) Nature 231, 229-231. 17. Kierns, J. J., Yang, C. S. & Gilmont, M. V. (1971) Biochem. Biophys. Res. Commun. 45, 835-841. 18. Martinosi, A. & Halpin, R. A. (1971) Arch. Biochem. Biophys. 144, 66-77. 19. Fisher, H. F. (1964) Proc. Nat. Acad. Sci. USA 51, 12851291. 20. Vanderkooi, G. & Capaldi, R. A. (1972) "Conference on Membrane Structure and Its Biological Implications," eds. Green, D. E. & Danielli, J. F., Ann. N.Y. Acad. Sci., in press.
Proc. Nat. Acad. Sci. USA 69
(1972)
21. Vanderkooi, G. & Sundaralingam, M. (1970) Proc. Nat. Acad. Sci. USA 67, 233-238. 22. Wilkins, M. H. F. (1972) "Conference on Membrane Structure and Its Biological Implications," eds. Green, D. E. & Danielli, J. F., Ann. N.Y. Acad. Sci., in press. 23. Blaurock, A. E. & Stoeckenius, W. (1971) Biophys. J. 11, 115abstr. 24. Blaurock, A. E. & Stoeckenius, W. (1971) Nature New Biol. 233, 152-155. 25. Vanderkooi, G., Senior, A. E., Capaldi, R. A. & Hayashi, H. Biochim. Biophys. Acta, in press. 26. Spatz, L. & Strittmatter, P. (1971) Proc. Nat. Acad. Sci. USA 68, 1042-1046. 27. Eng., L. P., Chao, F. C., Gerstl, B., Pratt, D. & Tavaststjema, M. G. (1968) Biochemistry 7, 4455-4465. 28. Sandermann, H., Jr. & Strominger, J. L. (1971) Proc. Nat. Acad. Sci. USA 68, 2441-2443. 29. Stoeckenius, W. & Kanau, W. H. (1968) J. Cell Biol. 38, 337-357. 30. Heller, J. (1968) Biochemistry 7, 2906-2920. 31. Thornber, J. M., Stewart, J. C., Hatton, M. W. C. & Bailey, J. L. (1967) Biochemistry 6, 2006-2014. 32. Thurkell, D. & Hunter, M. I. S. (1969) J. Gen. Microbiol. 58, 289-292. 33. Matsubara, H., Oiu, Y. & Okunuki, K. (1965) Biochim. Biophys. Acta 97, 61-67. 34. MacLennan, D. H., Seeman, P., Iles, G. H. & Yip, C. C. (1971) J. Biol. Chem. 246, 2702-2710.
35. Senior, A. E. (1971) Bioenergetics 2, 141-150. 36. MacLennan, D. H. & Wong, P. T. S. (1971) Proc. Nat. Acad. Sci. USA 68, 1231-1235. 37. Senior, A. E. & MacLennan, D. H. (1970) J. Biol. Chem. c 245, 5086-5095. 38. Oreland, L. (1971) Arch. L ochem. Biophys. 146, 410421. 39. Schnebli, H. P., Vatter, A. E. & Abrams, A. (1970) J. Biol. Chem. 245, 1122-1127.
