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Ann. Rev. Biophys. Bioeng. Copyright
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1977 by
1977. 6:195-238
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PROTEIN-LIPID
-:-9093
INTERACTIONSl Robert B. Gennis2 Department of Chemistry, School of Chemical Scien ces, U n iversity of Illinois,
Urbana. Illinois 6 1 80 I
Ana Jonas3 School of Basic Medical Sciences and Department of Biochemistry, School of Chemical Sc iences. Un iversity of Illinois, Urbana, Illinois 6 1 801
INTRODUCTION In re ce nt years it has become apparent that many critical functions of living cells
are performed by membrane-associated proteins; as a consequence, research in diverse areas of biochemistry has converged on the study of membrane structure and function. The fundamental questions regarding all membrane-associated processes depend on the clear understanding of the dynamics and structure of protein-lipid complexes. It is the purpose of this article to review the current state of research in this relatively new area of protein-lipid interactions and to discuss the concepts. experimental techniques. and difficulties within the context of modern biophysical lAbbreviations: HDL, serum high density lipoprotein; LDL. serum low density lipoprotein; VLDL. serum very low d en si ty l ipop rotei n ; apoLP - Glnl = apo-A-I, major protein component of HDL; apo LP-G \n II = apo-A-II, second most abundant p rotein of human HDL; apoLP Ala = C-IlI. protein component common to human HDL and VLDL with C-terminal ala; apoL P-Ser = C-l, protein component common to human HDL and VLDL wit h C-terminal ser; CMC. critical micelle concentration; Cl4"PC, di myr istoyl phosphatidylcholine; BLM. black lipid membrane; Cwlyso PC, palmitoyl-Iysophosphatidylcholine; PE, phosphat i dyl
ethanolamine; SDS, sodium dodecyl sulfate; CI4NMe]CI. tetradecyltri methylammonium chlo ride; BSA, bovine serum albumin; BDH, fj-hydroxybutyrate dehydrogenase; CD. circular
dichroism; Tm. melting or critical temperature; DOC, deoxycholate; CL, cardiolipin; TPP,
thiamin pyrophosphate; DSC . differential scanning calorimet ry. IThis work was completed with the support of grants from the NIH, HL16101, and the Illinois Heart Association, N-2. RBG is also grat efu l for suppo rt from Career Development
Award PHS K04 H L00040. 3Established Investigator of the American Heart Association. This work was comp le ted under the support of grants PHS-HEW H L 1 6059 and IIlinos Illinois Heart Association N-2A. 195
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196
GENNIS & JONAS
chemistry. Published work on protein-lipid interactions consists of a collection of studies on a number of distinct biochemical systems where the specific research questions are often unique and the methods of investigation are varied. Hopefully, the more detailed the information that becomes available for a g reater number of systems, the easier it will be to make generalizations on protein-lipid interactions and their role in membrane and lipoprotein structure and function. A major hindrance to progress in the area of protein-lipid interactions has been the paucity of well-characterized systems for study. Purified proteins that have biochemically relevant lipid-binding properties are the first prerequisite for these studies. Obviously most of the work in this area concerns membrane proteins; significant exceptions are the serum lipoproteins, soluble lipid transfer proteins, and soluble lipid metabolizing enzymes . The membrane proteins that have received the most attention are those involved in energy transduction, for example, flavo proteins and cytochromes involved in electron transport or ATPases involved in intercon verting chemical and osmotic free energy.
Fluid Mosaic Model The conceptual framework for much of membrane biochemistry today is the fluid mosaic model of Singer & Nicolson (I, 2). The basic premise is that the structural determinant for biological membranes is the phospholipid bilayer. Membrane pro teins are largely viewed as globular entities that can float on or in the lipid sea; they are pictured as being amphipathic, i.e. having distinct polar and apolar regions. The polar surfaces are exposed to the aqueous phase or may be involved in protein protein interactions whereas the hydrophobic surfaces bind to the lipids. In contrast to some earlier membrane models, the fluid mosaic model really has little to say concerning the details of protein-lipid interactions; rather, attention has been di rected quite successfully at the dynamic aspects of membranes, in particular at the lateral mobility of membrane components (3). Much of this work has concerned the study of thermally induced liquid crystal phase transitions in both model mem branes and biomembranes. This topic was recently reviewed by Melchior & Steim (4). The concept of the membrane as a two-dimensional fluid has been useful in describing these phase transition phenomena (5, 6), and this concept has been important in formulating some of our general ideas about membrane dynamics, structure, and function.
Membrane Proteins The fluid mosaic model is compatible with most arrangements for membrane pro teins. These proteins may be located on the surface of the membrane, may be immersed to varying degrees, or may penetrate the bilayer completely. Membrane proteins are divided into two operational categories: peripheral (or extrinsic) pro teins and integral (or intrinsic) proteins. Peripheral proteins are those removed from the membrane by relatively gentle techniques, such as sonication or changing ionic strength, whereas those proteins requiring more drastic methods for solubilization, such as the use of detergents or organic s olvents, are termed integral membrane proteins. It is becoming clear that this classification does not imply a clear division
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PROTEIN-LIPID I NTERACTIONS
197
between those proteins that bind to the membrane by electrostatic interaction with the lipid polar head groups and those that interact through hydrophobic forces with the core of the lipid bilayer. Singer (2) has suggested that peripheral membrane proteins bind primarily to integral proteins in the bilayer rather than directly to the lipid. For example, cytochrome c, the most intensively studied peripheral protein, can bind to specific membrane proteins in the mitochondrial membrane (7,8). One general principle that applies is that the binding between lipids and proteins in most naturally occurring protein-lipid systems is noncovalent. Notable exceptions are the murein lipoprotein from the Escherichia coli envelope (9, 10) and the penicillinase from Bacillus licheniformis (11). General aspects of membrane protein s have re cently been reviewed by Guidotti (2). A very important consideration in working with most lipid-binding protei ns is their strong tendency to aggregate. Presumably, the hydrophobic surfaces normally
in contact with lipids in vivo will result in protein aggregation when the lipids are removed. In some cases aggregates are limited in size and are water soluble; how ever, more frequently detergents must be used to maintain the protein in a water soluble form. Serum Apolipoprotein s
Serum lipoproteins (13, 14) are isolated and characterized according to their buoy ant densities. They are globular structures containing polar lipids, neutral lipids, and proteins in distinct arrangements. The major classes are named appropriately HDL, LDL, and VLDL. The delipidated protein components, called apolipoproteins, have in many instances been isolated, fully characterized, and even sequenced. The most intensively studied are the two main components ofHDL, apoLp-Gln-I and apoLp GIn-II, where the C-terminal residue is used for identification. These are commonly called apo-A-I and apo-A-II and have been purified from the blood of various animals. The second most intensively studied apolipoproteins are the "C-proteins," which are components of both human HDL and VLDL. Most of the work has focused on apoLp-Ala (C-III), which actually consists of two prqteins differing only in their sialic acid content. ApoLp-Ser or C-I is another protein of this class that has received some attention. The apolipoproteins are water soluble in the absence of detergents, but like the water-soluble integral membrane proteins they have a tendency to aggregate. The lipid binding properties of severai proteins that do not associate with lipids in vivo, e.g. ribonuclease and lysozyme. have been studied. For convenience, these proteins will be termed hydrophilic proteins. Serum albumin will also be included in this group. Lipid Self-Association
To study the dynamics and structure of a protein-lipid complex, it is evident that the solution properties of the lipids must be understood. Most of the lipids of structural importance in biological systems are amphipathic (amphiphilic); that is, they contain both a polar and nonpolar portion. The solution thermodynamics of amphipathic molecules has been discussed by Shinoda et al ( 15), Elworthy et al ( 1 6),
198
GENNIS & JONAS
and recently Tanford (17, 18). At sufficiently low concentrations these molecules are soluble in aqueous solution as monomers. As the lipid concentration is increased the solubility limit for the monomeric lipid is reached. If the amount of lipid present exceeds this value then a second phase is formed consisting of lipid aggregates, which may remain dispersed in solution. The lipid aggregates are called micelles and the concentration above which micelles form is called the CMC. In all aqueous micelles the geometry is such that the nonpolar groups are aggregated to exclude
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water, and the polar groups on the amphiphile are exposed to the solvent. It is important to realize that micelles are in equilibrium with monomer lipids in solu tion, and that to a first approximation the concentration of monomer at lipid concentrations above the CMC will equal the CMC. This is analogous to the situation of adding salt to a saturated salt solution; the amount dissolved does not change, only the amount in the solid phase changes. For phospholipids found in biomembranes, the CMC is so low that the concentration of free monomeric lipid is completely negligible. The CMC of dipalmitoyl phosphatidylcholine (CwPC) has been measured at 4.7 X IO-IOM
(19). The CMCs of the commonly used synthetic
lecithins dimyristoyl (CwPC), dilauroyl (C1rPC), and longer chain (C1S-C24), natu
rally occurring phosphatidylcholines have not been reported but they are sufficiently
low so that monomer concentrations in aqueous solution can be neglected and only the micelles need be considered. However, for detergents used in lipid binding studies with proteins, the monomeric lipid concentration is often considerable; CMCs range from
10-6 to 10-2 M ( 16,20,2 1). A complete classification of lipids in
terms of their behavior in water is given by Small (22,23). Detergents generally have a high relative balance of polar versus nonpolar character, which results in greater solubility in water and also results in smaller, globular micelles. Hence detergents are referred to as soluble amphiphiles. Insoluble amphiphiles are either present as a separate solid or liquid phase in bulk water (e.g. di- and triglycerides, sterol esters, cholesterol) or swell in water to form liquid crystalline phases that can be dispersed further in solution (e.g. physphoglycerides and sphingolipids). A number of compre hensive reviews cover the preparative methods and properties of various forms of aggregates of insoluble, swelling amphiphiles in the presence of water: multilamellar vesicles (24-26), single-walled vesicles (27,28), BLM (29, 30), and monolayers (31, 32). Multilamellar vesicles and single-walled vesicles are often referred to as lipo somes. Multilamellar vesicles are formed when some phospholipids, e.g. lecithin, are dispersed in water. The bilayer in this case takes the form of a series of concentric spheres; sonication yields single-walled vesicles where the bilayer forms a spherical shell with a diameter of several hundred
A.
BLMs are usually formed by painting
a hydrocarbon solution containing the phospholipid over a small aperture. Drainage of the solvent results in the formation of a phospholipid bilayer, although some of the solvent, such as decane, may remain in the film. Monolayers are formed by spreading insoluble amphiphiles in a monomolecular layer between air anC: an aqueous subphase.
The term micelle is used here in its broadest sense, referring to all amphipathic
lipid aggregates. Micelles may be found in a variety of sizes and shapes. They range
PROTEIN-LIPID INTERACTIONS
199
from compact spheres. as for SDS, to extended planar bilayers, as for many phos pholipids. The factors that determine the
CMC as well as micelle size and shape
have been described by ranford (17). To understand how proteins interact with self-aggregating lipids. it is important to understand the forces stabilizing the lipid micelle. A simple thermodynamic argument easily relates the CMC to the standard unitary free-energy difference between the monomeric lipid free in aqueous solution and the lipid molecule within the micelle. This
.6.GOM1C can be divided into two
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components, that which favors micelle formation and that which opposes micelle formation
(17). The driving force for micelle formation is the hydrophobic force,
i.e. the tendency of the nonpolar groups to self-aggregate and exclude water. The opposing force is largely due to repulsions between polar groups within the micelle. This may be electrostatic or may involve the interactions of bulky, hydrated polar groups. A parameter of critical importance to both components of
AGOM1C is the
surface area-to-volume ratio of the micelle. It can be assumed that there are no holes inside of a globular micelle, so the micelle volume is strictly determined by the number of molecules in the micel1e. Hence, the surface:volume ratio is a measure of the surface area per lipid molecule. This can be directly related to the repulsive free energy between head groups, as well as to the degree to which water is excluded from the nonpolar micelle interior. In computing the surface:volume ratio for a micelle it should be assumed that one of the dimensions describing the micelle, such as the sphere diameter or bilayer thickness, is equal to twice the extended length of the amphiphile molecule. Simple, charged detergents such as SDS require a large surface:volume ratio due to charge repulsions. Hence they fonn spherical micelles where the radius is the length of the alkyl chain. As the preferred surface:volume ratio decreases, the micelle shape distorts to become ellipsoid. The limiting form of the prolate ellipsoid is an infinite cylinder, and the limiting form of the oblate ellipsoid is the infinite planar bilayer. The presence of two long alkyl chains on the diacyl phospholipids or sphingoJipids is responsibiJe for the thermodynamic stability of the bilayer, which is the structure with the smallest possible surface:volume ratio. If only one chain is present, as in lyso-phosphatides. then the volume requirement per molecule is reduced and a globular micelle results. Very short alkyl chains accomplish the same result; for example. ell-pc does not form a bilayer. Short alkyl chains reduce the magnitude of the driving force for micelle formation and increase the
CMC. Bilayer stability is also a function of alkyl chain length (33). It is impor
tant to keep these considerations in mind when studying the interaction between proteins and these various lipids. The thermodynamics governing the
CMC as well as micelle size and shape may
be influenced by numerous agents. For example, specific counter-ion binding and ionic strength are important considerations in the solution properties of charged amphiphiles. Those proteins capable of converting phospholipid bilayers into globu lar phospholipid micelle-protein complexes. e.g. apoLp-Ala complexes with CwPC
(34), are essentially stabilizing a micellar form with a larger surface area per mole cule. It is likely that interactions with the micelle surface are an important feature of such complexes.
200
GENNIS & JONAS
Binding Studies The emphasis of modern physical biochemistry is on the study of molecular interac tions. The thermodynamic formalism is, of course, the same regardless of the nature of the interacting species and the pathway followed in reaching equilibrium. The
(a) stoichiometry of (b) the strength of the interaction, i.e.
usual information derived from equilibrium binding studies is the interacting components in the complex,
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binding free energy, and (c) evidence relating to the nature of the interaction when there is more than one ligand, i.e. cooperative or non-cooperative. Methods for the presentation of binding data and for the determination of binding parameters have been described by Klotz (35) and Weber (36, 37). Binding parameters can be readily determined for interactions of small ligands with proteins or nucleic acids, as well as for protein-protein and protein-nucleic acid interactions. There are serious difficulties, however, in performing and interpreting similar binding experiments with protein-lipid complexes. To analyze a solution using equilibrium thermodynamics, it is necessary to know the identity of all the species present in solution and to be able to determine their concentrations. This can be difficult or impossible when dealing with lipids that self-associate and proteins that have a tendency to self-aggregate. Another problem that is becoming apparent is how to recognize whether a solution is truly at equilibrium. There are numerous reports dealing with protein-lipid complexes where the nature of the complex formed is entirely dependent on the method of preparation and not on the final solution conditions. For example, a stable solution containing egg lecithin vesicles and a BSA-Iecithin complex can be obtained if the BSA is·allowed to interact with lecithin that has been coated on Celite (38, 39). If the lipid and protein are simply mixed in neutral solution, there is no evidence of complex formation. In dealing with synthetic phospholipids, it is clear that the rate of complex formation with proteins such as cytochrome
bs (40) or apoLp-Ala (41) is a very strong function of the
physical state of the lipid bilayer. In the case of another protein, Css-isoprenoid alcohol phosphokinase, the state of protein aggregation in the presence of lipids depends on the method of preparation rather than on the final solution conditions
(42). It is likely that different preparations involve distinct metastable states. Many procedures for both functional and structural reconstitution of protein phospholipid complexes involve the use of detergents or sonication to disrupt the protein and lipid self-aggregates and to facilitate complex formation. The purpose of most such experiments is not to rigorously study the thermodynamics of binding but rather to examine the structure or function of the isolated complex. Complexes of proteins with phospholipid bilayer vesicles are well suited for many kinds of experiments. One can utilize the inside-outside asymmetry of the sealed vesicle to assay for solute transport activity and to reconstitute ion pumps and segments of the electron transport and oxidative phosphorylation systems. Also, the protein-protein interactions and the distribution and the nature of the lipids in the bilayer near the prot.eins can be studied. However, there are some useful questions that are more easily answered by studying detergent binding to these proteins.
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PROTEIN-LIPID INTERACTIONS
201
Mild detergents, such as Triton X-IOO and DOC, appear to bind to proteins at biologically meaningful lipid-binding areas. Only proteins that form lipid complexes in vivo have a substantial affinity for these detergents (43--46) (see Table 2). Protein detergent complexes thus formed can be characterized and studied. In some cases, the biochemical activity is preserved so that one has some assurance that the native structure is intact. Studying such complexes has several advantages. The solutions are normally clear and are much more suitable for examining the optical properties of the protein than with phospholipid dispersions. Also, the high CMC values for most detergents compared with diacyl phospholipids permits one to study the interaction between the protein and monomeric amphiphile species. This can be useful for examining discrete binding sites or classes of lipid binding sites and permits a quantitative analysis of binding specificity as a function of lipid or deter gent structure. A distinct drawback to detergent binding experiments is that these protein-detergent complexes are clearly only an approximation of the situation in natural protein-lipid complexes. However, in conjunction with other approaches, detergent binding is a useful method that has yielded considerable quantitative data. The next two sections of this article are dedicated to the discussion of experimen tal techniques and systems used in the study of protein-detergent interactions and protein-phospholipid interactions. Most of the work on the binding of biologically important lipids with purified proteins has been performed using phospholipid liposomes; however, a short section on the use of BLM and monolayers has also been included. The last section of this article identifies the major concepts and problems relating to protein-lipid interactions in membranes and presents relevant experimen tal results.
PROTEIN-DETERGENT INTERACTIONS Because of their similarities with membrane lipids, their ability to displace lipids from protein-lipid complexes, and their moderate solubility in water, detergents have been employed to probe the amphiphile binding properties of serum apolipo proteins (47, 48-52) and of purified membrane proteins (43, 45, 5 3-61) in efforts to elucidate the lipid binding behavior of these proteins. Bin din g of Monom eric Detergen ts
There are only a few water-soluble proteins, namely serum albumin (see 62; 44, 63-66), 13-lactoglobulin (67-69), human high-density apolipoproteins A-I and A-II (48- 5 1 ), and pyruvate oxidase from E. coli (70-72), for which binding of detergent s to specific high-affinity sites has been demonstrated. The binding of detergents to these sites does not result in protein denaturation. Binding isotherms have been obtained for each one of t hese proteins with a number of ionic detergents below CMC and the binding parameters for the complexes are listed in Table I. The interpretation of some of t hese data may be subject to ambiguity when the binding isotherms for the strong binding sites and the initial cooperative binding sites are not clearly resolved.
202
GENNIS & JONAS
Table 1
High-affinity sites of proteins for detergents
Number
of sites
(r.,.)
Reference(s)
Sodium tetradecyl sulfate
8-10 10-11
9. 1 X 10-7 6.7 X 10-7 6.7 X 10-5 5 X 10-5 1.5 X 10-5
64 64 66 44 44
SDS
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BSAa
Kd
Detergent
Protein
C'4NMe3Cl
Triton X-IOO
4 4
DOC
4
67-69
f3-Lactoglobulin
SDS
2
Human Apo-A-I
SDS
4 4 4?
5 X 10-5 5 X 10-5 2.5 X 1O-7?
49 48
4
5 X 10-5 10-4
49 48
C'4NMe3C1
C16-lyso PC Human
Apo-A-II
Pyruvate oxidaseb
SDS C'4NMe3Cl SDS
10
2-4
Further
Quick links to online content
Ann. Rev. Biophys. Bioeng. Copyright
co.·
1977 by
1977. 6:195-238
Annual Reviews Inc. All rights reserved
Annu. Rev. Biophys. Bioeng. 1977.6:195-238. Downloaded from www.annualreviews.org by University of South Carolina - Columbia on 07/08/13. For personal use only.
PROTEIN-LIPID
-:-9093
INTERACTIONSl Robert B. Gennis2 Department of Chemistry, School of Chemical Scien ces, U n iversity of Illinois,
Urbana. Illinois 6 1 80 I
Ana Jonas3 School of Basic Medical Sciences and Department of Biochemistry, School of Chemical Sc iences. Un iversity of Illinois, Urbana, Illinois 6 1 801
INTRODUCTION In re ce nt years it has become apparent that many critical functions of living cells
are performed by membrane-associated proteins; as a consequence, research in diverse areas of biochemistry has converged on the study of membrane structure and function. The fundamental questions regarding all membrane-associated processes depend on the clear understanding of the dynamics and structure of protein-lipid complexes. It is the purpose of this article to review the current state of research in this relatively new area of protein-lipid interactions and to discuss the concepts. experimental techniques. and difficulties within the context of modern biophysical lAbbreviations: HDL, serum high density lipoprotein; LDL. serum low density lipoprotein; VLDL. serum very low d en si ty l ipop rotei n ; apoLP - Glnl = apo-A-I, major protein component of HDL; apo LP-G \n II = apo-A-II, second most abundant p rotein of human HDL; apoLP Ala = C-IlI. protein component common to human HDL and VLDL with C-terminal ala; apoL P-Ser = C-l, protein component common to human HDL and VLDL wit h C-terminal ser; CMC. critical micelle concentration; Cl4"PC, di myr istoyl phosphatidylcholine; BLM. black lipid membrane; Cwlyso PC, palmitoyl-Iysophosphatidylcholine; PE, phosphat i dyl
ethanolamine; SDS, sodium dodecyl sulfate; CI4NMe]CI. tetradecyltri methylammonium chlo ride; BSA, bovine serum albumin; BDH, fj-hydroxybutyrate dehydrogenase; CD. circular
dichroism; Tm. melting or critical temperature; DOC, deoxycholate; CL, cardiolipin; TPP,
thiamin pyrophosphate; DSC . differential scanning calorimet ry. IThis work was completed with the support of grants from the NIH, HL16101, and the Illinois Heart Association, N-2. RBG is also grat efu l for suppo rt from Career Development
Award PHS K04 H L00040. 3Established Investigator of the American Heart Association. This work was comp le ted under the support of grants PHS-HEW H L 1 6059 and IIlinos Illinois Heart Association N-2A. 195
Annu. Rev. Biophys. Bioeng. 1977.6:195-238. Downloaded from www.annualreviews.org by University of South Carolina - Columbia on 07/08/13. For personal use only.
196
GENNIS & JONAS
chemistry. Published work on protein-lipid interactions consists of a collection of studies on a number of distinct biochemical systems where the specific research questions are often unique and the methods of investigation are varied. Hopefully, the more detailed the information that becomes available for a g reater number of systems, the easier it will be to make generalizations on protein-lipid interactions and their role in membrane and lipoprotein structure and function. A major hindrance to progress in the area of protein-lipid interactions has been the paucity of well-characterized systems for study. Purified proteins that have biochemically relevant lipid-binding properties are the first prerequisite for these studies. Obviously most of the work in this area concerns membrane proteins; significant exceptions are the serum lipoproteins, soluble lipid transfer proteins, and soluble lipid metabolizing enzymes . The membrane proteins that have received the most attention are those involved in energy transduction, for example, flavo proteins and cytochromes involved in electron transport or ATPases involved in intercon verting chemical and osmotic free energy.
Fluid Mosaic Model The conceptual framework for much of membrane biochemistry today is the fluid mosaic model of Singer & Nicolson (I, 2). The basic premise is that the structural determinant for biological membranes is the phospholipid bilayer. Membrane pro teins are largely viewed as globular entities that can float on or in the lipid sea; they are pictured as being amphipathic, i.e. having distinct polar and apolar regions. The polar surfaces are exposed to the aqueous phase or may be involved in protein protein interactions whereas the hydrophobic surfaces bind to the lipids. In contrast to some earlier membrane models, the fluid mosaic model really has little to say concerning the details of protein-lipid interactions; rather, attention has been di rected quite successfully at the dynamic aspects of membranes, in particular at the lateral mobility of membrane components (3). Much of this work has concerned the study of thermally induced liquid crystal phase transitions in both model mem branes and biomembranes. This topic was recently reviewed by Melchior & Steim (4). The concept of the membrane as a two-dimensional fluid has been useful in describing these phase transition phenomena (5, 6), and this concept has been important in formulating some of our general ideas about membrane dynamics, structure, and function.
Membrane Proteins The fluid mosaic model is compatible with most arrangements for membrane pro teins. These proteins may be located on the surface of the membrane, may be immersed to varying degrees, or may penetrate the bilayer completely. Membrane proteins are divided into two operational categories: peripheral (or extrinsic) pro teins and integral (or intrinsic) proteins. Peripheral proteins are those removed from the membrane by relatively gentle techniques, such as sonication or changing ionic strength, whereas those proteins requiring more drastic methods for solubilization, such as the use of detergents or organic s olvents, are termed integral membrane proteins. It is becoming clear that this classification does not imply a clear division
Annu. Rev. Biophys. Bioeng. 1977.6:195-238. Downloaded from www.annualreviews.org by University of South Carolina - Columbia on 07/08/13. For personal use only.
PROTEIN-LIPID I NTERACTIONS
197
between those proteins that bind to the membrane by electrostatic interaction with the lipid polar head groups and those that interact through hydrophobic forces with the core of the lipid bilayer. Singer (2) has suggested that peripheral membrane proteins bind primarily to integral proteins in the bilayer rather than directly to the lipid. For example, cytochrome c, the most intensively studied peripheral protein, can bind to specific membrane proteins in the mitochondrial membrane (7,8). One general principle that applies is that the binding between lipids and proteins in most naturally occurring protein-lipid systems is noncovalent. Notable exceptions are the murein lipoprotein from the Escherichia coli envelope (9, 10) and the penicillinase from Bacillus licheniformis (11). General aspects of membrane protein s have re cently been reviewed by Guidotti (2). A very important consideration in working with most lipid-binding protei ns is their strong tendency to aggregate. Presumably, the hydrophobic surfaces normally
in contact with lipids in vivo will result in protein aggregation when the lipids are removed. In some cases aggregates are limited in size and are water soluble; how ever, more frequently detergents must be used to maintain the protein in a water soluble form. Serum Apolipoprotein s
Serum lipoproteins (13, 14) are isolated and characterized according to their buoy ant densities. They are globular structures containing polar lipids, neutral lipids, and proteins in distinct arrangements. The major classes are named appropriately HDL, LDL, and VLDL. The delipidated protein components, called apolipoproteins, have in many instances been isolated, fully characterized, and even sequenced. The most intensively studied are the two main components ofHDL, apoLp-Gln-I and apoLp GIn-II, where the C-terminal residue is used for identification. These are commonly called apo-A-I and apo-A-II and have been purified from the blood of various animals. The second most intensively studied apolipoproteins are the "C-proteins," which are components of both human HDL and VLDL. Most of the work has focused on apoLp-Ala (C-III), which actually consists of two prqteins differing only in their sialic acid content. ApoLp-Ser or C-I is another protein of this class that has received some attention. The apolipoproteins are water soluble in the absence of detergents, but like the water-soluble integral membrane proteins they have a tendency to aggregate. The lipid binding properties of severai proteins that do not associate with lipids in vivo, e.g. ribonuclease and lysozyme. have been studied. For convenience, these proteins will be termed hydrophilic proteins. Serum albumin will also be included in this group. Lipid Self-Association
To study the dynamics and structure of a protein-lipid complex, it is evident that the solution properties of the lipids must be understood. Most of the lipids of structural importance in biological systems are amphipathic (amphiphilic); that is, they contain both a polar and nonpolar portion. The solution thermodynamics of amphipathic molecules has been discussed by Shinoda et al ( 15), Elworthy et al ( 1 6),
198
GENNIS & JONAS
and recently Tanford (17, 18). At sufficiently low concentrations these molecules are soluble in aqueous solution as monomers. As the lipid concentration is increased the solubility limit for the monomeric lipid is reached. If the amount of lipid present exceeds this value then a second phase is formed consisting of lipid aggregates, which may remain dispersed in solution. The lipid aggregates are called micelles and the concentration above which micelles form is called the CMC. In all aqueous micelles the geometry is such that the nonpolar groups are aggregated to exclude
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water, and the polar groups on the amphiphile are exposed to the solvent. It is important to realize that micelles are in equilibrium with monomer lipids in solu tion, and that to a first approximation the concentration of monomer at lipid concentrations above the CMC will equal the CMC. This is analogous to the situation of adding salt to a saturated salt solution; the amount dissolved does not change, only the amount in the solid phase changes. For phospholipids found in biomembranes, the CMC is so low that the concentration of free monomeric lipid is completely negligible. The CMC of dipalmitoyl phosphatidylcholine (CwPC) has been measured at 4.7 X IO-IOM
(19). The CMCs of the commonly used synthetic
lecithins dimyristoyl (CwPC), dilauroyl (C1rPC), and longer chain (C1S-C24), natu
rally occurring phosphatidylcholines have not been reported but they are sufficiently
low so that monomer concentrations in aqueous solution can be neglected and only the micelles need be considered. However, for detergents used in lipid binding studies with proteins, the monomeric lipid concentration is often considerable; CMCs range from
10-6 to 10-2 M ( 16,20,2 1). A complete classification of lipids in
terms of their behavior in water is given by Small (22,23). Detergents generally have a high relative balance of polar versus nonpolar character, which results in greater solubility in water and also results in smaller, globular micelles. Hence detergents are referred to as soluble amphiphiles. Insoluble amphiphiles are either present as a separate solid or liquid phase in bulk water (e.g. di- and triglycerides, sterol esters, cholesterol) or swell in water to form liquid crystalline phases that can be dispersed further in solution (e.g. physphoglycerides and sphingolipids). A number of compre hensive reviews cover the preparative methods and properties of various forms of aggregates of insoluble, swelling amphiphiles in the presence of water: multilamellar vesicles (24-26), single-walled vesicles (27,28), BLM (29, 30), and monolayers (31, 32). Multilamellar vesicles and single-walled vesicles are often referred to as lipo somes. Multilamellar vesicles are formed when some phospholipids, e.g. lecithin, are dispersed in water. The bilayer in this case takes the form of a series of concentric spheres; sonication yields single-walled vesicles where the bilayer forms a spherical shell with a diameter of several hundred
A.
BLMs are usually formed by painting
a hydrocarbon solution containing the phospholipid over a small aperture. Drainage of the solvent results in the formation of a phospholipid bilayer, although some of the solvent, such as decane, may remain in the film. Monolayers are formed by spreading insoluble amphiphiles in a monomolecular layer between air anC: an aqueous subphase.
The term micelle is used here in its broadest sense, referring to all amphipathic
lipid aggregates. Micelles may be found in a variety of sizes and shapes. They range
PROTEIN-LIPID INTERACTIONS
199
from compact spheres. as for SDS, to extended planar bilayers, as for many phos pholipids. The factors that determine the
CMC as well as micelle size and shape
have been described by ranford (17). To understand how proteins interact with self-aggregating lipids. it is important to understand the forces stabilizing the lipid micelle. A simple thermodynamic argument easily relates the CMC to the standard unitary free-energy difference between the monomeric lipid free in aqueous solution and the lipid molecule within the micelle. This
.6.GOM1C can be divided into two
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components, that which favors micelle formation and that which opposes micelle formation
(17). The driving force for micelle formation is the hydrophobic force,
i.e. the tendency of the nonpolar groups to self-aggregate and exclude water. The opposing force is largely due to repulsions between polar groups within the micelle. This may be electrostatic or may involve the interactions of bulky, hydrated polar groups. A parameter of critical importance to both components of
AGOM1C is the
surface area-to-volume ratio of the micelle. It can be assumed that there are no holes inside of a globular micelle, so the micelle volume is strictly determined by the number of molecules in the micel1e. Hence, the surface:volume ratio is a measure of the surface area per lipid molecule. This can be directly related to the repulsive free energy between head groups, as well as to the degree to which water is excluded from the nonpolar micelle interior. In computing the surface:volume ratio for a micelle it should be assumed that one of the dimensions describing the micelle, such as the sphere diameter or bilayer thickness, is equal to twice the extended length of the amphiphile molecule. Simple, charged detergents such as SDS require a large surface:volume ratio due to charge repulsions. Hence they fonn spherical micelles where the radius is the length of the alkyl chain. As the preferred surface:volume ratio decreases, the micelle shape distorts to become ellipsoid. The limiting form of the prolate ellipsoid is an infinite cylinder, and the limiting form of the oblate ellipsoid is the infinite planar bilayer. The presence of two long alkyl chains on the diacyl phospholipids or sphingoJipids is responsibiJe for the thermodynamic stability of the bilayer, which is the structure with the smallest possible surface:volume ratio. If only one chain is present, as in lyso-phosphatides. then the volume requirement per molecule is reduced and a globular micelle results. Very short alkyl chains accomplish the same result; for example. ell-pc does not form a bilayer. Short alkyl chains reduce the magnitude of the driving force for micelle formation and increase the
CMC. Bilayer stability is also a function of alkyl chain length (33). It is impor
tant to keep these considerations in mind when studying the interaction between proteins and these various lipids. The thermodynamics governing the
CMC as well as micelle size and shape may
be influenced by numerous agents. For example, specific counter-ion binding and ionic strength are important considerations in the solution properties of charged amphiphiles. Those proteins capable of converting phospholipid bilayers into globu lar phospholipid micelle-protein complexes. e.g. apoLp-Ala complexes with CwPC
(34), are essentially stabilizing a micellar form with a larger surface area per mole cule. It is likely that interactions with the micelle surface are an important feature of such complexes.
200
GENNIS & JONAS
Binding Studies The emphasis of modern physical biochemistry is on the study of molecular interac tions. The thermodynamic formalism is, of course, the same regardless of the nature of the interacting species and the pathway followed in reaching equilibrium. The
(a) stoichiometry of (b) the strength of the interaction, i.e.
usual information derived from equilibrium binding studies is the interacting components in the complex,
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binding free energy, and (c) evidence relating to the nature of the interaction when there is more than one ligand, i.e. cooperative or non-cooperative. Methods for the presentation of binding data and for the determination of binding parameters have been described by Klotz (35) and Weber (36, 37). Binding parameters can be readily determined for interactions of small ligands with proteins or nucleic acids, as well as for protein-protein and protein-nucleic acid interactions. There are serious difficulties, however, in performing and interpreting similar binding experiments with protein-lipid complexes. To analyze a solution using equilibrium thermodynamics, it is necessary to know the identity of all the species present in solution and to be able to determine their concentrations. This can be difficult or impossible when dealing with lipids that self-associate and proteins that have a tendency to self-aggregate. Another problem that is becoming apparent is how to recognize whether a solution is truly at equilibrium. There are numerous reports dealing with protein-lipid complexes where the nature of the complex formed is entirely dependent on the method of preparation and not on the final solution conditions. For example, a stable solution containing egg lecithin vesicles and a BSA-Iecithin complex can be obtained if the BSA is·allowed to interact with lecithin that has been coated on Celite (38, 39). If the lipid and protein are simply mixed in neutral solution, there is no evidence of complex formation. In dealing with synthetic phospholipids, it is clear that the rate of complex formation with proteins such as cytochrome
bs (40) or apoLp-Ala (41) is a very strong function of the
physical state of the lipid bilayer. In the case of another protein, Css-isoprenoid alcohol phosphokinase, the state of protein aggregation in the presence of lipids depends on the method of preparation rather than on the final solution conditions
(42). It is likely that different preparations involve distinct metastable states. Many procedures for both functional and structural reconstitution of protein phospholipid complexes involve the use of detergents or sonication to disrupt the protein and lipid self-aggregates and to facilitate complex formation. The purpose of most such experiments is not to rigorously study the thermodynamics of binding but rather to examine the structure or function of the isolated complex. Complexes of proteins with phospholipid bilayer vesicles are well suited for many kinds of experiments. One can utilize the inside-outside asymmetry of the sealed vesicle to assay for solute transport activity and to reconstitute ion pumps and segments of the electron transport and oxidative phosphorylation systems. Also, the protein-protein interactions and the distribution and the nature of the lipids in the bilayer near the prot.eins can be studied. However, there are some useful questions that are more easily answered by studying detergent binding to these proteins.
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PROTEIN-LIPID INTERACTIONS
201
Mild detergents, such as Triton X-IOO and DOC, appear to bind to proteins at biologically meaningful lipid-binding areas. Only proteins that form lipid complexes in vivo have a substantial affinity for these detergents (43--46) (see Table 2). Protein detergent complexes thus formed can be characterized and studied. In some cases, the biochemical activity is preserved so that one has some assurance that the native structure is intact. Studying such complexes has several advantages. The solutions are normally clear and are much more suitable for examining the optical properties of the protein than with phospholipid dispersions. Also, the high CMC values for most detergents compared with diacyl phospholipids permits one to study the interaction between the protein and monomeric amphiphile species. This can be useful for examining discrete binding sites or classes of lipid binding sites and permits a quantitative analysis of binding specificity as a function of lipid or deter gent structure. A distinct drawback to detergent binding experiments is that these protein-detergent complexes are clearly only an approximation of the situation in natural protein-lipid complexes. However, in conjunction with other approaches, detergent binding is a useful method that has yielded considerable quantitative data. The next two sections of this article are dedicated to the discussion of experimen tal techniques and systems used in the study of protein-detergent interactions and protein-phospholipid interactions. Most of the work on the binding of biologically important lipids with purified proteins has been performed using phospholipid liposomes; however, a short section on the use of BLM and monolayers has also been included. The last section of this article identifies the major concepts and problems relating to protein-lipid interactions in membranes and presents relevant experimen tal results.
PROTEIN-DETERGENT INTERACTIONS Because of their similarities with membrane lipids, their ability to displace lipids from protein-lipid complexes, and their moderate solubility in water, detergents have been employed to probe the amphiphile binding properties of serum apolipo proteins (47, 48-52) and of purified membrane proteins (43, 45, 5 3-61) in efforts to elucidate the lipid binding behavior of these proteins. Bin din g of Monom eric Detergen ts
There are only a few water-soluble proteins, namely serum albumin (see 62; 44, 63-66), 13-lactoglobulin (67-69), human high-density apolipoproteins A-I and A-II (48- 5 1 ), and pyruvate oxidase from E. coli (70-72), for which binding of detergent s to specific high-affinity sites has been demonstrated. The binding of detergents to these sites does not result in protein denaturation. Binding isotherms have been obtained for each one of t hese proteins with a number of ionic detergents below CMC and the binding parameters for the complexes are listed in Table I. The interpretation of some of t hese data may be subject to ambiguity when the binding isotherms for the strong binding sites and the initial cooperative binding sites are not clearly resolved.
202
GENNIS & JONAS
Table 1
High-affinity sites of proteins for detergents
Number
of sites
(r.,.)
Reference(s)
Sodium tetradecyl sulfate
8-10 10-11
9. 1 X 10-7 6.7 X 10-7 6.7 X 10-5 5 X 10-5 1.5 X 10-5
64 64 66 44 44
SDS
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BSAa
Kd
Detergent
Protein
C'4NMe3Cl
Triton X-IOO
4 4
DOC
4
67-69
f3-Lactoglobulin
SDS
2
Human Apo-A-I
SDS
4 4 4?
5 X 10-5 5 X 10-5 2.5 X 1O-7?
49 48
4
5 X 10-5 10-4
49 48
C'4NMe3C1
C16-lyso PC Human
Apo-A-II
Pyruvate oxidaseb
SDS C'4NMe3Cl SDS
10
2-4
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