Lipid-assisted Protein Folding

phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol cardiolipin phosphatidylserine lipopolysaccharide monosialoganglioside Although there has been significant progress in our understanding of how water-soluble proteins fold (1Fenton W.A. Horwich A. Protein Sci. 1997; 6: 743-760Crossref PubMed Scopus (329) Google Scholar, 2Ellis R.J. Hartl F.U. Curr. Opin. Struct. Biol. 1999; 9: 102-110Crossref PubMed Scopus (269) Google Scholar), the factors and mechanism driving correct folding of integral membrane proteins are largely unknown. The folding of membrane proteins, like their soluble counterparts, is dictated by their amino acid sequence and their environment (Fig. 1). Integral membrane proteins can also interact with other proteins within the membrane and with the hydrophobic and hydrophilic components of the lipid bilayer itself during and after attainment of native structure. The role of lipids as an important structure-forming environment was elucidated during the last decade (3Marsh D. Watts A. New Comprehensive Biochemistry: Protein-Lipid Interactions. 25. Elsevier Science Publishers B.V., Amsterdam1993: 41-66Google Scholar). However, the role individual lipids play as part of the protein folding machinery has been largely ignored. Are individual lipids mobilized to protect and guide the nascent polypeptide chain during its membrane assembly? Do lipids act as specific molecular chaperones or transient ligands during the assembly of a membrane protein? The main experimental approach to study soluble protein folding has been to investigate refolding of denatured (unfolded) protein back to the native state (4Anfinsen C.B. Sheraga H.A. Adv. Protein Chem. 1975; 29: 205-300Crossref PubMed Scopus (812) Google Scholar). The discovery of protein molecular chaperones indicated that in vivo folding of proteins is a more complex process than initially thought (5Pelham H.R. Cell. 1986; 46: 959-961Abstract Full Text PDF PubMed Scopus (1146) Google Scholar). Similarly, the folding of membrane proteins during in vitro renaturation may greatly differ from in vivo folding, which involves interaction with the phospholipid bilayer. Therefore, detergent/lipid micelles and lipid vesicles have been included in the renaturation solution used to dilute the denaturant to non-denaturating conditions (6Huang K.S. Bayley H. Liao M.-J. London E. Korana H. J. Biol. Chem. 1981; 256: 3802-3809Abstract Full Text PDF PubMed Google Scholar) because they mimic the properties of the lipid bilayer. Using this approach, the α-helical protein bacteriorhodopsin was the first membrane protein refolded from the denatured state in detergent to a folded state in detergent/lipid micelles (7London E. Korana H.G. J. Biol. Chem. 1982; 257: 7003-7011Abstract Full Text PDF PubMed Google Scholar) and lipid vesicles (8Curran A.R. Templer R.H. Booth P.J. Biochemistry. 1999; 38: 9328-9336Crossref PubMed Scopus (111) Google Scholar, 9Booth P.J. Curran A.R. Curr. Opin. Struct. Biol. 1999; 9: 115-121Crossref PubMed Scopus (96) Google Scholar). α-Helix formation (the rate-limiting step in folding) is much slower for bacteriorhodopsin relative to that of soluble proteins. Folding, as well as membrane insertion, is slowed even more as the proportion of dimyristoyl-PC1 is increased in dimyristoyl-PC/dihexanoyl-PC mixed vesicles or as the non-bilayer-forming lipid phosphatidylethanolamine (PE) is added to PC vesicles. These results indicate that an increase in the radius of curvature of the bilayer (or departure from a flat bilayer) may slow α-helix formation within or inhibit insertion of polypeptide into the membrane bilayer. The kinetics of refolding of the Escherichia coli membrane protein, OmpA, in lipid vesicles has also been studied (10Kleinschmidt J.H. Tamm L.K. Biochemistry. 1996; 35: 12993-13000Crossref PubMed Scopus (149) Google Scholar, 11Surrey T. Jahnig F. J. Biol. Chem. 1995; 270: 28199-28203Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar). Surprisingly, the yield in renaturation of this β-barrel membrane protein was considerably higher in dimyristoyl-PC/dimyristoyl-PG (80/20) mixed bilayers than in mixtures of 1-palmitoyl 2-oleoyl-PE/1-palmitoyl 2-oleoyl-PG (80/20) mixed bilayers that more closely mimic the lipid composition of the E. coli inner cytoplasmic membrane (13Jahnig F. Surrey T. von Heijne G. Membrane Protein Assembly. R. G. Landes Co., Austin, TX1997: 83-98Google Scholar). This protein resides in the outer membrane of E. coli but must pass through the inner membrane where presumably it must maintain a more unfolded state. In these studies the folding of integral membrane proteins has been investigated as a function of the collective properties of lipid bilayers, i.e. as a function of the hydrophobicity (14Carpenter K.A. Wilkes B.C. De Lean A. Fournier A. Schiller P.W. Biopolymers. 1997; 42: 37-48Crossref PubMed Google Scholar), electrostatics (15Rankin S.E. Watts A. Pinheiro T.J.T. Biochemistry. 1998; 37: 12588-12595Crossref PubMed Scopus (48) Google Scholar), rigidity (16Booth P.J. Riley M.L. Flitsch S.L. Templer R.H. Faroog A. Curran A.R. Chadborn N. Wright P. Biochemistry. 1997; 36: 197-203Crossref PubMed Scopus (105) Google Scholar), and intrinsic radius of curvature (9Booth P.J. Curran A.R. Curr. Opin. Struct. Biol. 1999; 9: 115-121Crossref PubMed Scopus (96) Google Scholar). No systematic investigation of the possible role of specific lipids or the chemical properties of membrane lipids on the yield of refolding, formation of refolding intermediates, or alteration of the refolding pathway was included in these studies. However, these reports suggest that membrane protein folding is more complex than interaction with the lipid bilayer approximated as a simple hydrophobic core bounded by water interfaces. The role of protein molecular chaperones in directing protein folding, preventing protein misfolding (17Frydman J. Hartl F.U. Science. 1996; 272: 1497-1502Crossref PubMed Scopus (219) Google Scholar), and even unfolding proteins (18Shtilerman M. Lorimer G.H. Englander S.W. Science. 1999; 284: 822-825Crossref PubMed Scopus (265) Google Scholar) is now well established for the conformational maturation of soluble and membrane proteins (19Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (164) Google Scholar, 20Tatu U. Helenius A. J. Cell Biol. 1997; 136: 555-565Crossref PubMed Scopus (187) Google Scholar). The presence of protein molecular chaperones in refolding protocols can significantly increase the yield of native structure. Evidence is now accumulating that specific lipids may also participate as molecular chaperones in the folding and possibly the unfolding of integral membrane proteins. The development of mutants of E. coli (21Dowhan W. Annu. Rev. Biochem. 1997; 66: 199-232Crossref PubMed Scopus (786) Google Scholar, 22Dowhan W. Biochim. Biophys. Acta. 1998; 1376: 446-455Crossref Scopus (24) Google Scholar) in which the membrane phospholipid composition can be readily altered has made it possible to separate phospholipid-assisted and -unassisted folding events and study the role of individual phospholipids in protein folding. The absence of the major phospholipid PE in one such mutant results in the misfolding of the polytopic membrane protein lactose permease (LacY) without affecting its insertion into the membrane. Refolding dependent on phospholipid of unfolded LacY was monitored using the Eastern-Western blotting procedure (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). This procedure involves the transfer of proteins from a SDS-polyacrylamide gel by standard Western blotting methodology to a solid support coated with phospholipids that interact with partially denatured proteins as they refold. These refolding experiments have uncovered a role for PE as a non-protein molecular chaperone. LacY initially assembled in the presence of PE but not in its absence has full biological function (25Bogdanov M. Dowhan W. J. Biol. Chem. 1995; 270: 732-739Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and displays a continuous epitope (4B1) recognized by the conformation-dependent monoclonal antibody 4B1 (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). This epitope is in a periplasmic loop near the middle of LacY between transmembrane helices VII and VIII. The proper conformation of this epitope is required for full function of LacY as a transporter (26Sun J. Wu J. Carrasco N. Kaback H.R. Biochemistry. 1996; 35: 990-998Crossref PubMed Scopus (85) Google Scholar) and depends on exposure to PE during assembly (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar,24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). Sufficient conformational information was retained to detect epitope 4B1 after partial unfolding of LacY by SDS, complete removal of PE, and refolding in the absence of PE. Therefore, once information is imparted by PE during LacY folding in vivo, the presence of PE is no longer required to maintain proper conformation of this epitope consistent with the primary definition of a molecular chaperone (27Ellis R.J. Biochem. Biophys. Res. Commun. 1997; 238: 687-692Crossref PubMed Scopus (53) Google Scholar). LacY lacking recognition by monoclonal antibody 4B1, because of initial assembly in the absence of PE, can be induced to form this epitope if partially denatured and then renatured in the presence of PE or phosphatidylserine (PS) specifically; other phospholipids such as PC, PG, or cardiolipin (CL) do not support the regain of epitope 4B1 during refolding. Thus, the misfolded conformation of LacY can be corrected by specific lipids (absent during in vivoassembly) by interaction with non-native LacY, also consistent with an important criterion in the definition of a molecular chaperone (27Ellis R.J. Biochem. Biophys. Res. Commun. 1997; 238: 687-692Crossref PubMed Scopus (53) Google Scholar). Refolding of denatured full-length protein is not an ideal model because the folding of the nascent peptide in vivo begins during translation (28Fedorov A.N. Baldwin T.O. J. Biol. Chem. 1997; 272: 32715-32718Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Moreover, molecular chaperones were shown to interact differently with denatured proteins than with their newly translated counterparts (17Frydman J. Hartl F.U. Science. 1996; 272: 1497-1502Crossref PubMed Scopus (219) Google Scholar). Molecular chaperones can bind transiently to nascent proteins and prevent premature folding (28Fedorov A.N. Baldwin T.O. J. Biol. Chem. 1997; 272: 32715-32718Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) or can assist in folding by binding post-translationally at a later stage of assembly (29Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1470) Google Scholar). The liposome-induced conformational transition of colicin E1 occurs concomitantly with insertion into membranes (30Mel S.F. Stroud R.M. Biochemistry. 1993; 32: 2082-2089Crossref PubMed Scopus (37) Google Scholar, 31van der Goot F.G. Gonzalez-Manas J.M. Lakey J.H. Pattus F. Nature. 1991; 354: 408-410Crossref PubMed Scopus (421) Google Scholar). The rate of unfolding of acetylcholine esterase from Torpedo californica was greatly enhanced in the presence of PC vesicles with concomitant insertion of the protein into the lipid bilayer (32Shin I. Kreimer D. Silman I. Weiner Sci. U. A. 1997; PubMed Scopus Google Scholar). β-barrel membrane proteins, folding and membrane insertion are that (10Kleinschmidt J.H. Tamm L.K. Biochemistry. 1996; 35: 12993-13000Crossref PubMed Scopus (149) Google J.H. T. Tamm L.K. Biochemistry. 1999; 38: PubMed Scopus (123) Google Scholar). However, by using mutants lacking PE with the to PE post-translationally to LacY assembled in the the process of membrane insertion was shown to be separate from a step of conformational maturation specific PE M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). In of LacY in the presence of membranes lacking PE with or of PE in the of the epitope In of PE in membranes lacking PE and in vivo LacY also in the of epitope PE to be required in a step of conformational maturation after membrane insertion and for the transition from near native to the native state. This stage is to the of of protein molecular chaperones J. Biol. 1999; Scholar) and is consistent with the that LacY in the absence of PE is partially as a vivo and in (25Bogdanov M. Dowhan W. J. Biol. Chem. 1995; 270: 732-739Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). LacY and assembled in the absence of PE also full function and epitope 4B1 after of PE in P. and W. PE can act as a non-protein molecular chaperone a that the stage folding of the for phospholipids as molecular chaperones is there molecular of lipids other than PE that are transiently in the folding of integral membrane proteins. reports on the refolding, and assembly of outer membrane of E. coli suggest a molecular chaperone role for lipopolysaccharide Refolding of denatured in the presence of and not with other lipids M. I. W. U. J. Biochem. PubMed Scopus Google Scholar). form of was in the inner membrane that a conformational interaction with the protein membrane R. H. I. U. J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar). 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This interaction the of into an form or into the This is the first of molecular chaperone to a form and to protein into a These to a of that lipids may bind to of unfolded proteins, the of misfolding or Therefore, lipids act in an to protein molecular to the of non-protein chaperones (27Ellis R.J. Biochem. Biophys. Res. Commun. 1997; 238: 687-692Crossref PubMed Scopus (53) Google Scholar), which and A. T. N. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, W. A. G. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). lipids chemical in the acid with the major individual molecular in the to Dowhan W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In lipids such as the or R.M. Biochim. Biophys. Acta. 1998; 1376: PubMed Scopus Google Scholar). 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