Irreversible misfolding of diacylglycerol kinase is independent of aggregation and occurs prior to trimerization and membrane association

Escherichia coli diacylglycerol kinase (DAGK) is a homotrimeric helical integral membrane protein in which a number of single-site mutations to cysteine are known to promote misfolding. Here, effects of other amino acid replacements have been explored using a folding assay based on the dilution of acidic urea/DAGK stock solutions into detergent/lipid mixed micelles. DAGK with an I110P or I110R mutation in the third transmembrane helix could not be purified because its expression was toxic to the E. coli host, most likely because of severe folding defects. Other mutations at Ile110 enhanced irreversible misfolding to varying degrees that generally correlated both with the polarity of the inserted amino acid and with the degree of protein destabilization. However, the I110W mutant was an exception in that it was highly misfolding prone while at the same time being more stable than the wild-type protein. This contrasts with I110Y, which also exhibited enhanced stability but folded with an efficiency similar to that of the wild type. For most mutants, the critical step leading to irreversible misfolding occurred for monomeric DAGK prior to trimerization and independent of association with mixed micelles. Misfolding of DAGK evidently involves the formation of incorrect monomer tertiary structure. Mutations appear to enhance misfolding by disfavoring the formation of correct structure rather than by directly stabilizing the misfolded state. Finally, when urea-solubilized DAGK was diluted into detergent/lipid-free buffer, it retained a significant degree of folding competency over a period of minutes. This property may be relevant to membrane protein folding in cells under conditions where the usual machinery associated with membrane integration is saturated, dysregulated, or dysfunctional.     

Reconstitutive refolding of diacylglycerol kinase, an integral membrane protein

While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason.     

Use of amphipathic polymers to deliver a membrane protein to lipid bilayers

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.     

Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane protein

Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.     

Micelle-vesicle transition in phospholipid-bile salt mixtures. A study by precision scanning calorimetry

A systematic study of the micelle-vesicle transformation in phospholipid-bile salt mixtures using differential scanning calorimetry (DSC) indicates that the lipid undergoes a variety of changes in its thermal properties as mixed micellar solutions are diluted to concentrations at which vesicles form. In the experiments, micellar solutions of 50 mg/mL total lipid, containing sodium taurocholate (TC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), are diluted to concentrations corresponding to differing extents of aggregation of the TC with phospholipid. Turbidity and equilibrium dialysis measurements are used to establish boundaries between where micelles persist and where vesicles are formed and to determine the extent of aggregation of the TC with DPPC. At molar ratios Re of bound TC to DPPC greater than 0.3, micellar solutions are formed, while at Re less than 0.15 vesicles are evident upon dilution. As the transformation from micelles to vesicles occurs, the thermal transitions in the lipid change from broad, low Cp (max) peaks in the micelle region to multiple peaks of high cooperativity in regions of composition where lamellar structures and vesicles form. The DSC curves show that in the composition region corresponding to where bilayer micelles exist a new thermal phase forms, which has a melting transition near 32 degrees C, if the solutions are allowed to equilibrate for 48 h at 21 degrees C. Furthermore, at compositions between Re = 0 and 0.25, there is metastability in the lipid when equilibrated at 21 degrees C, but heating the lipid through the thermal transitions leads to reversible behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ 19F NMR studies of an E. coli membrane protein

In this report, (19)F spin incorporation in a specific site of a specific membrane protein in E. coli was accomplished via trifluoromethyl-phenylalanine ((19) F-tfmF). Site-specific (19)F chemical shifts and longitudinal relaxation times of diacylglycerol kinase (DAGK), an E. coli membrane protein, were measured in its native membrane using in situ magic angle spinning (MAS) solid state nuclear magnetic resonance (NMR). Comparing with solution NMR data of the purified DAGK in detergent micelles, the in situ MAS-NMR data illustrated that (19)F chemical shift values of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid or lipid mimic environments, while (19)F side chain longitudinal relaxation values were probably affected by different interactions of DAGK with planar lipid bilayer versus globular detergent micelles.     

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.     

Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: homologous sites in the transmembrane domain

This work represents the first stage of thiol-based cross-linking studies to map the oligomeric interface of the homotrimeric membrane protein diacylglycerol kinase (DAGK). A total of 53 single-cysteine mutants spanning DAGK's three transmembrane segments and the first part of a cytoplasmic domain were purified and subjected to catalytic oxidation in mixed micelles. Four mutants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer disulfide bond formation between homologous sites on adjacent subunits. To establish whether the homologous sites are proximal in the ground-state conformation of DAGK or whether the disulfide bonds formed as a result of motions that brought normally distal sites into transient proximity, additional cross-linking experiments were carried out in three different milieus of varying fluidity [mixed micelles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles, and Escherichia coli membranes]. Cross-linking experiments included disulfide bond formation under three different catalytic conditions [Cu(II)-phenanthroline oxidation, I(2) oxidation, and thionitrobenzoate-based thiol exchange] and reactions with a set of bifunctional thiol-reactive chemical cross-linkers presenting two different reactive chemistries and several spacer lengths. On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a result of protein motions. Results for position 74 were ambiguous. In lipid vesicles and mixed micelles DAGK appears to execute motions that are not present in native membranes, with mobility also being higher for DAGK in mixed micelles than in POPC vesicles.     

In vitro Unfolding and Refolding of the Small Multidrug Transporter EmrE

The composition of the lipid bilayer is increasingly being recognised as important for the regulation of integral membrane protein folding and function, both in vivo and in vitro. The folding of only a few membrane proteins, however, has been characterised in different lipid environments. We have refolded the small multidrug transporter EmrE in vitro from a denatured state to a functional protein and monitored the influence of lipids on the folding process. EmrE is part of a multidrug resistance protein family that is highly conserved amongst bacteria and is responsible for bacterial resistance to toxic substances. We find that the secondary structure of EmrE is very stable and only small amounts are denatured even in the presence of unusually high denaturant concentrations involving a combination of 10 M urea and 5% SDS. Substrate binding by EmrE is recovered after refolding this denatured protein into dodecylmaltoside detergent micelles or into lipid vesicles. The yield of refolded EmrE decreases with lipid bilayer compositional changes that increase the lateral chain pressure within the bilayer, whilst conversely, the apparent rate of folding seems to increase. These results add further weight to the hypothesis that an increased lateral chain pressure hinders protein insertion across the bilayer. Once the protein is inserted, however, the greater pressure on the transmembrane helices accelerates correct packing and final folding. This work augments the relatively small number of biophysical folding studies in vitro on helical membrane proteins.     

Influence of total lipid concentration, bile salt:lecithin ratio, and cholesterol content on inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile.

We modified classic equilibrium dialysis methodology to correct for dialysant dilution and Donnan effects, and have systematically studied how variations in total lipid concentration, bile salt (taurocholate):lecithin (egg yolk) ratio, and cholesterol content influence inter-mixed micellar/vesicular (non-lecithin-associated) concentrations (IMC) of bile salts (BS) in model bile. To simulate large volumes of dialysant, the total volume (1 ml) of model bile was exchanged nine times during dialysis. When equilibrium was reached, dialysate BS concentrations plateaued, and initial and final BS concentrations in the dialysant were identical. After corrections for Donnan effects, IMC values were appreciably lower than final dialysate BS concentrations. Quasielastic light scattering was used to validate these IMC values by demonstrating that lipid particle sizes and mean scattered light intensities did not vary when model biles were diluted with aqueous BS solutions of the appropriate IMC. Micelles and vesicles were separated from cholesterol-supersaturated model bile, utilizing high performance gel chromatography with an eluant containing the IMC. Upon rechromatography of micelles and vesicles using an identical IMC, there was no net transfer of lipid between micelles and vesicles. To simulate dilution during gel filtration, model biles were diluted with 10 mM Na cholate, the prevailing literature eluant, resulting in net transfer of lipid between micelles and vesicles, the direction of which depended upon total lipid concentration and BS/lecithin ratio. Using the present methodology, we demonstrated that inter-mixed micellar/vesicular concentrations (IMC) values increased strongly (5 to 40 mM) with increases in both bile salt (BS):lecithin ratio and total lipid concentration, whereas variations in cholesterol content had no appreciable effects. For model biles with typical physiological biliary lipid compositions, IMC values exceeded the critical micellar concentration of the pure BS, implying that in cholesterol-supersaturated biles, simple BS micelles coexist with mixed BS/lecithin/cholesterol micelles and cholesterol/lecithin vesicles. We believe that this methodology allows the systematic evaluation of IMC values, with the ultimate aim of accurately separating micellar, vesicular, and potential other cholesterol-carrying particles from native bile.     

Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease.

Previously we presented evidence that phosphatidylethanolamine (PE) acts as a molecular chaperone in the folding of the polytopic membrane protein lactose permease (LacY) of Escherichia coli. Here we provide more definitive evidence supporting the chaperone properties of PE. Membrane insertion of LacY prevents its irreversible aggregation, and PE participates in a late step of conformational maturation. The temporal requirement for PE was demonstrated in vitro using a coupled translation-membrane insertion assay that allowed the separation of membrane insertion from phospholipid-assisted folding. LacY was folded properly, as assessed by recognition with conformation-specific monoclonal antibodies, when synthesized in the presence of PE-containing inside-out membrane vesicles (IOVs) or in the presence of IOVs initially lacking PE but supplemented with PE synthesized in vitro either co- or post-translationally. The presence of IOVs lacking PE and containing anionic phospholipids or no addition of IOVs resulted in misfolded or aggregated LacY, respectively. Therefore, critical folding steps occur after membrane insertion dependent on the interaction of LacY with PE to prevent illicit interactions which lead to misfolding of LacY.     

In vitro folding of KvAP, a voltage-gated K+ channel

In this contribution, we report in vitro folding of the archaebacterial voltage-gated K(+) channel, K(v)AP. We show that in vitro folding of the K(v)AP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature, and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature, and almost quantitative refolding of the K(v)AP channel is observed at 80 °C. To differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition, suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the K(v)AP channel provides a good model system for investigating the folding of a multidomain integral membrane protein.     

A critical residue in the folding pathway of an integral membrane protein

Although a number of common diseases are a direct consequence of membrane protein misfolding, studies of membrane protein folding and misfolding lag well behind those of soluble proteins. Here it is shown that an interfacial residue, Tyr16, of the integral membrane protein diacylglycerol kinase (DAGK) plays a critical role in the folding pathway of this protein. Properly folded Y16C exhibits kinetic parameters and stability similar to wild-type DAGK. However, when unfolded and then allowed to spontaneously fold in the presence of model membranes, Y16C exhibits dramatically lower rates and efficiencies of functional assembly compared to the wild-type protein. The Y16C mutant represents a class of mutations which may be commonly found in disease-related membrane proteins.     

Functionality of a membrane protein in bicelles

Bicelles are bilayered discoidal lipid-detergent assemblies which are useful as model membranes. To date, there has been no direct demonstration of functional viability for an integral membrane protein reconstituted into bicelles. In this contribution, the catalytic activity of diacylglycerol kinase (DAGK) was measured following reconstitution into several different bicelle systems and compared to activities measured in traditional mixed micelles and vesicles. For the most optimal bicelle systems tested, DAGK activities approached those observed in mixed micelles or vesicles. For some other bicellar mixtures tested, activities were much lower, with steady-state kinetic data indicating reduced V(max) rather than perturbations in substrate K(m). Catalytically, DAGK showed a strong preference for bicelles containing 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) as the detergentcomponent relative to short-chained phosphatidylcholine.DAGK also exhibited a preference for dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine bicelles relative to those of dilauroylphosphatidylcholine.     

Secondary and tertiary structure formation of the beta-barrel membrane protein OmpA is synchronized and depends on membrane thickness

The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of beta-sheet secondary structure and closure of the beta-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other beta-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of alpha-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins.     

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.     

Synergetics of the Membrane Self-Assembly: A Micelle-to-Vesicle Transition

A model approach is developed to study intermediate steps and transientstructures in a course of the membrane self-assembly. The approach isbased on investigation of mixed lipid/protein-detergent systems capable ofthe temperature induced transformation from a solubilized micellar stateto closed membrane vesicles. We performed a theoretical analysis ofself-assembling molecular structures formed in binary mixtures ofdimyristoylphosphatidylcholine (DMPC) and sodium cholate (NaC). Thetheoretical model is based on the Helfrich theory of curvature elasticity,which relates geometrical shapes of the structures to their free energy inthe Ginzburg-Landau approximation. The driving force for the shapetransformation is spontaneous curvature of amphiphilic aggregates which isnonlinearly dependent on the lipid/detergent composition. An analysis ofthe free energy in the regular solution approximation shows that theformation of mixed structures of different shapes (discoidal micelles,rod-like micelles, multilayer membrane structures and vesicles) ispossible in a certain range of detergent/lipid ratios. A transition fromthe flat discoidal micelles to the rod-like cylindrical micelles isinduced by curvature instabilities resulting from acyl chain melting andinsertion of detergent molecules into the lipid phase. Nonideal mixing ofthe NaC and DMPC molecules results in formation of nonideal cylindricalaggregates with elliptical cross section. Further dissolution of NaCmolecules in DMPC may be accompanied with a change of their orientation inthe lipid phase and leads to temperature-induced curvature instabilitiesin the highly curved cylindrical geometry. As a result the rod-likemicelles fuse into less curved bilayer structures which transformeventually to the unilamellar and multilamellar membrane vesicles. Thetheoretical analysis performed shows that a sequence of shapetransformations in the DMPC/NaC mixed systems is determined by thesynergism of four major factors: detergent/lipid ratio, temperature (acylchain melting), DMPC and NaC mixing, and reorientation of NaC molecules inmixed aggregates.     

Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol

The spontaneous incorporation of the polyene antibiotic amphotericin B from a micellar solution into phospholipid vesicles was examined as a function of the lipid composition of the vesicles and their physical state. Virtually no insertion of the antibiotic into egg phosphatidylcholine vesicles was observed even when cholesterol was also present in the bilayer. In contrast, rapid incorporation occurred into systems containing an anionic phospholipid such as phosphatidylglycerol or phosphatidylserine with the fastest rates observed for lipids containing the saturated dimyristoyl fatty acyl species. Insertion of amphotericin B into vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3 mole ratio) was rapid either above, below or within the gel-to-liquid-crystalline phase transition temperature (23 degrees C). The ability of amphotericin B to intercalate into lipid vesicles is discussed in relation to their relative bilayer stabilities.     

Kinetics of an individual transmembrane helix during bacteriorhodopsin folding

The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.     

Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.