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.     

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.     

Kinetic study of folding and misfolding of diacylglycerol kinase in model membranes

Despite the relevance of membrane protein misfolding to a number of common diseases, our understanding of the folding and misfolding of membrane proteins lags well behind soluble proteins. Here, the overall kinetics of membrane insertion and folding of the homotrimeric integral membrane protein diacylglycerol kinase (DAGK) is addressed. DAGK was purified into lipid/detergent-free urea and guanidinium solutions and subjected to general structural characterization. In urea, the enzyme was observed to be monomeric but maintained considerable tertiary structure. In guanidinium, it was also monomeric but exhibited much less tertiary structure. Aliquots of these DAGK stock solutions were diluted 200-fold into lipid vesicles or into detergent/lipid mixed micelles, and the rates and efficiencies of folding/insertion were monitored. Reactions were also carried out in which micellar DAGK solutions were diluted into vesicular solutions. Productive insertion of DAGK from denaturant solutions into mixed micelles occurred much more rapidly than into lipid vesicles, suggesting that bilayer transversal represents the rate-limiting step for DAGK assembly in vesicles. The efficiency of productive folding/insertion into vesicles was highest in reactions initiated with micellar DAGK stock solutions (where DAGK maintains a nativelike fold and oligomeric state) and lowest in reactions starting with guanidinium stocks (where DAGK is an unfolded monomer). Moreover, the final ratio of irreversibly misfolded DAGK to reversibly misfolded enzyme was highest following reactions initiated with guanidinium stock solutions and lowest when micellar stocks were used. Finally, it was also observed that very low concentrations of detergents were able to both enhance the bilayer insertion rate and suppress misfolding.     

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 final stages of folding of the membrane protein bacteriorhodopsin occur by kinetically indistinguishable parallel folding paths that are mediated by pH

The folding of the transmembrane protein bacteriorhodopsin that occurs during the binding of its retinal cofactor is investigated in a membrane-like environment. Changes in the retinal absorption band reveal two transient retinal-protein intermediate states, with apparent absorption maxima at 380 nm and 440 nm, respectively. Studies on a bacteriorhodopsin mutant of Lys216, which cannot bind retinal covalently, add to evidence that retinal is non-covalently bound in these intermediate states. The two retinal-protein intermediates are genuine intermediate states that form in parallel, each with an observed rate constant of 1.1 s-1. Meanwhile no formation of the folded state is detected. Folded bacteriorhodopsin, with all trans retinal covalently bound, forms from both retinal-bound intermediates with the same apparent rate constant of 0.0070 s-1 that is independent of retinal concentration. Retinal isomerisation then occurs with a rate constant of 0.00033 s-1 to give bacteriorhodopsin containing all trans and 13 cis-retinal. These results provide experimental evidence for multiple folding routes for a membrane protein that are pH dependent, with pH conditions determining the apparent folding route. These observed parallel folding paths are kinetically indistinguishable, which contrasts with most other observations of parallel folding pathways where only pathways with different kinetics have been reported. Furthermore, together with previous work, this study shows that bacteriorhodopsin has to populate at least two folding intermediates, during folding in the mixed lipid micelles investigated here, before the final fold is attained.     

Molecular dynamics of individual alpha-helices of bacteriorhodopsin in dimyristol phosphatidylocholine. I. Structure and dynamics.

Understanding the role of the lipid bilayer in membrane protein structure and dynamics is needed for tertiary structure determination methods. However, the molecular details are not well understood. Molecular dynamics computer calculations can provide insight into these molecular details of protein:lipid interactions. This paper reports on 10 simulations of individual alpha-helices in explicit lipid bilayers. The 10 helices were selected from the bacteriorhodopsin structure as representative alpha-helical membrane folding components. The bilayer is constructed of dimyristoyl phosphatidylcholine molecules. The only major difference between simulations is the primary sequence of the alpha-helix. The results show dramatic differences in motional behavior between alpha-helices. For example, helix A has much smaller root-mean-squared deviations than does helix D. This can be understood in terms of the presence of aromatic residues at the interface for helix A that are not present in helix D. Additional motions are possible for the helices that contain proline side chains relative to other amino acids. The results thus provide insight into the types of motion and the average structures possible for helices within the bilayer setting and demonstrate the strength of molecular simulations in providing molecular details that are not directly visualized in experiments.     

Manipulating the folding of membrane proteins: using the bilayer to our advantage

The folding mechanisms of integral membrane proteins have largely eluded detailed study. This is owing to the inherent difficulties in folding these hydrophobic proteins in vitro, which, in turn, reflects the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is, however, a large body of information on lipid properties and, in particular, on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to develop efficient in vitro lipid-bilayer folding systems for the membrane protein, bacteriorhodopsin. Furthermore, we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency appear to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer, and the lateral pressure that the lipid chains exert on the their neighbouring folding proteins. These are generic properties of the bilayer that can be achieved with simple mixtures of biological lipids, and are not specific to the lipids studied here. These bilayer properties also seem to be important in modulating the function of several membrane proteins, as well as the function of membranes in vivo. Thus, it seems likely that careful manipulations of lipid properties will shed light on the forces that drive membrane protein folding, and will aid the development of bilayer folding systems for other membrane proteins.     

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

Effective detergent/lipid ratios in the solubilization of phosphatidylcholine vesicles by Triton X-100.

Effective detergent:lipid ratios (i.e. molar ratios in the mixed aggregates, vesicles or micelles) have been estimated for the solubilization of phosphatidylcholine vesicles by Triton X-100. Effective molar ratios are given for both the onset and the completion of bilayer solubilization; small unilamellar, large unilamellar and multilamellar vesicles have been used. Effective detergent:lipid ratios are independent of phospholipid concentration, and their use allows a deeper understanding of membrane-surfactant interactions.     

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.     

Structure and plasticity of the human immunodeficiency virus gp41 fusion domain in lipid micelles and bilayers

A thorough understanding of the structure of fusion domains of enveloped viruses in changing lipid environments helps us to formulate mechanistic models on how they might function in mediating viral entry by membrane fusion. We have expressed the N-terminal fusion domain of HIV-1 gp41 as a construct that is water-soluble in the absence of membranes, but that also binds with high affinity to lipid micelles and bilayers in their presence. We have solved the structure and studied the dynamics of this domain bound to dodecylphosphocholine micelles by homo- and heteronuclear NMR spectroscopy. The fusion peptide forms a stable hydrophobic helix from Ile(4) to Ala(14), but is increasingly more disordered and dynamic in a segment of intermediate polarity that stretches from Ala(15) to Ser(23). When bound to lipid bilayers at low concentration, the HIV fusion domain is also largely alpha-helical, as determined by CD and FTIR spectroscopy. However, at higher protein/lipid ratios, the domain is partially converted to form beta-structures in lipid bilayers. Controlled lipid mixing occurs at concentrations that support the alpha-helical, but not the beta-strand conformation.     

Spontaneous transmembrane insertion of membrane proteins into lipid vesicles facilitated by short-chain lecithins

Functional reconstitution of the membrane protein bacteriorhodopsin into lipid vesicles is achieved by mixing aqueous suspensions of long-chain lecithins and purple membrane with the short-chain lecithin diheptanoylphosphatidylcholine (20 mol % of total lipid). The membrane protein is transmembranously inserted in the lipid bilayer of the vesicle and highly active as a light-energized proton pump. This rapid, easy, and gentle procedure might allow functional reconstitution of other membrane systems and isolated membrane proteins as well.     

Kinetics of dye efflux and lipid flip-flop induced by delta-lysin in phosphatidylcholine vesicles and the mechanism of graded release by amphipathic, alpha-helical peptides

Delta-lysin is a 26-residue, amphipathic, alpha-helical peptide of bacterial origin. Its specificity is to some extent complementary to that of antimicrobial peptides. Therefore, understanding its mechanism is important for the more general goal of understanding the interaction of amphipathic peptides with membranes. In this article, we show that delta-lysin induces graded efflux of the contents of phosphatidylcholine vesicles. In view of this finding, carboxyfluorescein efflux kinetics were re-examined. In addition, peptide-induced lipid flip-flop was directly measured using fluorescence energy transfer between two lipid fluorophores initially placed on opposite leaflets of the bilayer. Carboxyfluorescein efflux and lipid flip-flop occur with essentially identical rate constants. On the basis of a detailed, quantitative analysis of the kinetics of peptide-vesicle interactions, we conclude that the peptide translocates across the bilayer as a small, transient aggregate, most likely a trimer. Dye efflux and lipid flip-flop occur concomitantly with the transient peptide-induced perturbation of the membrane. The experimental data are interpreted by comparing the predictions of the available models for the mechanism of action of amphipathic alpha-helical peptides. We demonstrate how the combination of the quantitative kinetic analysis, graded efflux, and reversibility of the peptide-vesicle interaction can be used to reject several models for this particular peptide. Two models are compatible with the data, the toroidal pore model and the sinking raft model. On the basis of the small aggregate size, a trimer, the latter appears to be more plausible. Some significant modifications are introduced in the sinking raft model to take into account the new finding of graded dye release. Furthermore, we present an explanation for the phenomenon of graded release in general, which, contrary to all-or-none efflux, has not been well-understood.     

Ion permeation across the bilayer of annealed phosphatidylcholine vesicles at elevated temperatures. Concentration dependence and the micelle-bilayer dynamic equilibrium

The relative stability of the lipid bilayer toward ions above the crystalline to liquid-crystalline phase transition temperature has been studied under isotonic conditions for small annealed vesicles of dilauroyl (DLPC), dimyristoyl (DMPC), diplamitoyl (DPPC), and distearoyl (DSPC) phosphatidylcholine by using lanthanide ions as a probe. The bilayer stability increased as the chain length of the lipid fatty acid increased, and a rapid translocation of ions across the bilayer started at about 60, 70, and 80° C for DMPC, DPPC, and DSPC vesicles, respectively. The bilayer of DLPC vesicles is apparently permeable for the tested ions even at room temperature. Two other important phenomena concomitant with the observed translocation of ions were found. Firstly, the ion leakage occurred in an “an-or-none” fashion, i.e. as soon as the vesicles start to become permeable toward ions, the concentration of ions in the intra-and extravesicular media are equalized within a short time. Secondly, the rate of the relative number of inward facing lipid molecules which become exposed to extravesicularly added paramagnetic lanthanide is a function of the inverse phosphatidylcholine concentration. This feature explicitly excludes the possibilities that the observed ion leakage occurs through a diffusion, pore formation, or through the rupture of vesicle walls induced by vesicle-vesicle collisions. We instead propose as the most probable mechanism that a dynamic equilibrium between the various states of the phosphatidylcholine molecules in water, such as monomers, micelles, vesicles, and multilamellar liposomes, is in fact responsible for the observed phenomena.     

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.     

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.     

Unravelling the folding of bacteriorhodopsin

The folding mechanism of integral membrane proteins has eluded detailed study, largely as a result of the inherent difficulties in folding these proteins in vitro. The seven-transmembrane helical protein bacteriorhodopsin has, however, allowed major advances to be made, not just on the folding of this particular protein, but also on the factors governing folding of transmembrane alpha-helical proteins in general. This review focusses on kinetic and equilibrium studies of bacteriorhodopsin folding in vitro. It covers what is currently known about secondary and tertiary structure formation as well as the events accompanying retinal binding, for protein in detergent and lipid systems, including native membrane samples.     

Circular dichroic analysis of the secondary structure of myelin basic protein and derived peptides bound to detergents and to lipid vesicles.

In aqueous solution bovine myelin basic protein exhibits no significant alpha-helical or beta-pleated sheet structure. However, in vivo this protein is associated largely with the myelin membrane: experiments have therefore been performed to determine the structure of the protein when bound to lipid bilayers. Circular dichroism spectra show that this protein undergoes a major conformational change on binding to lipid bilayer vesicles formed from diacylphosphatidylserine or diacylphosphatidic acid, and on binding to micelles of several detergents. Association with diacylphosphatidylcholine failed to induce a structural change: this observation is interpreted in terms of an earlier report that lysophosphatidylcholine does increase the alpha-helical content of basic protein. These circular dichroism measurements and studies of the binding to the bilayer-forming lipids appear to provide support for significant hydrophobic lipid-protein interactions. Similar studies using two peptides produced by cleavf basic protein indicate that a major structure-forming region in the middle of the protein has been disrupted by this scission.