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.     

Interaction of DnaK with native proteins and membrane proteins correlates with their accessible hydrophobicity

Molecular chaperones are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. They interact specifically with hydrophobic sequences that are exposed in unfolded proteins, and buried in native proteins. We have studied the interaction of DnaK with native water-soluble proteins and membrane proteins. DnaK–native protein interactions are characterized by dissociation constants between 1 and 50 μM (compared with 0.01–1 μM for unfolded proteins). This affinity is within the range of most intracellular protein concentrations, suggesting that DnaK interacts with a greater number of native proteins than previously suspected. We found a correlation between the affinity of native proteins for DnaK and their affinity for hydrophobic-interaction chromatography adsorbents, suggesting that DnaK interacts with exposed hydrophobic groups in native proteins. The interaction between DnaK and membrane proteins is characterized by DnaK's high affinity for detergent-solubilized membrane proteins, and its lower affinity for membrane proteins inserted in lipid bilayers, suggesting that the chaperone can interact with the hydrophobic sequences of the former, while it cannot penetrate the hydrophobic core of lipid bilayers. Thus, the specificity of DnaK for hydrophobic sequences is involved in its interaction with not only unfolded proteins, but also native water-soluble proteins and membrane proteins. All proteins interact with DnaK according to their exposed hydrophobicity.     

The role of the lipid matrix for structure and function of the GPCR rhodopsin

Photoactivation of rhodopsin in lipid bilayers results within milliseconds in a metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium that is very sensitive to the lipid composition. It has been well established that lipid bilayers that are under negative curvature elastic stress from incorporation of lipids like phosphatidylethanolamines (PE) favor formation of MII, the rhodopsin photointermediate that is capable of activating G protein. Furthermore, formation of the MII state is favored by negatively charged lipids like phosphatidylserine and by lipids with longer hydrocarbon chains that yield bilayers with larger membrane hydrophobic thickness. Cholesterol and rhodopsin-rhodopsin interactions from crowding of rhodopsin molecules in lipid bilayers shift the MI-MII equilibrium towards MI. A variety of mechanisms seems to be responsible for the large, lipid-induced shifts between MI and MII: adjustment of the thickness of lipid bilayers to rhodopsin and adjustment of rhodopsin helicity to the thickness of bilayers, curvature elastic deformations in the lipid matrix surrounding the protein, direct interactions of PE headgroups and polyunsaturated hydrocarbon chains with rhodopsin, and direct or lipid-mediated interactions between rhodopsin molecules. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Single-Molecule Force Spectroscopy of Membrane Protein Folding

Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles.     

Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.     

Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.     

Bilayer Thickness and Curvature Influence Binding and Insertion of a pHLIP Peptide

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membrane’s hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.     

Folding of β-barrel membrane proteins in lipid bilayers — Unassisted and assisted folding and insertion

In cells, β-barrel membrane proteins are transported in unfolded form to an outer membrane into which they fold and insert. Model systems have been established to investigate the mechanisms of insertion and folding of these versatile proteins into detergent micelles, lipid bilayers and even synthetic amphipathic polymers. In these experiments, insertion into lipid membranes is initiated from unfolded forms that do not display residual β-sheet secondary structure. These studies therefore have allowed the investigation of membrane protein folding and insertion in great detail. Folding of β-barrel membrane proteins into lipid bilayers has been monitored from unfolded forms by dilution of chaotropic denaturants that keep the protein unfolded as well as from unfolded forms present in complexes with molecular chaperones from cells. This review is aimed to provide an overview of the principles and mechanisms observed for the folding of β-barrel transmembrane proteins into lipid bilayers, the importance of lipid–protein interactions and the function of molecular chaperones and folding assistants. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Membrane Morphology Is Actively Transformed by Covalent Binding of the Protein Atg8 to PE-Lipids

Autophagy is a cellular degradation pathway involving the shape transformation of lipid bilayers. During the onset of autophagy, the water-soluble protein Atg8 binds covalently to phosphatdylethanolamines (PEs) in the membrane in an ubiquitin-like reaction coupled to ATP hydrolysis. We reconstituted the Atg8 conjugation system in giant and nm-sized vesicles with a minimal set of enzymes and observed that formation of Atg8-PE on giant vesicles can cause substantial tubulation of membranes even in the absence of Atg12-Atg5-Atg16. Our findings show that ubiquitin-like processes can actively change properties of lipid membranes and that membrane crowding by proteins can be dynamically regulated in cells. Furthermore we provide evidence for curvature sorting of Atg8-PE. Curvature generation and sorting are directly linked to organelle shapes and, thus, to biological function. Our results suggest that a positive feedback exists between the ubiquitin-like reaction and the membrane curvature, which is important for dynamic shape changes of cell membranes, such as those involved in the formation of autophagosomes.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

The folding mechanism of outer membrane proteins (OMPs) of Gram-negative bacteria into lipid bilayers has been studied using OmpA of E. coli and FomA of F. nucleatum as examples. Both, OmpA and FomA are soluble in unfolded form in urea and insert and fold into phospholipid bilayers upon strong dilution of the denaturant urea. OmpA is a structural protein and forms a small ion channel, composed of an 8-stranded transmembrane beta-barrel domain. FomA is a voltage-dependent porin, predicted to form a 14 stranded beta-barrel. Both OMPs fold into a range of model membranes of very different phospholipid compositions. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers that demonstrated a highly synchronized mechanism of secondary and tertiary structure formation of beta-barrel membrane proteins. A study on FomA folding into lipid bilayers indicated the presence of parallel folding pathways for OMPs with larger transmembrane beta-barrels.     

Bicelle size modulates the rate of bacteriorhodopsin folding

The conformational equilibria of integral membrane proteins have proven extremely difficult to characterize within native lipid bilayers. To circumvent technical issues, investigations of the structure and stability of α‐helical membrane proteins are often carried out in mixed micelle or bicelle solvents that mimic the membrane and facilitate measurements of reversible folding. Under these conditions, the energetics of membrane protein folding are typically proportional to the mole fraction of an anionic detergent in the micelle. However, investigations of the folding and unfolding of bacteriorhodopsin (bR) surprisingly revealed that the folding rate is also highly sensitive to the bulk molar concentration of lipids and detergents. We show here that this rate enhancement coincides with changes in bicelle size and suggest this effect arises through restriction of the conformational search space during folding. In conjunction with previous mutagenic studies, these results provide additional evidence that a topological search limits the rate of bR folding. Furthermore, this finding provides insights into the manner by which micellar and bicellar environments influence the conformational stability of polytopic membrane proteins.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

Spontaneous Curvature,Differential Stress,and Bending Modulus of Asymmetric Lipid Membranes

Lipid bilayers can exhibit asymmetric states, in which the physical characteristics of one leaflet differ from those of the other. This most visibly manifests in a different lipid composition, but it can also involve opposing lateral stresses in each leaflet that combine to an overall vanishing membrane tension. Here, we use theoretical modeling and coarse-grained simulation to explore the interplay between a compositional asymmetry and a nonvanishing differential stress. Minimizing the total elastic energy leads to a preferred spontaneous curvature that balances torques due to both bending moments and differential stress, with sometimes unexpected consequences. For instance, asymmetric flat bilayers, whose specific areas in each leaflet are matched to those of corresponding tensionless symmetric flat membranes, still exhibit a residual differential stress because the conditions of vanishing area strain and vanishing bending moment differ. We also measure the curvature rigidity of asymmetric bilayers and find that a sufficiently strong differential stress, but not compositional asymmetry alone, can increase the bending modulus. The likely cause is a stiffening of the compressed leaflet, which appears to be related to its gel transition but not identical with it. We finally show that the impact of cholesterol on differential stress depends on the relative strength of elastic and thermodynamic driving forces: if cholesterol solvates equally well in both leaflets, it will redistribute to cancel both leaflet tensions almost completely, but if its partitioning free energy prefers one leaflet over the other, the resulting distribution bias may even create differential stress. Because cells keep most of their lipid bilayers in an asymmetric nonequilibrium steady state, our findings suggest that biomembranes are elastically more complex than previously thought: besides a spontaneous curvature, they might also exhibit significant differential stress, which could strongly affect their curvature energetics.     

Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.     

Beta-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane beta-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.     

Membrane protein folding and oligomerization: the two-stage model.

We discuss the view that the folding of many, perhaps most, integral membrane proteins can be considered as a two-stage process. In stage I, hydrophobic alpha-helices are established across the lipid bilayer. In stage II, they interact to form functional transmembrane structures. This model is suggested by the nature of transmembrane segments in known structures, refolding experiments, the assembly of integral membrane protein from fragments, and the existence of very small integral membrane protein subunits. It may extend to proteins with a variety of functions, including the formation of transmembrane aqueous channels. The model is discussed in the context of the forces involved in membrane protein folding and the interpretation of sequence data.     

Contributions of Gaussian curvature and nonconstant lipid volume to protein deformation of lipid bilayers

An elastic model for membrane deformations induced by integral membrane proteins is presented. An earlier theory is extended to account for nonvanishing saddle splay modulus within lipid monolayers and perturbations to lipid volume proximal to the protein. Analytical results are derived for the deformation profile surrounding a single cylindrical protein inclusion, which compare favorably to coarse-grained simulations over a range of protein sizes. Numerical results for multi-protein systems indicate that membrane-mediated interactions between inclusions are strongly affected by Gaussian curvature and display nonpairwise additivity. Implications for the aggregation of proteins are discussed.     

Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange

Although progress has been made in understanding the thermodynamic stability of water-soluble proteins, our understanding of the folding of membrane proteins is at a relatively primitive level. A major obstacle to understanding the folding of membrane proteins is the discovery of systems in which the folding is in thermodynamic equilibrium, and the development of methods to quantitatively assess this equilibrium in micelles and bilayers. Here, we describe the application of disulfide cross-linking to quantitatively measure the thermodynamics of membrane protein association in detergent micelles. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. The 19-46 alpha-helical transmembrane segment of the M2 protein from the influenza A virus was used as a model membrane protein system for this study. Previously it has been shown that transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. We used thiol-disulfide exchange to quantitatively measure the tetramerization equilibrium of this transmembrane protein in dodecylphosphocholine (DPC) detergent micelles. The association constants obtained agree remarkably well with those derived from analytical ultracentrifugation studies. The experimental method established herein should provide a broadly applicable tool for thermodynamic studies of folding, oligomerization and protein-protein interactions of membrane proteins.     

Membrane curvature affects the stability and folding kinetics of bacteriorhodopsin

Biological membrane is crucial for the function, stability and folding of membrane proteins. By studying the stability and folding kinetics of bacteriorhodopsin (bR) in lipid vesicles with different sizes, here we report the influence of membrane curvature (vesicle size) on the stability and folding kinetics of bR. The results show that both the stability and folding kinetics of bR can be significantly changed when reconstituted into mimic membranes with different curvatures. The stability of bR decreases and unfolding rate of bR increases with the growth of vesicle size, i.e. decrease of membrane curvature. Our results suggest that it is possible to regulate the properties of membrane proteins by changing the curvature of membranes.