The Formation of ß-Strand Nine (ß9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers.

Transmembrane proteins span lipid bilayer membranes and serve essential functions in all living cells. Membrane-inserted domains are of either α-helical or ß-barrel structure. Despite their biological importance, the biophysical mechanisms of the folding and insertion of proteins into membranes are not well understood. While the relative composition of the secondary structure has been examined by circular dichroism spectroscopy in folding studies for several outer membrane proteins, it is currently not known how individual ß-strands fold. Here, the folding and insertion of the ß-barrel assembly machinery protein A (BamA) from the outer membrane of Escherichia coli into lipid bilayers were investigated, and the formation of strand nine (ß9) of BamA was examined. Eight single-cysteine mutants of BamA were overexpressed and isolated in unfolded form in 8 M urea. In each of these mutants, one of the residues of strand ß9, from R572 to V579, was replaced by a cysteine and labeled with the fluorophore IAEDANS for site-directed fluorescence spectroscopy. Upon urea-dilution, the mutants folded into the native structure and were inserted into lipid bilayers of dilauroylphosphatidylcholine, similar to wild-type BamA. An aqueous and a membrane-adsorbed folding intermediate of BamA could be identified by strong shifts in the intensity maxima of the IAEDANS fluorescence of the labeled mutants of BamA towards shorter wavelengths, even in the absence of lipid bilayers. The shifts were greatest for membrane-adsorbed mutants and smaller for the inserted, folded mutants or the aqueous intermediates. The spectra of the mutants V573C-, L575C-, G577C-, and V579C-BamA, facing the lipid bilayer, displayed stronger shifts than the spectra recorded for the mutants R572C-, N574C-, T576C-, and K578C-BamA, facing the ß-barrel lumen, in both the membrane-adsorbed form and the folded, inserted form. This alternating pattern was neither observed for the IAEDANS spectra of the unfolded forms nor for the water-collapsed forms, indicating that strand ß9 forms in a membrane-adsorbed folding intermediate of BamA. The combination of cysteine scanning mutagenesis and site-directed fluorescence labeling is shown to be a valuable tool in examining the local secondary structure formation of transmembrane proteins.

The Thermodynamic Stability of Membrane Proteins in Micelles and Lipid Bilayers Investigated with the Ferrichrom Receptor FhuA.

Extraction of integral membrane proteins into detergents for structural and functional studies often leads to a strong loss in protein stability. The impact of the lipid bilayer on the thermodynamic stability of an integral membrane protein in comparison to its solubilized form in detergent was examined and compared for FhuA from Escherichia coli and for a mutant, FhuAΔ5-160, lacking the N-terminal cork domain. Urea-induced unfolding was monitored by fluorescence spectroscopy to determine the effective free energies [Formula: see text] of unfolding. To obtain enthalpic and entropic contributions of unfolding of FhuA, [Formula: see text] were determined at various temperatures. When solubilized in LDAO detergent, wt-FhuA and FhuAΔ5-160 unfolded in a single step. The 155-residue cork domain stabilized wt-FhuA by [Formula: see text]~ 40 kJ/mol. Reconstituted into lipid bilayers, wt-FhuA unfolded in two steps, while FhuAΔ5-160 unfolded in a single step, indicating an uncoupled unfolding of the cork domain. For FhuAΔ5-160 at 35 °C, [Formula: see text] increased from ~ 5 kJ/mol in LDAO micelles to about ~ 20 kJ/mol in lipid bilayers, while the temperature of unfolding increased from TM ~ 49 °C in LDAO micelles to TM ~ 75 °C in lipid bilayers. Enthalpies [Formula: see text]were much larger than free energies [Formula: see text], for FhuAΔ5-160 and for wt-FhuA, and compensated by a large gain of entropy upon unfolding. The gain in conformational entropy is expected to be similar for unfolding of FhuA from micelles or bilayers. The strongly increased TM and [Formula: see text] observed for the lipid bilayer-reconstituted FhuA in comparison to the LDAO-solubilized forms, therefore, very likely arise from a much-increased solvation entropy of FhuA in bilayers.

Kinetics of Insertion and Folding of Outer Membrane Proteins by Gel Electrophoresis.

To examine the mechanisms of folding and insertion of TMPs into membranes, kinetic studies are instrumental, for example, for the analysis of folding steps and involved intermediates or for the determination of activation energies. For many ß-barrel transmembrane proteins (ß-TMPs) it has been shown that the folded, functional form can be separated from the unfolded form by a simple electrophoretic mobility assay. The only requirements for a separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are that the folded form is sufficiently stable and that the samples are not heat-denatured before the electrophoresis is performed. Many folded ß-TMPs resist the treatment with SDS at room temperature and are stable against forces during electrophoresis. On the other side, SDS also binds to unfolded forms of ß-TMPs and prevents their folding into ß-barrel structure. These observations have been used to develop a simple assay to monitor the kinetics of ß-barrel tertiary structure formation in a membrane environment by electrophoresis. A folding reaction of a ß-TMP is initiated by dilution of the denaturant in the presence of preformed lipid bilayers, proteoliposomes or membrane vesicles. At selected times, samples are taken from the reaction. In these samples, folding is stopped by addition of SDS. At the end of the entire folding reaction, all samples are analyzed by SDS-PAGE and the fractions of folded ß-TMP that they contain are determined by densitometry.An advantage of this kinetic assay is that it not only allows a direct determination of fractions of folded and unfolded forms at a selected time during folding of the ß-TMP into a membrane, but also facilitates the determination of the impact of folding factors (e.g., molecular chaperones) or folding machinery that most often have a different molecular mass and electrophoretic mobility. The assay has been very useful to examine how folding and insertion is affected by the structure of the phospholipids in the lipid bilayer and how folding machinery compensates for the presence of membrane lipids that retard folding and insertion of ß-TMPs.

Folding of ß-Barrel Membrane Proteins into Lipid Membranes by Site-Directed Fluorescence Spectroscopy.

Protein-lipid interactions are important for folding and membrane insertion of integral membrane proteins that are composed either of α-helical or of ß-barrel structure in their transmembrane domains. While α-helical transmembrane proteins fold co-translationally while they are synthesized by a ribosome, ß-barrel transmembrane proteins (ß-TMPs) fold and insert posttranslationally-in bacteria after translocation across the cytoplasmic membrane, in cell organelles of eukaryotes after import across the outer membrane of the organelle. ß-TMPs can be unfolded in aqueous solutions of chaotropic denaturants like urea and spontaneously refold upon denaturant dilution in the presence of preformed lipid bilayers. This facilitates studies on lipid interactions during folding into lipid bilayers. For several ß-TMPs, the kinetics of folding has been reported as strongly dependent on protein-lipid interactions. The kinetics of adsorption/insertion and folding of ß-TMPs can be monitored by fluorescence spectroscopy. These fluorescence methods are even more powerful when combined with site-directed mutagenesis for the preparation of mutants of a ß-TMP that are site-specifically labeled with a fluorophore or a fluorophore and fluorescence quencher or fluorescence resonance energy acceptor. Single tryptophan or single cysteine mutants of the ß-TMP allow for the investigation of local protein-lipid interactions, at specific regions within the protein. To examine the structure formation of ß-TMPs in a lipid environment, fluorescence spectroscopy has been used for double mutants of ß-TMPs that contain a fluorescent tryptophan and a spin-label, covalently attached to a cysteine as a fluorescence quencher. The sites of mutation are selected so that the tryptophan is in close proximity to the quencher at the cysteine only when the ß-TMP is folded. In a folding experiment, the evolution of fluorescence quenching as a function of time at specific sites within the protein can provide important information on the folding mechanism of the ß-TMP. Here, we report protocols to examine membrane protein folding for two ß-TMPs in a lipid environment, the outer membrane protein A from Escherichia coli, OmpA, and the voltage-dependent anion-selective channel, human isoform 1, hVDAC1, from mitochondria.

Correction To: Lipid-Protein Interactions.

This chapter was inadvertently published with the expansion of the term "MNB" printed incorrectly as "N-Methyl-N-nitrosobenzamide" under section 2.5. Instead, it should have been "Methyl 4- nitrobenzenesulfonate." This correction has been updated in the chapter.

Folding of ß-barrel membrane proteins in lipid bilayers - Unassisted and assisted folding and insertion.

In cells, ß-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 ß-sheet secondary structure. These studies therefore have allowed the investigation of membrane protein folding and insertion in great detail. Folding of ß-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 ß-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.

Folding and stability of integral membrane proteins in amphipols.

Amphipols (APols) are a family of amphipathic polymers designed to keep transmembrane proteins (TMPs) soluble in aqueous solutions in the absence of detergent. APols have proven remarkably efficient at (i) stabilizing TMPs, as compared to detergent solutions, and (ii) folding them from a denatured state to a native, functional one. The underlying physical-chemical mechanisms are discussed.

The lipid bilayer-inserted membrane protein BamA of Escherichia coli facilitates insertion and folding of outer membrane protein A from its complex with Skp.

Folding of ß-barrel membrane proteins, either from a urea-unfolded form or from chaperone-bound aqueous forms, has been characterized for pure lipid bilayers. The impact of preinserted integral proteins from biomembranes has not been examined in biophysical comparisons, but this knowledge is important for the characterization of protein assembly machinery in membranes to distinguish specific effects from unspecific effects. Here, folding was studied for a ß-barrel membrane protein, outer membrane protein A (OmpA) from Escherichia coli, in the absence and presence of two other preinserted integral proteins, BamA of the ß-barrel assembly machinery complex (BAM) from E. coli and FomA from Fusobacterium nucleatum. Three different preformed lipid membranes of phosphatidylcholine were prepared to compare the folding kinetics of OmpA, namely, proteoliposomes containing either BamA or FomA and pure liposomes. Urea-unfolded OmpA folded faster into phosphatidylcholine bilayers containing FomA than into pure lipid bilayers, but the kinetics of OmpA folding and insertion were fastest for bilayers containing BamA. Incorporation of BamA into lipid bilayers composed of phosphatidylcholine and phosphatidylethanolamine greatly weakened the inhibiting effect of phosphatidylethanolamine on the folding of OmpA. Folding of OmpA from its complex with the periplasmic chaperone Skp into bilayers composed of phosphatidylethanolamine and phosphatidylcholine was inhibited in the absence of BamA but facilitated when BamA was present, indicating an interaction of Skp-OmpA complexes with BamA.

Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35.

Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured ß-barrel membrane proteins, like outer membrane proteins OmpA from Escherichia coli and FomA from Fusobacterium nucleatum, can be folded by dilution of the denaturant urea in the presence of amphipol A8-35. Here, the folding kinetics and stability of OmpA in A8-35 have been investigated. Folding is well described by two parallel first-order processes, whose half-times, ~5 and ~70 min, respectively, are independent of A8-35 concentration. The faster process contributed ~55-64 % to OmpA folding. Folding into A8-35 was faster than into dioleoylphosphatidylcholine bilayers and complete at ratios as low as ~0.17 g/g A8-35/OmpA, corresponding to ~1-2 A8-35 molecules per OmpA. Activation energies were determined from the temperature dependence of folding kinetics, monitored both by electrophoresis, which reports on the formation of stable OmpA tertiary structure, and by fluorescence spectroscopy, which reflects changes in the environment of tryptophan side chains. The two methods yielded consistent estimates, namely ~5-9 kJ/mol for the fast process and ~29-37 kJ/mol for the slow one, which is lower than is observed for OmpA folding into dioleoylphosphatidylcholine bilayers. Folding and unfolding titrations with urea demonstrated that OmpA folding into A8-35 is reversible and that amphipol-refolded OmpA is thermodynamically stable at room temperature. Comparison of activation energies for folding and unfolding in A8-35 versus detergent indicates that stabilization of A8-35-trapped OmpA against denaturation by urea is a kinetic, not a thermodynamic phenomenon.

ß-Barrel scaffolds for the grafting of extracellular loops from G-protein-coupled receptors.

Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) ß-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural systems.

Association of neighboring ß-strands of outer membrane protein A in lipid bilayers revealed by site-directed fluorescence quenching.

We present a detailed study on the formation of neighboring ß-strands during the folding of a monomeric integral membrane protein of the ß-barrel type. ß-Strand and ß-barrel formations were investigated for the eight-stranded transmembrane domain of outer membrane protein A (OmpA) with single-tryptophan (W), single-cysteine (C) OmpA mutants. Based on the OmpA structure, W and C were introduced in two neighboring ß-strands oriented toward the hydrocarbon core of the membrane. Replaced residue pairs were closer to either the periplasmic turns (named cis-side) or the outer loops (named trans-side) of the strand. W(n)C(m) OmpA mutants containing W at position n and C at position m along the polypeptide chain were labeled at the C by a nitroxyl spin label, which is a short-range fluorescence quencher. To monitor the association of neighboring ß-strands, we determined the proximity between fluorescent W and labeled C in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native ß-strand contacts in folding experiments required the lipid membrane. Residues in the trans-side of strands ß(1), ß(2), and ß(3), represented by mutants W(15)C(35) (ß(1)ß(2), trans) and W(57)C(35) (ß(3)ß(2), trans), reached close proximity prior to residues in the N(ß(1))- and C(ß(8))-terminal strands as examined for mutants W(15)C(162) (ß(1)ß(8), trans) and W(7)C(170) (ß(1)ß(8), cis). Tryptophan and cysteine converged slightly faster in W(15)C(162) (ß(1)ß(8), trans) than in W(7)C(170) (ß(1)ß(8), cis). The last folding step was observed for residues at the cis-ends of strands ß(1) and ß(2) for the mutant W(7)C(43) (ß(1)ß(2), cis). The data also demonstrate that the neighboring ß-strands associate upon insertion into the hydrophobic core of the lipid bilayer.

The periplasmic chaperone Skp facilitates targeting, insertion, and folding of OmpA into lipid membranes with a negative membrane surface potential.

The basic biochemical and biophysical principles by which chaperone-bound membrane proteins are targeted to the outer membrane of Gram-negative bacteria for insertion and folding are unknown. Here we compare spontaneous folding of outer membrane protein A (OmpA) of Escherichia coli from its urea-unfolded form and from the complex with its periplasmic chaperone Skp into lipid bilayers. Skp facilitated folding of OmpA into negatively charged membranes containing dioleoylphosphatidylglycerol (DOPG). In contrast, Skp strongly inhibited folding of OmpA when bilayers were composed of dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine (DOPC). These results indicate that the positively charged Skp targets OmpA to a negatively charged membrane, which facilitates the release of OmpA from its complex with Skp for subsequent folding and membrane insertion. The dual functionality of Skp as a chaperone and as a targeting protein is ideal to mediate the transport of OmpA and other outer membrane proteins across the periplasm in a folding-competent form to the outer membrane, which is negatively charged on its periplasmic side. OmpA (pI 5.5) folded most efficiently above its isoelectric point. In the absence of Skp and in contrast to folding into DOPC bilayers, insertion and folding of OmpA were retarded for membranes containing DOPG at neutral or basic pH because of electrostatic repulsion. When folding of OmpA was performed near its isoelectric point, urea dilution led to a more compact aqueous form of OmpA previously characterized by fluorescence, which folded at a much slower rate. Under conditions where two different aqueous conformations of OmpA coexisted, e.g., in the titration region of OmpA, the last step of OmpA folding could be well described by two parallel pseudo-first-order kinetic phases. In this kinetic model, the contribution of the faster folding process, but not the changes in the rate constants, determined the folding yields obtained at different pH. The faster phase dominated when the experimental conditions favored the less compact form of aqueous OmpA.

Binding regions of outer membrane protein A in complexes with the periplasmic chaperone Skp. A site-directed fluorescence study.

Periplasmic Skp facilitates folding and membrane insertion of many outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. We have examined the binding sites of outer membrane protein A (OmpA) from Escherichia coli in its complexes with the membrane protein chaperone Skp and with Skp and lipopolysaccharide (LPS) by site-directed fluorescence spectroscopy. Single-Trp OmpA mutants, W(n)-OmpA, with tryptophan at position n in the polypeptide chain were isolated in the unfolded form in 8 M urea. In five beta(x)W(n)-OmpA mutants, the tryptophan was located in beta-strand x, in four l(y)W(n)-OmpA mutants, in outer loop y, and in three t(z)W(n)-OmpA mutants in turn z of the beta-barrel transmembrane domain (TMD) of OmpA. PDW(286)-OmpA contained tryptophan in the periplasmic domain (PD). After dilution of the denaturant urea in aqueous solution, spectra indicated a more hydrophobic environment of the tryptophans in beta(x)W(n) mutants in comparison to l(y)W(n)-OmpA and t(z)W(n)-OmpA, indicating that the loops and turns form the surface of hydrophobically collapsed OmpA, while the strand regions are less exposed to water. Addition of Skp increased the fluorescence of all OmpA mutants except PDW(286)-OmpA, demonstrating binding of Skp to the entire beta-barrel domain but not to the PD of OmpA. Skp bound the TMD of OmpA asymmetrically, displaying much stronger interactions with strands beta(1) to beta(3) in the N-terminus than with strands beta(5) to beta(7) in the C-terminus. This asymmetry was not observed for the outer loops and the periplasmic turns of the TMD of OmpA. The fluorescence results demonstrated that all turns and loops l(1), l(2), and l(4) were as strongly bound to Skp as the N-terminal beta-strands. Addition of five negatively charged LPS per one preformed Skp.W(n)-OmpA complex released the C-terminal loops l(2), l(3), and l(4) of the TMD of OmpA from the complex, while its periplasmic turn regions remained bound to Skp. Our results demonstrate that interactions of Skp.OmpA complexes with LPS change the conformation of OmpA in the Skp complex for facilitated insertion and folding into membranes.

Omp85(Tt) from Thermus thermophilus HB27: an ancestral type of the Omp85 protein family.

Proteins belonging to the Omp85 family are involved in the assembly of beta-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria, and chloroplasts. The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered, including an outer membrane that is not well characterized. Neither the precise lipid composition nor much about integral membrane proteins is known. The genome of HB27 encodes one Omp85-like protein, Omp85(Tt), representing an ancestral type of this family. We overexpressed Omp85(Tt) in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified Omp85(Tt) existed mainly as a monomer, composed of two stable protease-resistant modules. Circular dichroism spectroscopy indicated predominantly beta-sheet secondary structure. Electron microscopy of negatively stained lipid-embedded Omp85(Tt) revealed ring-like structures with a central cavity of approximately 1.5 nm in diameter. Single-channel conductance recordings indicated that Omp85(Tt) forms ion channels with two different conducting states, characterized by conductances of approximately 0.4 nS and approximately 0.65 nS, respectively.

The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions.

The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various OMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltage-dependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDAC1 nor for soluble bovine serum albumin. The Skp trimer formed 1:1 complexes, OMP.Skp(3), with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP.Skp(3) complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp(3) with YaeT displayed the smallest dissociation constants, complexes with NalP the largest. OMP binding to Skp(3) was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and stability of OMP.Skp(3) complexes. Light-scattering and circular dichroism experiments demonstrated that Skp(3) remained a stable trimer from pH 3 to pH 11. In the OmpA.Skp(3) complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA.Skp(3) complexes at low stoichiometries. Acrylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpA were located closer to the surface than in binary OmpA.Skp(3) complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS.

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.

Amphipathic polymers: tools to fold integral membrane proteins to their active form.

Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

Curvature elasticity and refolding of OmpA in large unilamellar vesicles.

The stability of OmpA in large unilamellar vesicles of dilauroyl phosphatidylcholine was studied using different concentrations of urea. The effective energy of unfolding, as determined from refolding experiments, is greater than that for small sonicated unilamellar vesicles by an amount that is compatible with estimates of the elastic energy of highly curved vesicles. The on-rate for refolding and insertion is slower for large unilamellar vesicles than for small unilamellar vesicles, which indicates a contribution of vesicle strain also to the free energy of the transition state.

Membrane elastic fluctuations and the insertion and tilt of beta-barrel proteins.

Folding of porin-like beta-barrel outer membrane proteins can be achieved in the presence of phospholipid vesicles, and takes place concurrently with incorporation into the membrane. The pronounced dependence found for the insertion of the protein OmpA on membrane thickness (Kleinschmidt, J. H., and L. K. Tamm. 2002. J. Mol. Biol. 324:319-330) is analyzed in terms of the effects of out-of-plane elastic fluctuations on the area dilation modulus (Evans, E., and W. Rawicz. 1990. Phys. Rev. Lett. 64:2094-2097). For unstrained large unilamellar vesicles, the elastic free energy for membrane insertion is predicted to depend on the fourth power of the membrane thickness. The influence of thermally induced bending fluctuations on the effective tilt of the OmpA beta-barrel in disaturated phosphatidylcholine membranes of different thicknesses (Ramakrishnan, M., J. Qu, C. L. Pocanschi, J. H. Kleinschmidt, and D. Marsh. 2005. Biochemistry. 44:3515-3523) is also considered. A contribution to the orientational order parameter that scales as the inverse second power of the membrane thickness is predicted.