2022-10, Pocanschi, Cosmin L., Kleinschmidt, Jörg

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.

2013-03, Pocanschi, Cosmin L., Popot, Jean-Luc, Kleinschmidt, Jörg

Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured β-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.

2009, Qu, Jian, Behrens-Kneip, Susanne, Holst, Otto, Kleinschmidt, Jörg

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, Wn-OmpA, with tryptophan at position n in the polypeptide chain were isolated in unfolded form in 8 M urea. In five βxWn-OmpA mutants, the tryptophan was located in β-strand x, in four lyWn-OmpA mutants, in outer loop y, and in three tzWn-OmpA mutants in turn z of the β-barrel transmembrane domain (TMD) of OmpA. PDW286-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 βxWn mutants in comparison to lyWn-OmpA and tzWn-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 PDW286-OmpA, demonstrating binding of Skp to the entire β-barrel domain, but not to the PD of OmpA. Skp bound the TMD of OmpA asymmetrically, displaying much stronger interactions with strands β1 to β3 in the N- than with strands β5 to β7 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 l1, l2, and l4 were as strongly bound to Skp as the N-terminal β-strands. Addition of five negatively charged LPS per one preformed Skp-Wn-OmpA complex released the C-terminal loops l2, l3, and l4 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.

2008, Nesper, Jutta, Brosig, Alexander, Ringler, Philippe, Patel, Geetika J., Müller, Shirley A., Kleinschmidt, Jörg, Boos, Winfried, Diederichs, Kay, Welte, Wolfram

Proteins belonging to the Omp85 family are involved in the assembly of β-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, Omp85Tt, representing an ancestral type of this family. We overexpressed Omp85Tt in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified Omp85Tt existed mainly as a monomer, composed of two stable protease-resistant modules. Circular dichroism spectroscopy indicated predominantly β-sheet secondary structure. Electron microscopy of negatively stained lipid-embedded Omp85Tt revealed ring-like structures with a central cavity of ~1.5 nm in diameter. Single-channel conductance recordings indicated that Omp85Tt forms ion channels with two different conducting states, characterized by conductances of ~0.4 nS and ~0.65 nS, respectively.

2014, Sharma, Meenakshi, Patel, Geetika J., Kleinschmidt, Jörg

BamD (YfiO) [1] is an essential component of the β-barrel assembly machine of the outer membrane of Escherichia coli. BamD is synthesized in the cytosol and translocated across the cytopasmic membrane in unfolded form for processing and transport to the outer membrane by the LOL system [2].

2011-07-25, Walser, Reto, Kleinschmidt, Jörg, Zerbe, Oliver

Graft order: The putative binding epitopes of peptides of the NPY family at their cognate GPCR, the Y1 receptor, were grafted onto a stable β‐barrel scaffold. We demonstrate the synthetic feasibility of such an approach and discuss its potential benefits. The integrity of the scaffold as well as its interaction with NPY was studied by using solution NMR spectroscopy.

2009, Patel, Geetika J., Behrens-Kneip, Susanne, Holst, Otto, Kleinschmidt, Jörg

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 dioleolylphosphatidylglycerol (DOPG). In contrast, Skp strongly inhibited folding of OmpA when bilayers were composed of dioleoylphosphatidylethanolamine and dioleoyl¬phosphatidyl¬choline (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 was 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.

2013-06-11, Patel, Geetika J., Kleinschmidt, Jörg

Folding of β-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 β-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 β-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.

2011-03-25, Kleinschmidt, Jörg, Bulieris, Paula V., Qu, Jian, Dogterom, Marileen, den Blaauwen, Tanneke

2008, Kleinschmidt, Jörg, Marsh, Derek, Vijay, N., Anbazhagan, Veerappan

FomA, the major outer membrane protein of Fusobacterium nucleatum, was expressed and purified in Escherichia coli and reconstituted from detergent in bilayer membranes of phosphatidylcholines with chain lengths from C(12:0) to C(17:0). The conformation and orientation of membrane-incorporated FomA were determined from polarized, attenuated total reflection, infrared (IR) spectroscopy, and lipid-protein interactions with FomA were characterized by using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids. Approximately 190 residues of membranous FomA are estimated to be in a !-sheet configuration from IR band fitting, which is consistent with a 14-strand transmembrane !-barrel structure. IR dichroism of FomA indicates that the !-strands are tilted by !45° relative to the sheet/barrel axis and that the order parameter of the latter displays a discontinuity corresponding to hydrophobic matching with fluid C(13:0) lipid chains. The stoichiometry (Nb ) 23 lipids/monomer) of lipid-protein interaction from EPR demonstrates that FomA is not trimeric in membranes of diC(14:0) phosphatidylcholine and is consistent with a monomeric !-barrel of 14-16 strands. The pronounced selectivity of interaction found with anionic spin-labeled lipids places basic residues of the protein in the vicinity of the polar-apolar membrane interfaces, consistent with current topology models. Comparison with similar data from the 8- to 22-stranded E. coli outer membrane proteins, OmpA, OmpG, and FhuA, supports the above conclusions.