[show abstract][hide abstract] ABSTRACT: 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.
Biophysics of Structure and Mechanism 02/2013; · 2.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.
Journal of Molecular Biology 02/2006; 355(3):548-61. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The outer-membrane proteins OmpA and FhuA of Escherichia coli are monomeric beta-barrels of widely differing size. Polarized attenuated total reflection infrared spectroscopy has been used to determine the orientation of the beta-barrels in phosphatidylcholine host matrices of different lipid chain lengths. The linear dichroism of the amide I band from OmpA and FhuA in hydrated membranes generally increases with increasing chain length from diC(12:0) to diC(17:0) phosphatidylcholine, in both the fluid and gel phases. Measurements of the amide I and amide II dichroism from dry samples are used to deduce the strand tilt (beta = 46 degrees for OmpA and beta = 44.5 degrees for FhuA). These values are then used to deduce the order parameters, P(2)(cos alpha), of the beta-barrels from the amide I dichroic ratios of the hydrated membranes. The orientational ordering of the beta-barrels and their assembly in the membrane are discussed in terms of hydrophobic matching with the lipid chains.
[show abstract][hide abstract] ABSTRACT: The outer membrane proteins OmpA and FhuA of Escherichia coli were reconstituted in lipid bilayer membranes in order to study the lipid-protein interactions using spin label ESR and the barrel orientation in lipid membranes using polarised attenuated total reflection infrared spectroscopy (ATR). The proteins were reconstituted in phosphatidylglycerol and phosphatidylcholine membranes for ESR and ATR studies, respectively. The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the β-barrel, in addition to that from the fluid bilayer lipids. FhuA exhibits more pronounced lipid selectivity than does OmpA, which correlates with the predominance of positively charged residues facing the lipids in the extensions of the β-sheet and shorter interconnecting loops on the extracellular side of FhuA. The amide I dichroism of OmpA and FhuA obtained from ATR measurements for the hydrated membranes generally increases with increasing chain length from diC(12:0) to diC(17:0) phosphatidylcholine, in both the fluid and gel phases. Measurements of the amide I and amide II dichroism from dry samples were used to deduce the strand tilt (β = 46° for OmpA, and β = 44.5° for FhuA). These values are then used to deduce the orientational order parameter of the β-barrels from the amide I dichroic ratios of the hydrated membranes.
[show abstract][hide abstract] ABSTRACT: Amphipathic polymers (amphipols) are a new class of non-detergent surfactants to stabilize integral membrane proteins in their native form in aqueous solution and to preserve their activity [1, 2]. Whether denatured membrane proteins can be refolded into fully synthetic amphipols was previously not investigated.
Here we report the successful refolding of the outer membrane proteins OmpA from E. coli [3, 4] and FomA from F. nucleatum into amphipol A8-35, upon dilution of the denaturant urea. Folding of the 8-stranded β-barrel OmpA was verified by fluorescence and circular dichroism spectroscopy, by electrophoretic mobility measurements, and by single channel conductivity experiments. CD spectra of OmpA refolded in A8-35 and of OmpA refolded in LDAO-detergent were identical, indicating equivalent β-sheet secondary structure. Trypsin digestion and analysis by SDS-PAGE led to the characteristic 24 kDa fragment, indicating that A8-35 protected the OmpA transmembrane domain. OmpA in A8-35 could be delivered to black lipid films and single channel conductivity measurements indicated OmpA activity.
At 40 °C, the folding kinetics of OmpA into A8-35 were of second order and fitted to a pseudo-first order rate-law. Below 40 °C, folding kinetics were more complex and two folding phases were observed. The kinetics could be fitted to double-exponential functions. Activation energies were ~ 5-6 kJ/mol for the fast step and ~27-35 kJ/mol for the slower step. Equilibrium unfolding revealed that OmpA folding/unfolding into A8-35 is fully reversible and that amphipol-refolded OmpA is thermodynamically stable at room temperature.
 Popot JL, et al. 2003, Cell Mol Life Sci 60, 1559-1574.
 Kleinschmidt JH, 2003,Cell Mol Life Sci 60, 1547-1558.
[show abstract][hide abstract] ABSTRACT: The interaction of spin-labeled lipids with beta-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for alpha-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the beta-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane beta-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the beta-sheet and shorter interconnecting loops on the extracellular side of FhuA.
[show abstract][hide abstract] ABSTRACT: Detergent–micelle extraction of integral membrane proteins for structural and functional studies often leads to a strong loss in protein activity suggesting that the protein becomes thermodynamically less stable. To quantitatively assess the destabilization, we have performed equilibrium unfolding studies using the integral membrane protein FhuA as an example. Unfolding was monitored by fluorescence spectroscopy and the stabilities of FhuA in lipid bilayers and in detergent micelles were determined.
Wild-type (wt) FhuA consists of a 22-stranded β-barrel domain and an N-terminal cork domain (residues 1-160) inside the barrel. The free energies required for the first unfolding transition of FhuAΔ5-160 were ΔGo = 7 kJ/mol in LDAO micelles and ΔGo = 19 kJ/mol in DOPC/DOPG bilayers (containing 10% PG) at 40°C. FhuAΔ5-160 was most stable close to its isoelectric point at pH 5 and considerably destabilized at pH 3 or at pH 10. Urea unfolding of LDAO-solubilized wt-FhuA and FhuAΔ5-160, respectively, indicated that the β-barrel domain of wt-FhuA is stabilized by interactions with the cork domain. However, this effect was smaller than the strong impact of the lipid bilayer on the stability of FhuA.
[show abstract][hide abstract] ABSTRACT: FhuA (M=78kDa) is an active outer membrane transporter required for efficient iron uptake into Gram-negative bacteria. The protein consists of a 22-stranded β-barrel transmembrane domain (554 residues) and a 160 residue cork domain. Native FhuA, isolated in 0.1% LDAO, can be completely denatured in 8M urea.
We have studied the stability of wild type FhuA and of a deletion mutant lacking the cork domain (FhuAΔ5-160). The stability of wt-FhuA and FhuAΔ5-160 in detergent micelles and in lipid bilayers was investigated by fluorescence spectroscopy and electrophoresis as a function of the urea concentration. A comparison of the free energies of unfolding of wt-FhuA (ΔG = –17.4 kJ/mol) and FhuAΔ5-160 (ΔG = –2.0 kJ/mol) indicated that the cork has a stabilizing effect on the protein in detergent micelles. Moreover, the free energy of unfolding from a reconstituted state in phospholipid bilayers was dependent on the bilayer composition. FhuAΔ5-160 was stabilized in lipid bilayers (ΔG = –19.5 kJ/mol). Our results provide evidence that the structure of phospholipid bilayers increases the stability of FhuAΔ5-160 in comparison to a detergent micelle environment. wt-FhuA and FhuAΔ5-160 both develop β-sheet secondary structure, if the denaturant urea is strongly diluted in presence of preformed phospholipid bilayers.
[show abstract][hide abstract] ABSTRACT: Ferric hydroxamate uptake protein component A (FhuA) of the outer membrane membrane of E. coli is an iron siderophore receptor (78 kDa) for TonB dependent iron uptake. The protein consists of a 22-stranded b-barrel transmembrane domain (554 residues) and a 160 residue cork domain that was previously found to be instrumental in the uptake of ferrichrome iron .
In the present study, we have used circular dichroism and fluorescence spectroscopy to investigate the folding of the wild-type and of a separately isolated mutant of FhuA (FhuAD5-160) that corresponds to the amino acid sequence of the barrel domain of wild-type FhuA. For folding studies, wt-FhuA and FhuAD5-160 were isolated as described  and subsequently denatured in an 8 M urea solution. Fluorescence spectra of urea denatured FhuAD5-160 had a maximum at 355 nm. Circular dichroism spectra of the denatured protein indicated the complete loss of secondary structure in both proteins. When FhuAD5-160 was refolded into phospholipid bilayers, the fluorescence maximum shifted to 341 nm and fluorescence intensity increased by approx. 100 %. The fluorescence time courses of the folding of FhuAD5-160 into phospholipid bilayers at temperatures between 2 and 40 °C showed a strong temperature dependence. Refolding of FhuAD5-160 was also successful at pH 7 in the presence of micelles of N,N-dimethyl-N-dodecylamine-N-oxide at 40 °C as indicated by CD spectra. However, when attempts were made to refold wt-FhuA at the same conditions, wt-FhuA precipitated. Interestingly, when refolding of the wt-FhuA was performed at pH 9 or at pH 3, wt-FhuA did not precipitate and circular dichroism spectra indicated the formation of predominantly b-sheet secondary structure. Our data on the folding of FhuA and FhuAD5-160 indicates that the presence of the cork domain changes the in vitro folding pathways of the barrel domain in wt-FhuA.
1. Ferguson A. D., et al., 1998, Science 282, 2215-20.
2. Ferguson, A. D. et al., 1998, Protein Science 7, 1636-1638.