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.
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Notes
The molecular mass of A8-35 was previously reported to be 8–10 kDa, depending on the source of polyacrylic acid. This estimate was based on size exclusion chromatography analyses in aqueous solvent using polyethyleneglycol polymers as standards of molecular mass (Gohon et al. 2006; Tribet et al. 1996). A recent reexamination of this issue, using absolute calibration, has yielded the revised number-averaged mass 〈M n〉 ≈ 4.3 kDa (Giusti and Rieger, personal communication). This value will be used in the present article. This revision does not affect any of the conclusions that have been published thus far regarding the mass, composition and properties of A8-35 particles and TMP/A8-35 complexes.
Abbreviations
- A8-35:
-
A specific type of amphipol
- APol:
-
Amphipol
- Borate:
-
Sodium tetraborate-10-hydrate
- diC10PC:
-
1,2-Dicapryl-sn-glycero-3-phosphocholine
- diC12PC:
-
1,2-Dilauroyl-sn-glycero-3-phosphocholine
- diC12PG:
-
1,2-Dilauroyl-sn-glycero-3-phosphoglycerol
- DOPC:
-
1,2-Dioleoyl-sn-glycero-3-phosphocholine
- EDTA:
-
Ethylenediaminetetraacetic acid
- FomA:
-
Major outer membrane protein from Fusobacterium nucleatum
- KTSE:
-
Kinetics of tertiary structure formation determined by electrophoresis
- LDAO:
-
N-Lauryl-N,N-dimethylammonium-N-oxide
- MOMP:
-
Major outer membrane protein from Chlamydia trachomatis
- OMP:
-
Outer membrane protein
- OmpA:
-
Outer membrane protein A from Escherichia coli
- PAGE:
-
Polyacrylamide gel electrophoresis
- SDS:
-
Sodium dodecyl sulfate
- TMP:
-
Transmembrane protein
- Tris:
-
Tris-(hydroxymethyl)-aminomethane
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Acknowledgments
Particular thanks are due to Fabrice Giusti (CNRS UMR 7099, Paris) for synthesizing the batches of A8-35 used in the present work, as well as for information about the revised estimate of its average molecular mass. This work was supported by grants KL 1024/2-5 and 2-6 from the Deutsche Forschungsgemeinschaft to J.H.K.
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Special issue: Structure, function, folding and assembly of membrane proteins—insight from Biophysics.
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Pocanschi, C.L., Popot, JL. & Kleinschmidt, J.H. Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35. Eur Biophys J 42, 103–118 (2013). https://doi.org/10.1007/s00249-013-0887-z
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DOI: https://doi.org/10.1007/s00249-013-0887-z