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

Eur Biophys J. 2013 Mar;42(2-3):103-18. doi: 10.1007/s00249-013-0887-z. Epub 2013 Feb 1.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / chemistry*
  • Detergents / chemistry
  • Detergents / pharmacology
  • Escherichia coli*
  • Kinetics
  • Lipid Bilayers / pharmacology
  • Micelles
  • Polymers / pharmacology*
  • Propylamines / pharmacology*
  • Protein Folding / drug effects*
  • Protein Refolding / drug effects
  • Protein Stability / drug effects
  • Temperature

Substances

  • Bacterial Outer Membrane Proteins
  • Detergents
  • Lipid Bilayers
  • Micelles
  • Polymers
  • Propylamines
  • amphipol A8-35
  • OMPA outer membrane proteins