Journal
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
Volume 42, Issue 2-3, Pages 103-118Publisher
SPRINGER
DOI: 10.1007/s00249-013-0887-z
Keywords
Membrane protein folding; Amphipols; Kinetics; Thermodynamic stability; Outer membrane protein; A8-35
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Funding
- Deutsche Forschungsgemeinschaft [KL 1024/2-5, 2-6]
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Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured beta-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, similar to 5 and similar to 70 min, respectively, are independent of A8-35 concentration. The faster process contributed similar to 55-64 % to OmpA folding. Folding into A8-35 was faster than into dioleoylphosphatidylcholine bilayers and complete at ratios as low as similar to 0.17 g/g A8-35/OmpA, corresponding to similar 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 similar to 5-9 kJ/mol for the fast process and similar to 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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