The Thermodynamic Stability of Membrane Proteins in Micelles and Lipid Bilayers Investigated with the Ferrichrom Receptor FhuA

Abstract

AbstractExtraction 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 $$ \Delta G{^\text{o}_{\rm u}} $$ Δ G u o of unfolding. To obtain enthalpic and entropic contributions of unfolding of FhuA, $$ \Delta G{^\text{o}_{\rm u}} $$ Δ G u o 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 $$ \Delta\Delta G{^\text{o}_{\rm u}} $$ Δ Δ G u o ~ 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, $$ \Delta G{^\text{o}_{\rm u}} $$ Δ G u o 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 $$\Delta H{_{\rm M}^\text{o}}$$ Δ H M o were much larger than free energies $$ \Delta G{^\text{o}_{\rm u}} $$ Δ G u o , 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 $$\Delta H{_{\rm M}^\text{o}}$$ Δ H M o 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. Graphical abstract