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

Abstract

Extraction 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

The Formation of β-Strand Nine (β9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers

“…BamA is composed of a 16-stranded β -barrel domain and a similarly sized periplasmic domain ( Figure 1 A), which fold independently from one another [ 46 , 47 ]. To date, the folding of the secondary structure of the transmembrane β -barrel domains of outer membrane proteins has been examined either by circular dichroism [ 7 , 9 , 15 , 21 , 23 , 25 , 27 , 28 , 48 ] or by infrared spectroscopy [ 32 ]. Both methods report on the overall composition of the secondary structure but do not reveal a location of the β -structure along the polypeptide chain.…”

“…However, in contrast to the absolute maxima of fluorescence spectra, the intensity-weighted average fluorescence emission maxima correlate to the mole fractions of the folding states of each X n C-BamA in a folding or unfolding reaction; see, e.g., refs. [ 48 , 57 , 58 ].…”

Gating of β-Barrel Protein Pores, Porins, and Channels: An Old Problem with New Facets

“…Remarkably, the β strands are connected through a network of numerous hydrogen bonds between the amide and carbonyl groups of the polypeptide backbone. This dense hydrogen bonding distribution is the fundamental molecular mechanism by which a β-barrel scaffold attains its unusually high mechanical and thermodynamic stability [1]. In addition, β barrels have aromatic side chains at the water-membrane interface, thus forming stabilizing contacts with the polar headgroups and hydrophobic tails of surrounding lipids.…”