The Journal of Membrane Biology ( IF 2.3 ) Pub Date : 2022-05-13 , DOI: 10.1007/s00232-022-00238-w Cosmin L Pocanschi 1 , Jörg H Kleinschmidt 1, 2
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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 \( \Delta G{^\text{o}_{\rm u}} \) of unfolding. To obtain enthalpic and entropic contributions of unfolding of FhuA, \( \Delta G{^\text{o}_{\rm u}} \) 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 \( \Delta\Delta G{^\text{o}_{\rm u}} \)~ 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, \( \Delta G{^\text{o}_{\rm u}} \) 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 \(\Delta H{_{\rm M}^\text{o}}\)were much larger than free energies \( \Delta G{^\text{o}_{\rm u}} \), 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 \(\Delta H{_{\rm M}^\text{o}}\) 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
中文翻译：
用 Ferrichrom 受体 FhuA 研究胶束和脂质双分子层中膜蛋白的热力学稳定性
将完整的膜蛋白提取到洗涤剂中用于结构和功能研究通常会导致蛋白质稳定性的严重损失。脂质双层对整合膜蛋白的热力学稳定性的影响与其在洗涤剂中的溶解形式相比进行了检查，并与来自大肠杆菌的 FhuA和突变体 FhuAΔ5-160（缺乏 N 末端软木结构域）进行了比较。通过荧光光谱监测尿素诱导的展开，以确定展开的有效自由能\( \Delta G{^\text{o}_{\rm u}} \)。为了获得 FhuA 展开的焓和熵贡献，\( \Delta G{^\text{o}_{\rm u}} \) 在不同的温度下确定。当溶解在 LDAO 洗涤剂中时，wt-FhuA 和 FhuAΔ5-160 一步展开。155 个残基的软木结构域通过\( \Delta\Delta G{^\text{o}_{\rm u}} \) ~ 40 kJ/mol稳定了 wt-FhuA 。重组为脂质双层后，wt-FhuA 分两步展开，而 FhuAΔ5-160 一步展开，表明软木结构域的解偶联展开。对于 35 °C 下的 FhuAΔ5-160，\( \Delta G{^\text{o}_{\rm u}} \)从 LDAO 胶束中的 ~ 5 kJ/mol 增加到脂质双层中的约 ~ 20 kJ/mol ，而展开温度从 LDAO 胶束中的T M ~ 49 °C 增加到脂质双层中的T M ~ 75 °C。焓\(\Delta H{_{\rm M}^\text{o}}\)远大于自由能\( \Delta G{^\text{o}_{\rm u}} \) ，对于 FhuAΔ5-160 和 wt-FhuA，并在展开时通过大量熵增益进行补偿. FhuA 从胶束或双层展开时，构象熵的增加预计是相似的。因此，与 LDAO 溶解形式相比，脂质双层重组 FhuA 观察到的T M和\(\Delta H{_{\rm M}^\text{o}}\)显着增加，很可能来自双层中 FhuA 的溶剂化熵大大增加。
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