The spontaneous insertion of helical transmembrane (TM) polypeptides into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion. A bottleneck for understanding these three steps is the lack of experimental approaches to perturb membrane-bound hydrophobic polypeptides out of equilibrium rapidly and reversibly. Here, we report on a 24-residues-long hydrophobic α-helical polypeptide, covalently coupled to an azobenzene photoswitch (KCALP-azo), which displays a light-controllable TM/MI equilibrium in hydrated lipid bilayers. FTIR spectroscopy reveals that trans KCALP-azo folds as a TM α-helix (TM topology). After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), the helical structure of KCALP-azo is maintained, but its helix tilt increased from 32 ± 5° to 79 ± 8°, indication of a reversible TM-to-MI transition. Further analysis indicates that this transition is incomplete, with cis KCALP-azo existing in a ∼90% TM and ∼10% MI mixture.

螺旋跨膜 (TM) 多肽自发插入脂质双层由三个连续平衡驱动：溶液-膜界面 (MI) 分区、非结构化到螺旋折叠和 MI 到 TM 螺旋插入。理解这三个步骤的一个瓶颈是缺乏快速和可逆地扰乱膜结合疏水性多肽平衡的实验方法。在这里，我们报告了 24 个残基长的疏水性 α-螺旋多肽，它与偶氮苯光开关（KCALP-azo）共价偶联，在水合脂质双层中显示出光可控的 TM/MI 平衡。FTIR 光谱显示反式KCALP-偶氮折叠为 TM α-螺旋（TM 拓扑）。后反式-到-顺偶氮苯部分用紫外线光异构化（用蓝光反转），保持 KCALP-偶氮的螺旋结构，但其螺旋倾斜从 32 ± 5° 增加到 79 ± 8°，表明 TM-to-MI 可逆过渡。进一步的分析表明这种转变是不完全的，顺式KCALP-偶氮存在于约 90% 的 TM 和约 10% 的 MI 混合物中。