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首页> 外文期刊>Biochimica et biophysica acta. Biomembranes >Comparison of lipid-dependent bilayer insertion of pHLIP and its P20G variant
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Comparison of lipid-dependent bilayer insertion of pHLIP and its P20G variant

机译：脂质依赖性双层插入PHLIP及其P20G变体的比较

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Abstract The ability of the pH-Low Insertion Peptide (pHLIP) to insert into lipid membranes in a transbilayer conformation makes it an important tool for targeting acidic diseased tissues. pHLIP can also serve as a model template for thermodynamic studies of membrane insertion. We use intrinsic fluorescence and circular dichroism spectroscopy to examine the effect of replacing pHLIP's central proline on the pH-triggered lipid-dependent conformational switching of the peptide. We find that the P20G variant (pHLIP-P20G) has a higher helical propensity than the native pHLIP (pHLIP-WT), in both water:organic solvent mixtures and in the presence of lipid bilayers. Spectral shifts of tryptophan fluorescence reveal that with both pHLIP-WT and pHLIP-P20G, the deeply penetrating interfacial form (traditionally called State II) is populated only in pure phosphocholine bilayers. The presence of either anionic lipids or phosphatidylethanolamine leads to a much shallower penetration of the peptide (referred to here as State II S , for “shallow”). This novel state can be differentiated from soluble state by a reduction in accessibility of tryptophans to acrylamide and by FRET to vesicles doped with Dansyl-PE, but not by a spectral shift in fluorescence emission. FRET experiments indicate free energies for interfacial partitioning range from 6.2 to 6.8kcal/mol and are marginally more favorable for pHLIP-P20G. The effective pKa for the insertion of both peptides depends on the lipid composition, but is always higher for pHLIP-P20G than for pHLIP-WT by approximately one pH unit, which corresponds to a difference of 1.3kcal/mol in free energy of protonation favoring insertion of pHLIP-P20G. Graphical abstract Display Omitted Highlights ? Membrane insertion of pHLIP is modulated by lipid composition. ? The unstructured State II is only present in pure phosphatidylcholine membranes. ? A new shallow and unstructured interfacial State II S is identified. ? Interfacial partitioning free energy for both peptides ranges from 6 to 7kcal/mol. ? Protonation-dependent insertion ΔG is 1.3kcal/mol more favorable for pHLIP-P20G than pHLIP-WT.
机译：摘要pH-低插入肽（PHLIP）在转跨包构象中插入脂质膜的能力使其成为靶向酸性患病组织的重要工具。 Phlip还可以用作膜插入热力学研究的模型模板。我们使用内在荧光和圆形二色性光谱检查替代Phlip中脯氨酸对肽的pH-触发的脂质依赖性构象切换的影响。我们发现P20G变体（PHLIP-P20G）具有比天然Phlip（Phlip-WT）在水中的螺旋倾斜度更高，包括有机溶剂混合物和脂质双层的存在。色氨酸荧光的光谱偏移显示，通过Phlip-Wt和Phlip-p20g，仅在纯的磷光啉双层中填充深度渗透界面形式（传统称为状态II）。阴离子脂质或磷脂酰乙醇胺的存在导致肽的较浅的渗透（在此称为状态II S，用于“浅”）。这种新的状态可以通过降低色氨酸对丙烯酰胺的可及性和通过掺杂掺杂丹酰PE的囊泡的囊泡来区分可溶性状态来区分溶解状态，但不是通过荧光发射的光谱移位。 FRET实验表明界面分区的自由能量为6.2至6.8kcal / mol，并且对plip-p20g进行了略微更有利的。用于插入两种肽的有效PKA取决于脂质组合物，但PHLIP-P20G总是高于磷脂-WT的脂质组合物，其对应于对应于质子化的自由能量的1.​​3kcal / mol的差异。插入phlip-p20g。图形抽象显示省略了亮点？通过脂质组合物调节Phlip的膜插入。还非结构化状态II仅存在于纯磷脂酰胆碱膜中。还确定了一种新的浅和非结构化界面状态II S.还两种肽的界面分区可自由能为6至7kcal / mol。还质子化依赖性插入ΔG比plip-wt更有利于phlip-p20g。

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