Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520, United States.
Biochemistry. 2013 Jul 9;52(27):4595-604. doi: 10.1021/bi400252k. Epub 2013 Jun 27.
We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIP's resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation.
我们使用 pHlow 插入肽（pHLIP）家族来研究天冬氨酸嵌入深度在 pH 依赖性跨膜肽插入中的作用。pHLIP 在中性 pH 下作为一个基本上无结构的单体结合在脂质双层的表面上。当 pH 值降低时，pHLIP 会自发插入跨膜作为一个跨越的α-螺旋。当 pH 值调整回中性值时，pHLIP 插入是可逆的。促进 pHLIP 插入的一个关键事件是肽的跨越结构域中天冬氨酸的质子化：这些残基的负侧链转化为不带电的极性形式，通过改变跨越结构域的疏水性来促进插入。为了进一步研究这种质子化机制，我们创建了 pHLIP 序列变体，其中两个跨越的天冬氨酸（D14 和 D25）在序列中向上或向下移动。我们假设插入状态中天冬氨酸的深度会直接影响酸性侧链的质子亲和力，从而改变 pH 依赖性插入的 pKa。为此，我们还突变了位置 11 的精氨酸，以确定精氨酸潜水是否通过影响天冬氨酸深度来调节插入 pKa。我们的结果表明，这两种类型的突变都改变了插入 pKa，支持了天冬氨酸深度是决定 pH 依赖性的参与参数的观点。我们还表明，pHLIP 的聚集抗性可以通过我们的突变来改变，这为改善 pHLIP 在靶向酸性疾病组织（如癌症和炎症）时的体内性能提供了一个新的标准。
