Scott Haden L, Westerfield Justin M, Barrera Francisco N
Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee.
Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee.
Biophys J. 2017 Aug 22;113(4):869-879. doi: 10.1016/j.bpj.2017.06.065.
The pH-low insertion peptide (pHLIP) is a leading peptide technology to target the extracellular acidosis that characterizes solid tumors. The pHLIP binds to lipid membranes, and responds to acidification by undergoing a coupled folding/membrane insertion process. In the final transmembrane state, the C terminus of pHLIP gets exposed to the cytoplasm of the target cell, providing a means to translocate membrane-impermeable drug cargoes across the plasma membrane of cancer cells. There exists a need to develop improved pHLIP variants to target tumors with greater efficiency. Characterization of such variants typically relies on determining the pK parameter, the pH midpoint of peptide insertion into the lipid bilayer. Here we report that the value of the pK can be strongly dependent on the method used for its determination. Membrane insertion of pHLIP involves at least four intermediate states, which are believed to be linked to the staggered titration of key acidic residues. We propose that some spectroscopic methods are influenced more heavily by specific membrane folding intermediates, and as a result yield different pK values. To address this potential problem, we have devised an assay to independently monitor the environment of the two termini of pHLIP. This approach provides insights into the conformation pHLIP adopts immediately before the establishment of the transmembrane configuration. Additionally, our data indicate that the membrane translocation of the C terminus of pHLIP, the folding step more directly relevant to drug delivery, occurs at more acidic pH values than previously considered. Consequently, such a pK difference could have substantial ramifications for assessing the translocation of drug cargoes conjugated to pHLIP.
pH 低插入肽（pHLIP）是一种针对实体瘤所特有的细胞外酸中毒的领先肽技术。pHLIP 与脂质膜结合，并通过耦合折叠/膜插入过程对酸化作出反应。在最终的跨膜状态下，pHLIP 的 C 末端暴露于靶细胞的细胞质中，为将膜不可渗透的药物货物转运穿过癌细胞的质膜提供了一种手段。需要开发改进的 pHLIP 变体以更高效地靶向肿瘤。此类变体的表征通常依赖于确定 pK 参数，即肽插入脂质双层的 pH 中点。在此我们报告，pK 的值可能强烈依赖于用于确定它的方法。pHLIP 的膜插入涉及至少四个中间状态，据信这些状态与关键酸性残基的交错滴定有关。我们提出，一些光谱方法受特定膜折叠中间体的影响更大，因此会产生不同的 pK 值。为了解决这个潜在问题，我们设计了一种测定方法来独立监测 pHLIP 两个末端的环境。这种方法提供了对 pHLIP 在跨膜构型建立之前立即采用的构象的深入了解。此外，我们的数据表明，pHLIP 的 C 末端的膜转运，即与药物递送更直接相关的折叠步骤，发生在比以前认为的更酸性的 pH 值下。因此，这样的 pK 差异可能对评估与 pHLIP 偶联的药物货物的转运产生重大影响。
