Physics Department, University of Rhode Island, Kingston, Rhode Island, USA.
Biophys J. 2012 Apr 18;102(8):1846-55. doi: 10.1016/j.bpj.2012.03.021.
The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.
pH（低）插入肽（pHLIP）的膜相关折叠/展开为研究序列变化如何影响肽插入双层膜的动力学和途径提供了机会。在这里，我们介绍了带有不同数量带电残基的几种 pHLIP 变体、在肽的膜插入端带有附加极性货物的 pHLIP 变体以及在跨膜螺旋的开始、中间和末端处具有三个单个色氨酸变体的稳态和动力学研究结果。每个 pHLIP 变体都表现出与脂质双层的 pH 依赖性相互作用。虽然插入端可质子化残基的数量不会影响跨膜形成螺旋结构的最终形成，但它与肽插入的时间、折叠途径上中间状态的数量以及解折叠和退出的速率相关。在肽的插入端存在极性货物会导致插入途径上出现中间状态。货物极性与插入速率降低相关。我们得出结论，折叠和展开途径上中间状态的存在不是必需的，并且在非带电和非极性插入端的简单多肽的情况下，折叠和展开似乎是全或无的转变。我们提出了一种与膜相关的插入/折叠和出口/解折叠的模型，并讨论了这些观察结果对设计用于直接跨细胞膜转运极性治疗和诊断货物分子的新型输送剂的重要性。
