pH-Triggered Membrane Insertion of Proteins

描述（由申请人提供）：我的研究是为了寻找两个问题的答案：蛋白质如何跨膜，以及它们如何插入脂质双层？在细胞中，这些重要功能是通过复杂的多蛋白质组装来实现的。但在酸性条件下，白喉毒素 T 结构域 (DTT) 本身会插入膜中，并将催化结构域跨过脂质双层。低 pH 值还参与膜联蛋白 12 (ANX) 的跨双层插入，膜联蛋白 12 是一个大蛋白家族的代表，与多种膜功能和多种人类疾病有关。 pH 触发的 DTT 和其他蛋白质（例如肉毒杆菌毒素）的重折叠-插入-易位不仅具有固有的重要性，而且还可以揭示膜蛋白组装和稳定性的一般物理化学原理。该资助的目的是完善这些原理，开发用于跟踪膜中蛋白质折叠转变的实验工具，并将这些原理和工具应用于 pH 诱导的 DTT 和 ANX 膜插入问题。这些新工具将利用寿命荧光方法，特别适合分析异质构象状态。螺旋肽模型将用于确定膜界面上质子化和折叠的能量学。各种光谱方法，主要是 DTT 和 ANX 的位点选择性荧光标记，将用于检验以下假设：非组成型膜蛋白的膜插入途径包含必需的界面中间状态；该中间体的形成和随后的跨双层插入是由蛋白质与膜界面之间的疏水性和静电相互作用的微妙平衡介导的。具体目标是：（1）表征DTT和ANX膜插入途径上的折叠中间体。 (2)阐明pH诱导DTT在膜上重折叠的机制。 (3) 细化使用模型螺旋肽预测膜蛋白插入/折叠的物理化学规则。 (4) 推进膜蛋白插入/折叠研究荧光方法的发展。

DESCRIPTION (provided by applicant): My research is driven by a search for answers to two questions: how do proteins cross membranes, and how do they insert into lipid bilayers? In the cell, these vital functions are achieved by complex multi-protein assemblies. But the diphtheria toxin T-domain (DTT) by itself inserts into the membrane under acidic conditions and translocates the catalytic domain across the lipid bilayer. Low pH is also involved in transbilayer insertion of annexin 12 (ANX), a representative of a large protein family that has been implicated in a variety of membrane functions and in a number of human diseases. The pH-triggered refolding-insertion-translocation of DTT and other proteins (e.g., botulinum toxin) is not only of inherent importance, but also can reveal general physicochemical principles underlying membrane protein assembly and stability. The objectives of this grant are to refine these principles, develop experimental tools for following protein folding transitions in membranes, and apply these principles and tools to the problem of pH-induced membrane insertion of DTT and ANX. These new tools will take advantage of the lifetime fluorescence methodology, uniquely suited to analyzing heterogeneous conformational states. Model helical peptides will be used to determine the energetics of protonation and folding on membrane interfaces. Various spectroscopic approaches, chiefly the site-selective fluorescence labeling of DTT and ANX, will be used to test the following hypothesis: Membrane insertion pathways for non-constitutive membrane proteins contain an obligatory interfacial intermediate state; formation of this intermediate and subsequent transbilayer insertion is mediated by a subtle balance of hydrophobic and electrostatic interactions between proteins and the membrane interface. The specific aims are: (1) Characterize folding intermediates on the membrane insertion pathway of DTT and ANX. (2) Elucidate the mechanism of pH-induced refolding of DTT on membranes. (3) Refine the physicochemical rules for predicting membrane protein insertion/folding using model helical peptides. (4) Advance the development of fluorescence methods for insertion/folding studies of membrane proteins.