pH-Triggered Membrane Insertion of Proteins

描述(申请人提供)：我的研究是为了寻找两个问题的答案：蛋白质是如何跨膜的，以及它们是如何插入脂类双层的？在细胞中，这些重要功能是通过复杂的多蛋白质组装实现的。但白喉毒素T结构域(DTT)本身在酸性条件下插入到膜中，并使催化结构域跨过脂质双层。低pH也与膜联蛋白12(ANX)的跨双层插入有关，ANX是一个大的蛋白质家族的代表，与多种膜功能和许多人类疾病有关。PH触发的DTT和其他蛋白质(如肉毒杆菌毒素)的重折叠-插入-易位不仅具有内在的重要性，而且可以揭示膜蛋白组装和稳定性的一般物理化学原理。这笔赠款的目标是提炼这些原理，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的膜插入问题。这些新工具将利用寿命荧光方法，特别适合于分析非均相构象状态。模型螺旋肽将被用来确定质子化和膜界面折叠的能量学。不同的光谱方法，主要是DTT和ANX的位置选择性荧光标记，将被用来检验以下假设：非构成膜蛋白的膜插入途径包含一个必需的界面中间态；这个中间层和随后的跨双层插入的形成是由蛋白质和膜界面之间疏水和静电相互作用的微妙平衡所介导的。具体目的是：(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在膜上折叠的机理。(3)完善了用螺旋肽模型预测膜蛋白插入/折叠的物理化学规则。(4)发展用于膜蛋白插入/折叠研究的荧光方法。

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.