pH-Triggered Membrane Insertion of Proteins

pH 触发的蛋白质膜插入

• 批准号: 7470181
• 负责人: ALEXEY LADOKHIN
• 金额: $ 11.76万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7470181
• 关键词: Advanced Development; Affect; Annexins; Binding; Botulinum Toxins; Catalytic Domain; Cells; Complex; Condition; Cysteine; DNA Sequence Rearrangement; Diphtheria Toxin; Electrostatics; Endosomes; Environment; Equilibrium; Fluorescence; Free Energy; Grant; Helix (Snails); Histidine; Kinetics; Knowledge; Label; Lipid Bilayers; Lipids; Measurement; Mediating; Membrane; Membrane Proteins; Methodology; Methods; Mitochondria; Modeling; Molecular Conformation; Mutation; Nature; Numbers; Pathway interactions; Peptides; Protein Family; Protein translocation; Proteins; Research; Side; Site; TOM translocase; Testing; Toxin; Water; Work; aspartylglutamate; ear helix; experience; human disease; interfacial; mutant; novel; peptide I; protein folding; protonation; response; tool

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 transbflayer 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.

我的研究是由两个问题的答案驱动的：蛋白质如何交叉 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。但白喉毒素T结构域（DTT）本身 在酸性条件下插入膜中，并将催化结构域转移到膜上， 脂质双层。低pH也参与膜联蛋白12（ANX）的跨双层插入， 它是一个大的蛋白质家族的代表，与多种膜 功能，并在一些人类疾病。pH触发的折叠-插入-易位 DTT和其他蛋白质（例如肉毒杆菌毒素）的相互作用不仅具有固有的重要性，而且可以 揭示了膜蛋白组装和稳定性的一般物理化学原理。 这项资助的目标是完善这些原则，开发实验工具， 在膜中的蛋白质折叠转变之后，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 寿命荧光方法的优点，特别适合于分析异质 构象状态模型螺旋肽将用于确定 质子化和膜界面上的折叠。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： 非组成性膜蛋白的膜插入途径包含一个强制性的 界面中间状态;该中间状态和随后的transbf层的形成 插入是由疏水和静电相互作用之间的微妙平衡介导的， 蛋白质和膜界面。具体目的是：（1）表征折叠 DTT和ANX的膜插入途径上的中间体。(2)及其机制 pH诱导的DTT在膜上的重折叠。(3)细化物理化学规则， 使用模型螺旋肽预测膜蛋白插入/折叠。(4)推进 开发用于膜蛋白插入/折叠研究的荧光方法。