pH-Triggered Membrane Insertion of Proteins

pH 触发的蛋白质膜插入

• 批准号: 7931127
• 负责人: ALEXEY LADOKHIN
• 金额: $ 9.4万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931127
• 关键词: Advanced Development; Affect; Annexins; Binding; Botulinum Toxins; Catalytic Domain; Cells; Complex; Cysteine; DNA Sequence Rearrangement; Diphtheria Toxin; Electrostatics; Endosomes; Environment; Equilibrium; Fluorescence; Free Energy; Grant; Histidine; Kinetics; Knowledge; Label; Lipid Bilayers; Lipids; Measurement; Mediating; Membrane; Membrane Proteins; Methodology; Methods; Mitochondria; Modeling; Molecular Conformation; Mutation; Nature; Pathway interactions; Peptides; Protein Family; Protein translocation; Proteins; Research; Side; Site; TOM translocase; Testing; Toxin; Water; Work; aspartylglutamate; experience; human disease; interfacial; mutant; novel; peptide I; protein folding; protonation; response; tool

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.

描述（由申请人提供）：我的研究是由两个问题的答案搜索驱动的：蛋白质如何穿过膜，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。但是白喉毒素T结构域（DTT）本身在酸性条件下插入膜中，并将催化结构域转位穿过脂质双层。低pH还涉及膜联蛋白12（ANX）的跨双层插入，所述膜联蛋白12（ANX）是已经涉及多种膜功能和许多人类疾病的大蛋白质家族的代表。DTT和其他蛋白质（例如，肉毒杆菌毒素）不仅具有固有的重要性，而且可以揭示膜蛋白组装和稳定性的一般物理化学原理。这项资助的目的是完善这些原则，开发实验工具，以跟踪膜中蛋白质折叠的转变，并将这些原则和工具应用于DTT和ANX的pH诱导的膜插入问题。这些新工具将利用寿命荧光方法，独特地适合于分析异构构象状态。模型螺旋肽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，主要是位点选择性荧光标记的DTT和ANX，将被用来测试以下假设：膜插入途径的非组成性膜蛋白包含一个强制性的界面中间状态，形成这个中间和随后的transbilayer插入介导的蛋白质和膜界面之间的疏水和静电相互作用的微妙平衡。具体目的是：（1）对DTT和ANX膜插入途径上的折叠中间体进行表征。(2)阐明pH诱导DTT在膜上复性的机制。(3)使用模型螺旋肽改进预测膜蛋白插入/折叠的物理化学规则。(4)推进膜蛋白插入/折叠研究的荧光方法的发展。