Elucidating the Molecular Mechanisms of Conformational Switching during Protein Insertion into Membranes

阐明蛋白质插入膜过程中构象转换的分子机制

• 批准号: 10737458
• 负责人: ALEXEY LADOKHIN
• 金额: $ 60.19万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737458
• 关键词: Address; Affect; Amino Acids; Apoptosis; Apoptotic; BCL2 gene; Bacterial Toxins; Bacteriophages; Base Sequence; Behavior; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular biology; Charge; Cholesterol; Circular Dichroism; Collaborations; Complex; Coupled; Data; Development; Divalent Cations; Drug Targeting; Environment; Family; Fluorescence; Goals; Health; Human; Individual; Ions; Knowledge; Letters; Lipid Bilayers; Lipids; Liquid substance; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Modeling; Molecular; Molecular Conformation; NMR Spectroscopy; Organelles; Outcome; Pathogenicity; Peptides; Physiological; Play; Process; Property; Protein Conformation; Protein Family; Proteins; Protons; Publications; Regulation; Role; Structure; System; Testing; Therapeutic; Thermodynamics; Tissues; United States National Institutes of Health; Variant; Vesicle; Water; Work; cancer cell; cancer therapy; design; experimental study; improved; insight; interfacial; macromolecule; membrane model; molecular dynamics; molecular modeling; monomer; novel; protein structure; protonation; tool; tumor

Conformational switching associated with the conversion of a protein structure from a water-soluble to a membrane-inserted form is a critical step in several cellular processes; our ability to predict and manipulate such switching can be beneficial to human health. Notable examples of conformational switching include tumor targeting by the pH Low Insertion Peptide (pHLIP), cellular entry of bacterial toxins, and membrane-induced activation of the Bcl-2 family of apoptotic regulators. While these processes are of fundamental biomedical importance, the mechanism of conformational switching needs to be better understood. Specifically, the role of lipid composition and divalent cations in modulating membrane insertion remains largely unexplored, despite the mounting evidence of their physiological importance. We will test our hypothesis that changes in lipid composition, Ca2+ and Mg2+ concentrations, and pH play key roles in modulating conformational switching on membrane interfaces. In Aim 1, we will decipher the thermodynamic rules for predicting protein-membrane interactions by characterizing the protonation- and Ca2+/Mg2+-dependent bilayer partitioning of model peptides. We will use our findings to advance the existing tools for sequence-based predictions of membrane interactions and make them readily applicable to more realistic descriptions of the complex cellular environment. Specifically, we strive to predict the effects of divalent cations and the changes in pKas of titratable amino acid residues on membrane interfaces, which are critical for understanding membrane-dependent conformational rearrangements that underlie the functioning of many systems of biomedical importance. In Aim 2, we will determine the role of lipid composition in modulating the pH-dependent and Ca2+/Mg2+-dependent folding and transmembrane insertion of pHLIP. This will include (a) deciphering the coupled effect of Ca2+/Mg2+ and lipid composition on pHLIP targeting to model membranes, (b) determining the conformation of pHLIP-lipid complexes by molecular dynamics (MD) simulations and spectroscopic experiments, (c) establish the role of individual acidic residues in the conformational switching of pHLIP and (d) determining the protonation of anionic residues in membrane- inserted pHLIP by NMR spectroscopy. In Aim 3, we will test whether the molecular determinants identified in Aim 2 are sufficient to describe the behaviors of pHLIP in complex lipid systems containing de-mixed liquid domains, and in mammalian cells (i.e., we will determine the effect of pH, divalent cations, PS, and cholesterol on the interaction of pHLIP with cellular membranes).

与蛋白质结构从水溶性到非水溶性的转化相关的构象转换 膜插入形式是几个细胞过程中的关键步骤;我们预测和操纵这种形式的能力 转换可以有益于人类健康。构象转换的显著例子包括肿瘤 通过pH低插入肽（pHLIP）的靶向、细菌毒素的细胞进入和膜诱导 凋亡调节因子Bcl-2家族的激活。虽然这些过程是基本的生物医学 重要的是，构象转换的机制需要更好地理解。具体而言， 脂质组成和二价阳离子在调节膜插入仍然在很大程度上未被探索，尽管 越来越多的证据表明它们在生理上的重要性我们将检验我们的假设， 组成，Ca 2+和Mg 2+浓度和pH在调节构象转换中起关键作用。 膜界面在目标1中，我们将破译预测蛋白质膜的热力学规则 通过表征模型肽的质子化和Ca 2 +/Mg 2+依赖性双层分配来确定相互作用。 我们将利用我们的发现来推进现有的基于序列的膜相互作用预测工具 并且使它们容易地适用于复杂蜂窝环境的更真实的描述。具体地说， 我们努力预测二价阳离子和可滴定氨基酸残基的pKas变化对 膜界面，这是理解膜依赖性构象重排的关键 是许多重要生物医学系统的基础。在目标2中，我们将确定 调节pH依赖性和Ca 2 +/Mg 2+依赖性折叠和跨膜的脂质组成 插入pHLIP。这将包括（a）破译Ca 2 +/Mg 2+和脂质组成对细胞内Ca 2 +/Mg 2+的偶联作用。 pHLIP靶向模型膜，（B）通过分子生物学方法测定pHLIP-脂质复合物的构象， 动力学（MD）模拟和光谱实验，（c）建立单个酸性残基在 pHLIP的构象转换和（d）确定膜中阴离子残基的质子化- 插入的pHLIP通过NMR光谱。在目标3中，我们将测试在 目的2足以描述pHLIP在含有分层液体的复杂脂质体系中的行为 结构域，以及在哺乳动物细胞中（即，我们将确定pH值、二价阳离子、PS和胆固醇的影响 pHLIP与细胞膜的相互作用）。