pH-Triggered Membrane Insertion of Proteins

pH 触发的蛋白质膜插入

基本信息

批准号：
8513343
负责人：
Melanie J Cocco
金额：
$ 32.47万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8513343
关键词：
Apoptotic BCL2 gene Bacterial Toxins Biological Assay Catalytic Domain Cell physiology Cells Cellular biology Charge Code Collaborations Color Complement Complex Computers Cysteine Data Diphtheria Toxin Disease Electrostatics Endosomes Environment Equilibrium Experimental Designs Family Fluorescence Fluorescence Resonance Energy Transfer Foundations Free Energy Funding Goals Head Histidine Hydrophobic Interactions Kinetics Label Link Lipid Bilayers Lipids Measurement Mediating Medicine Membrane Membrane Proteins Methodology Methods Modeling Molecular Molecular Conformation Mutagenesis Nature Pathway interactions Physiological Process Property Proteins Research Resolution Roentgen Rays Role Scheme Series Signal Transduction Site Solutions Structure Supervision Tail Techniques Testing Tetanus Thermodynamics Toxin Water Work aqueous aspartylglutamate botulinum cancer therapy cellular targeting colicin college innovation insight interfacial medical schools membrane model molecular dynamics mutant protein folding protein structure protonation research study response simulation stop flow technique targeted delivery tool

项目摘要

DESCRIPTION (provided by applicant): This project is focused on deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the diphtheria toxin T-domain (DTT), which is considered to be a paradigm for cell entry of other toxins (e.g., tetanus and botulinum) and has a potential for targeted delivery of anti-cancer therapies. The pH-triggered insertion of DTT will also reveal general physicochemical principles underlying membrane protein assembly and signalyng on membrane interfaces. This first competing renewal of the project will capitalize on our progress in identifying key intermediate states along the insertion pathway, in establishing the concept of conformational switching for DTT action and in developing new methodologies for structural, kinetic and thermodynamic characterization of membrane protein refolding/insertion. The innovation of this proposal resides in the unique way that molecular dynamics (MD) simulations and sophisticated spectroscopic experiments will be brought together in order to understand molecular mechanisms which will bring clarity to a complex field. MD simulations will be used for (a) building atomic models consistent with low resolution spectroscopic data, and (b) guiding the experimental design to further verify them. Site-specific labeling of single-cysteine mutants and a battery of spectroscopic approaches (including FCS, fluorescence lifetime quenching, FRET, stopped-flow kinetic measurements) will be utilized to test the interface-directed refolding/insertion hypothesis, which assigns a special role to the bilayer interfacial region in modulating transmembrane insertion by assisting the formation of key intermediate states, shifting the balance of electrostatic and hydrophobic interactions and altering protonation properties of titratable residues. The nature of the conformational switching resulting in refolding, insertion and translocation transitions of DTT will be established through mutagenesis of His, Asp and Glu residues, guided by Thermodynamic Integration calculations. Various DTT mutants will be used to ascertain whether protonation of histidines assists in the unfolding of the protein in solution and promotes formation of a previously identified insertion-competent intermediate on the membrane interface, through electrostatic interactions with anionic lipids, while protonation of acidic residues enables transmembrane insertion. To gain insights into the pH-triggered membrane action of DTT, thus establishing the general physicochemical principles of membrane-protein interactions, we will pursue the following goals: (1) determine molecular details of the structural organization of key intermediate and final inserted states; (2) determine the free energy profile of transitions along the insertion pathway and determine how the properties of the bilayer modulate structural, thermodynamic and kinetic parameters of the DTT insertion; and (3) identify key residues responsible for pH-triggered functional conformational switching.

描述（由申请人提供）：该项目的重点是解释pH依赖性的重新折叠和膜插入的分子机制，对二骨毒素T域（DTT）（DTT），这被认为是其他毒素的细胞进入细胞的范式（例如，Tetanus和botulapies and Ternipe ternies ternie ternie cancered terni-cance andi-cance andi-cance ternie cancane ternie。 DTT的pH触发插入还将揭示膜蛋白质组件和膜界面上信号的一般物理化学原理。该项目的第一个竞争性更新将利用我们在识别插入途径的关键中间状态方面的进步，以建立DTT动作的构象切换的概念，并开发用于膜蛋白质重塑/插入的结构，动力学和热力学表征的新方法。该提案的创新以独特的方式，即分子动力学（MD）模拟和复杂的光谱实验将被汇总在一起，以了解分子机制，这将使清晰度变为复杂的领域。 MD模拟将用于（a）与低分辨率光谱数据一致的构建原子模型，以及（b）指导实验设计以进一步验证它们。 Site-specific labeling of single-cysteine mutants and a battery of spectroscopic approaches (including FCS, fluorescence lifetime quenching, FRET, stopped-flow kinetic measurements) will be utilized to test the interface-directed refolding/insertion hypothesis, which assigns a special role to the bilayer interfacial region in modulating transmembrane insertion by assisting the formation of key中间状态，改变了静电和疏水相互作用的平衡，并改变了可滴定残基的质子化特性。构象转换的性质，导致DTT的重折叠，插入和易位转变，将通过热力学整合计算引导，通过诱变的AS，ASP和GLU残基建立。各种DTT突变体将用于确定组氨酸的质子化是否有助于蛋白质中的蛋白质展开，并通过与阴离子脂质的静电相互作用，促进先前鉴定的插入式插入的中间体，同时酸性残基的蛋白质化，使酸性残留物的蛋白质化。为了深入了解DTT的pH触发膜作用，从而建立了膜 - 蛋白质相互作用的一般物理化学原理，我们将追求以下目标：（1）确定关键中间和最终插入状态的结构性组织的分子细节；（2）确定沿插入途径的过渡的自由能曲线，并确定双层的性质如何调节DTT插入的结构，热力学和动力学参数；（3）确定负责pH触发功能构象切换的关键残基。