pH-Triggered Membrane Insertion of Proteins

描述（由申请人提供）：该项目专注于破解白喉毒素t结构域（DTT）的ph依赖性重折叠和膜插入的分子机制，这被认为是其他毒素（例如破伤风和肉毒杆菌）进入细胞的范例，并具有靶向递送抗癌治疗的潜力。ph触发的DTT插入也将揭示膜蛋白组装和膜界面信号传导的一般物理化学原理。该项目的第一次竞争性更新将利用我们在确定插入途径的关键中间状态，建立DTT作用的构象转换概念以及开发膜蛋白重折叠/插入的结构，动力学和热力学表征的新方法方面的进展。该提案的创新之处在于将分子动力学（MD）模拟和复杂的光谱实验结合在一起的独特方式，以了解分子机制，从而为复杂的领域带来清晰度。MD模拟将用于(a)建立与低分辨率光谱数据一致的原子模型，以及(b)指导实验设计以进一步验证它们。单半胱氨酸突变体的位点特异性标记和一系列光谱方法（包括FCS，荧光寿命猝灭，FRET，停止流动动力学测量）将用于测试界面定向重折叠/插入假说，该假说通过协助关键中间态的形成，在调节跨膜插入中赋予双层界面区域特殊作用。改变静电和疏水相互作用的平衡，改变可滴定残基的质子化性质。在热力学积分计算的指导下，将通过His、Asp和Glu残基的突变来确定导致DTT重折叠、插入和易位转变的构象切换的性质。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在溶液中展开，并通过与阴离子脂质的静电相互作用促进在膜界面上形成先前确定的插入能力中间体，而酸性残基的质子化是否能够跨膜插入。为了深入了解ph触发DTT的膜作用，从而建立膜-蛋白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组织的分子细节；(2)确定沿插入路径跃迁的自由能分布，确定双层性质如何调节DTT插入的结构、热力学和动力学参数；(3)确定ph触发功能构象转换的关键残基。

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.