PHOTO-INDUCED UNFOLDING OF CANCER-SPECIFIC MEMBRANE RECEPTORS

光诱导癌症特异性膜受体的展开

• 批准号: 8357129
• 负责人: LORENZO BRANCALEON
• 金额: $ 6.69万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8357129
• 关键词: Adopted; Algorithms; Amino Acid Sequence; Amino Acids; Binding; Binding Sites; Blood capillaries; Charge; Chemistry; Circular Dichroism Spectroscopy; Computer Simulation; Computer software; Data; Diffuse; Docking; Electron Transport; Event; Extracellular Domain; Extracellular Structure; Feedback; Fluorescence Resonance Energy Transfer; Funding; Grant; Health; Heat-Shock Proteins 70; In Vitro; Investigation; Lasers; Ligands; Location; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Methods; Modeling; Modification; National Center for Research Resources; Optical Methods; Peptide Sequence Determination; Peptides; Porphyrins; Principal Investigator; Protein Conformation; Proteins; Research; Research Infrastructure; Resources; Role; Sampling; Source; Spectroscopy, Fourier Transform Infrared; Structural Protein; Structure; Testing; Time; United States National Institutes of Health; base; capillary; cost; flexibility; irradiation; liquid chromatography mass spectrometry; molecular dynamics; molecular mechanics; polypeptide; protein structure; purge; receptor; simulation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Aim 1.1 Characterize the binding between each porphyrin and each extracellular domain in vitro. + Our in vitro studies will establish the binding parameters of each porphyrin to HSP70 and the extracellular domain of ERa. Subsequently a combination of experimental (in vitro) and computational (in silico) methods will be adopted to characterize the location of the binding site and the interaction between ligand and polypeptide. + Experimental investigations of the binding will include Fluorescence Resonance Energy Transfer (FRET), Circular Dichroism Spectroscopy (CD), FTIR spectroscopy and mass spectrometry (LC/MS and C/MS/MS). These methods combined can provide a wealth of information on interactions, structural changes and distances between the ligand (PS) and regions of the polypeptide. + The evidence provided by the experimental methods will serve as feedback for the computational docking simulations which will be carried out with different software applications (Dock, Autodock, etc.). The most likely location of the binding site will be determined with these simulations and the use of several algorithms will enable the avoidance of "false positives" and allow some flexibility in the protein. Aim 1.2 Characterize conformational changes produced by laser irradiation of the PS on the structure of the extracellular portion of HSP70 and ERa. + Photochemical/photophysical mechanisms will be characterized optically and chemically (LC/MS, LC/MS/MS) by detecting photoproducts of irradiation in the ligand and/or the receptors. + The effects on the structure of the polypeptides will be characterized in vitro with: o CD, which will enable us to quantify the effect on the secondary structure of the receptors. o FRET, which will probe even slight conformational effect that could go undetected with CD. + The location and extent of the conformational effects will also be characterized using capillary LC/MS and LC/MS/MS. Since the tertiary structures of HSP70 and ERa are well established, one can locate the specific amino acid residue(s) within each protein where structural change(s) occur, by comparing the experimental masses of peptide products generated from irradiated samples with those from controls and the theoretical masses from in silico cleavage of the protein sequence. + The role of diffusing O2 in the photo-induced unfolding of the protein will be tested in samples thoroughly purged with N2 using optical methods and LC/MS. Aim 1.3 Use molecular dynamic (MD) simulations to model the photo-induced folding transformation of the receptors. + Based on the experimental methods the events (photochemical modifications, electron transfer, etc.) responsible to trigger the folding changes will be used as the starting condition of the MD simulations. Photochemical changes will be entered as new atomic compositions of specific groups, whereas charge transfer will be reproduced by placing an extra elementary charge (positive or negative as determined by our experimental data) at group(s) within the receptors. These point modifications will generate a new force field that will induce a strain in the structure of the protein. + MD simulation will be carried out using the Chemistry at Harvard Molecular Mechanics (CHARMM) force field. The effects of the initial conditions will be followed by probing, in silico, the folding trajectory and the energy landscape as a function of time until the new stable conformation of the protein is reached.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 目的1.1表征每种卟啉与每种细胞外基质之间的结合 体外结构域。 + 我们的体外研究将建立每种卟啉与 HSP 70和ER α的胞外结构域。随后， 将采用实验（体外）和计算（计算机模拟）方法， 表征结合位点的位置以及配体与 多肽。 + 结合的实验研究将包括荧光共振 能量转移（FRET）、圆二色谱（CD）、FTIR光谱和 质谱法（LC/MS和C/MS/MS）。这些方法结合起来可以提供一个 关于相互作用、结构变化和 配体（PS）和多肽的区域。 + 实验方法提供的证据将作为反馈， 将使用不同的软件进行计算对接模拟 应用程序（Dock、Autodock等）。结合位点最可能的位置是 确定这些模拟和使用几种算法将使 避免了“假阳性”，并允许蛋白质具有一定的灵活性。 目的1.2表征激光照射PS产生的构象变化 HSP 70和ER α的细胞外部分的结构。 + 光化学/光物理机制的特点是光学和 通过检测配体中辐照的光产物进行化学（LC/MS，LC/MS/MS 和/或受体。 + 对多肽结构的影响将在体外表征： O CD，这将使我们能够量化对二级结构的影响， 受体。 O FRET，这将探测甚至轻微的构象效应， 未发现CD + 构象效应的位置和程度也将被表征 采用毛细管LC/MS和LC/MS/MS。由于HSP 70和ER α的三级结构 已经被很好地确立，人们可以定位每个内的特定氨基酸残基， 发生结构变化的蛋白质，通过比较 从辐照样品产生的肽产物与来自对照的肽产物和来自辐照样品的肽产物相比， 来自蛋白质序列的计算机切割的理论质量。 + 将测试扩散O2在蛋白质的光诱导展开中的作用 使用光学方法和LC/MS，在用N2彻底吹扫的样品中。 目的1.3利用分子动力学（MD）模拟模拟光诱导折叠 受体的转化。 + 基于实验方法，事件（光化学修饰， 电子转移等）负责触发折叠变化的将被用作 MD模拟的起始条件。光化学变化将输入为 特定基团的新原子组成，而电荷转移将是 通过放置额外的基本电荷（正或负， 由我们的实验数据确定）。这些点 修改将产生一个新的力场，该力场将在结构中引起应变 蛋白质。 + MD模拟将使用哈佛分子生物学实验室的Chemistry进行。 力学（CHARMM）力场。初始条件的影响将被跟踪， 探测，在硅片上，折叠轨迹和能源景观作为一个功能， 直到蛋白质达到新的稳定构象的时间。