PHOTO-INDUCED UNFOLDING OF CANCER-SPECIFIC MEMBRANE RECEPTORS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 目标1.1表征每个卟啉与每个细胞外的结合 体外结构域。 +我们的体外研究将确定每种卟啉与 HSP70和ERA的胞外区。随后，组合了 将采用实验(体外)和计算(计算机)方法来 表征结合部位的位置和配基之间的作用 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)、傅里叶变换红外光谱(FTIR)和 质谱学(LC/MS和C/MS/MS)。这些方法结合在一起可以提供一个 关于相互作用、结构变化和两国之间距离的丰富信息 配基(PS)和多肽的区域。 +实验方法提供的证据将作为对 将使用不同的软件进行计算对接模拟 应用程序(Dock、Autodock等)。结合部位最有可能的位置是 通过这些模拟和几种算法的使用确定的将使 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 关于HSP70和ERA胞外部分的结构。 +光化学/光物理机制将从光学和 化学(LC/MS，LC/MS/MS)通过检测配体中辐射的光产物 和/或受体。 +对多肽结构的影响将通过以下方式在体外表征： O CD，使我们能够量化对二级结构的影响 感受器。 O fret，它将探测可能出现的轻微构象效应 CD未检测到。 +构象效应的位置和程度也将被表征 利用毛细管LC/MS和LC/MS/MS分析HSP70和Era的三级结构 都是公认的，人们可以定位特定的氨基酸残基(S)在每个 发生结构变化的蛋白质(S)，通过比较实验质量 从辐照样品中产生的多肽产品与来自对照和 来自蛋白质序列的电子切割的理论质量。 +将测试扩散O2在光诱导蛋白质去折叠中的作用 用光学方法和LC/MS用氮气彻底净化的样品。 目的1.3利用分子动力学(MD)模拟对光诱导折叠进行模拟 受体的转化。 +基于实验方法的事件(光化学修饰， 电子转移等)负责触发折叠更改的将用作 MD模拟的起始条件。光化学变化将被输入为 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(正电荷或负电荷，如 由我们的实验数据确定)在受体内的组(S)。这几点 修改将产生新的力场，该力场将在结构中引起应变 蛋白质的含量。 +MD模拟将使用哈佛分子化学进行 力学(CHARMM)力场。初始条件的影响之后将是 在Silico中，探索折叠轨迹和能量景观作为 蛋白质达到新的稳定构象的时间。

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.