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.

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