Biologically important modes of polypeptide assembly

多肽组装的生物学重要模式

• 批准号: 9973889
• 负责人: SAMUEL H. GELLMAN
• 金额: $ 36.11万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973889
• 关键词: Address; Adopted; Adoption; Alzheimer' s Disease; Alzheimer' s disease brain; Amino Acid Sequence; Amyloid; Amyloid Fibrils; Amyloid beta-Protein Precursor; Amyloidosis; Area; Biological; Biological Models; Biological Process; Biology; Cell Membrane Proteins; Cell Surface Receptors; Cell physiology; Cells; Chemicals; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Data; Deposition; Dimensions; Disease; Family; Foundations; Future; Generations; Geometry; Goals; Health; Human; Hydrophobic Interactions; Hydrophobicity; Immunologic Receptors; Lead; Learning; Link; Lipid Bilayers; Liquid substance; Mediating; Membrane; Methods; Micro Electron Diffraction; Modeling; Molecular Conformation; Pathogenicity; Pathologic; Peptides; Phase; Protein Family; Protein Region; Proteins; Receptor Activation; Receptor Protein-Tyrosine Kinases; Research; Resolution; Role; SAP-A Protein; Signal Transduction; Structure; System; T-Cell Receptor; TYROBP gene; Techniques; Testing; Work; X ray diffraction analysis; alpha helix; amyloid formation; base; beta pleated sheet; chemical synthesis; design; dimer; human disease; in vivo; interest; polypeptide; programs; protein aminoacid sequence; protein function; protein structure; receptor; sarcoma; solid state nuclear magnetic resonance; stereochemistry; synthetic peptide

SUMMARY We want to elucidate modes of polypeptide assembly that are important for biological function and associated with human disease but are difficult to characterize via standard experimental approaches. In each case, we wish to understand the non-covalent forces that underlie the assembly mode. Because of differences among the types of assembly we are studying, the experimental approaches we adopt are variable. One goal is to characterize quaternary structures formed by single-pass transmembrane (SPTM) α-helices that are constituents of cell-surface receptors, such as receptor tyrosine kinases (RTKs) and activating immune receptors. These receptors form oligomers, and rearrangement of oligomer geometry transmits the signal. Crystallographic data are available in only for the SPTM α-helix of immune receptor component DAP12. There is no high-resolution structural information for alternative geometries of SPTM α-helix assemblies that are thought to be associated with different receptor activation states. We are applying racemic crystallization and micro-electron diffraction (via collaboration) to this structural challenge, and we are exploring protein-based “picodiscs” as hosts for SPTM α-helix assemblies. A second goal is to understand how sequence, composition and dimensions influence stability of polypeptides in the amyloid state. The β-sheet-rich structures that are common to disease-associated amyloid fibrils are distinct from tertiary and quaternary structures commonly found among soluble proteins. The techniques commonly used to elucidate sequence-stability relationships among soluble proteins are not readily applied to amyloid fibrils; therefore, we are developing a soluble model system for the amyloid state, which we will exploit to ask fundamental questions about amyloid state stability. The third goal is to understand the forces that lead to liquid-liquid phase separation (LLPS) mediated by proteins in the FUS (“fused in sarcoma”) family. The loose associations between polypeptide chains in the protein-rich liquid phase are not well understood. Such phases can transition to amyloid-like assemblies, which forms a strong connection between our second and third goals. These more ordered assemblies are associated with illnesses such as ALS. We seek to model LLPS of FUS family proteins with synthetic peptides in order to conduct incisive tests of recent mechanistic proposals and to evaluate the role of amino acid sequence and stereochemistry in LLPS and the transition to more ordered and pathogenic assemblies.

总结 我们希望阐明多肽组装的模式，这对生物学的重要性， 功能和与人类疾病相关，但难以通过标准 实验方法。在每一种情况下，我们都希望了解非共价力， 是装配模式的基础。由于不同类型的装配， 研究中，我们采用的实验方法是可变的。 一个目标是表征通过单次跨膜形成的四级结构 （SPTM）α-螺旋，是细胞表面受体的组成部分，如受体酪氨酸 激酶（RTK）和活化免疫受体。这些受体形成寡聚体， 低聚物几何结构的重排传递信号。晶体学数据可在 仅针对免疫受体组分DAP 12的SPTM α-螺旋。没有高分辨率 SPTM α-螺旋组装体替代几何结构的结构信息， 与不同的受体激活状态有关。我们将外消旋结晶 和微电子衍射（通过合作）来应对这一结构挑战，我们正在 探索蛋白质为基础的“picodisks”作为SPTM α-螺旋组装的宿主。 第二个目标是了解序列、组成和维度如何影响 淀粉样状态多肽的稳定性。β-片层丰富的结构是常见的 疾病相关的淀粉样蛋白原纤维不同于三级和四级结构， 在可溶性蛋白质中发现。通常用于阐明序列稳定性的技术 可溶性蛋白质之间的关系不容易应用于淀粉样纤维;因此，我们 开发淀粉样蛋白状态的可溶性模型系统，我们将利用它来问 淀粉样蛋白状态稳定性的基本问题。 第三个目标是了解导致液-液相分离的力 （LLPS）由FUS（“融合在肉瘤中”）家族中的蛋白质介导。松散的联系 在富含蛋白质的液相中多肽链之间的相互作用还没有很好地理解。等 相可以转变为淀粉样蛋白组装，这在我们的大脑中形成了强烈的联系。 第二和第三个目标。这些更有序的组装与疾病有关， 人症我们试图用合成肽模拟FUS家族蛋白的LLPS，以便进行 对最近提出的机制进行了深入的测试，并评估了氨基酸序列的作用 和立体化学的LLPS和过渡到更有序和致病性组装。