Structural Studies of Amylin Fibrils Associated with Type 2 Diabetes

与 2 型糖尿病相关的胰淀素原纤维的结构研究

• 批准号: 7593510
• 负责人: ROBERT TYCKO
• 金额: $ 33.82万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593510
• 关键词: Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid Fibrils; Amyloid beta-Protein; Beta Cell; Biochemistry; Chemicals; Collaborations; Condition; Crystallography; Data; Decompression Sickness; Dimensions; Dipeptides; Electron Microscopy; Electrons; Environment; Gel Chromatography; Goals; In Vitro; Insulin; Label; Laboratories; Length; Manuscripts; Measurement; Measures; Molecular; Molecular Structure; Morphology; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Patients; Peptide Synthesis; Peptides; Phase; Plant Resins; Positioning Attribute; Preparation; Proteins; Protocols documentation; Resolution; Sampling; Scanning Transmission Electron Microscopy Procedures; Seeds; Site; Solid; Source; Staging; Structural Models; Structure; Structure of molecular layer of cerebellar cortex; Vertebral column; Work; amyloid formation; beta pleated sheet; chemical synthesis; desire; in vivo; islet; islet amyloid polypeptide; neutrophil; protocol development; solid state

In FY2007, we have obtained sufficient structural constraints from solid state NMR and electron microscopy to develop candidate structural models for amylin fibrils. This progress is the result of considerable efforts to develop protocols for efficient chemical synthesis of full-length amylin with isotopic labeling of specific residues, as well as protocols for preparation of structurally homogeneous amylin fibrils. Successful, high-yield synthesis depends on the incorporation of Hmb-protected amino acids or pseudoproline dipeptides at certain sites during solid phase peptide synthesis, in order to disrupt secondary structure formation on the synthesis resin. Preparation of structurally homogeneous fibrils results from isolation of monomeric amylin fractions by gel filtration chromatography prior to fibril formation, and from the use of fibril seeds to self-propagate the desired morphology. The morphology that results from these protocols is a "striated ribbon", comprised of multiple 5-nm-wide protofilaments, laterally associated in a strictly parallel manner. Approximately ten amylin fibril samples, isotopically labeled at positions that span the entire amylin sequence, were prepared in this way. Measurements of mass-per-length by scanning transmission electron microscopy (collaboration with R.D. Leapman, DBEPS, NIBIB) indicate a protofilament mass-per-length of approximately 20 kD/nm, implying a structure that consists of two molecular layers in a cross-beta structural motif. Solid state NMR spectra reveal a single set of 13C chemical shifts, implying that all molecules are in the same structural environment, and consequently that the protofilament has two-fold symmetry. Secondary structure determined from chemical shifts and from quantitative measurements of backbone 15N-15N distances indicates the presence of two beta-strand segments, separated by a bend that allows these segments to come in contact with one another. Additional solid state NMR data indicate that the beta-sheets have an in-register parallel organization. Thus, the overall structure is a four-layered beta-sheet, comprised of two molecular layers. This very closely resembles the structure of Alzheimer's beta-amyloid fibrils with similar morphologies, which we have previously characterized (Petkova et al., Biochemistry 2006). However, whereas beta-amyloid fibrils are stabilized exclusively by hydrophobic contacts between beta-sheets, amylin fibrils appear to be stabilized by a combination of hydrophobic and polar interactions. This work is described in a full-length manuscript that is currently under review for Biochemistry. Additional work currently in progress includes: (1) Preparation of amylin microcrystals, which we discovered fortuitously to form under conditions closely related to conditions we use to prepare amylin fibrils. Dimensions of these microcrystals are too small to permit conventional x-ray crystallography, but we plan to attempt electron diffraction measurements in our own laboratory, and x-ray diffraction measurements on a new micro-focussed x-ray source at Argonne National Labs. If successful, a crystal structure of amylin will provide a wealth of new information about interactions that stabilize amylin fibrils; (2) Preparation of uniformly 15N,13C-labeled amylin by bacterial expression. The sharp 13C NMR lines observed in our solid state NMR spectra of synthetic, selectively-labeled amylin fibrils suggest that measurements on uniformly-labeled samples may have sufficiently high resolution to be interpretable. This will allow us to measure additional structural constraints that will pin down the high-resolution amylin fibril structure.

2007财年，我们从固态核磁共振和电子显微镜中获得了足够的结构约束，以开发胰淀素原纤维的候选结构模型。 这一进展是为了开发具有特定残基同位素标记的全长胰岛淀粉样多肽的有效化学合成方案以及用于制备结构均质胰岛淀粉样多肽原纤维的方案所做的巨大努力的结果。 成功的高产率合成取决于在固相肽合成过程中在某些位点掺入 Hmb 保护的氨基酸或假脯氨酸二肽，以破坏合成树脂上二级结构的形成。 结构均质的原纤维的制备是通过在原纤维形成之前通过凝胶过滤色谱法分离单体胰淀素级分，以及使用原纤维种子自我繁殖所需的形态来实现的。 这些协议产生的形态是“条纹带”，由多个 5 nm 宽的原丝组成，以严格平行的方式横向关联。 以这种方式制备了大约十个胰岛淀粉样多肽原纤维样品，在跨越整个胰岛淀粉样多肽序列的位置进行同位素标记。 通过扫描透射电子显微镜（与 R.D. Leapman、DBEPS、NIBIB 合作）测量的每长度质量表明原丝的每长度质量约为 20 kD/nm，这意味着其结构由交叉 β 结构基序中的两个分子层组成。 固态核磁共振谱揭示了一组 13C 化学位移，这意味着所有分子都处于相同的结构环境中，因此原丝具有双重对称性。 根据化学位移和主链 15N-15N 距离的定量测量确定的二级结构表明存在两个 β 链片段，由弯曲分开，允许这些片段彼此接触。 额外的固态核磁共振数据表明，β-片层具有登记内平行组织。 因此，整体结构是四层β-折叠，由两个分子层组成。 这与阿尔茨海默病β-淀粉样原纤维的结构非常相似，具有相似的形态，我们之前已经对其进行了表征（Petkova 等人，Biochemistry 2006）。 然而，β-淀粉样蛋白原纤维仅通过β-折叠之间的疏水接触来稳定，而糊精原纤维似乎通过疏水和极性相互作用的组合来稳定。 这项工作在一份完整的手稿中进行了描述，目前正在接受《生物化学》的审查。 目前正在进行的其他工作包括：（1）胰岛淀粉样多肽微晶体的制备，我们偶然发现其形成条件与我们用于制备胰岛淀粉样多肽原纤维的条件密切相关。 这些微晶体的尺寸太小，无法进行传统的 X 射线晶体学分析，但我们计划在我们自己的实验室中尝试电子衍射测量，并在阿贡国家实验室的新型微聚焦 X 射线源上进行 X 射线衍射测量。 如果成功，胰岛淀粉样多肽的晶体结构将提供大量关于稳定胰岛淀粉样多肽原纤维的相互作用的新信息。 (2)通过细菌表达制备均匀15N,13C标记的胰淀素。 在合成的、选择性标记的胰淀素原纤维的固态 NMR 谱中观察到的尖锐 13C NMR 谱线表明，对均匀标记的样品进行测量可能具有足够高的分辨率以进行解释。 这将使我们能够测量额外的结构约束，从而确定高分辨率的胰淀素原纤维结构。