Structural Studies of Amylin Fibrils Associated with Type 2 Diabetes

在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amylin原纤维的候选结构模型。这一进展是大量努力的结果，以开发有效的化学合成全长胰淀素与同位素标记特定残基的方案，以及制备结构均匀的胰淀素原纤维的方案。成功、高产的合成取决于在固相肽合成过程中，在某些位点加入受hmb保护的氨基酸或假脯氨酸二肽，以破坏合成树脂上的二级结构形成。制备结构均匀的原纤维的结果是在原纤维形成之前通过凝胶过滤层析分离单体的淀粉酶组分，并使用原纤维种子自我繁殖所需的形态。这些方案产生的形态是一个“条纹带”，由多个5纳米宽的原丝组成，横向以严格平行的方式联系在一起。用这种方法制备了大约十个胰淀素原纤维样品，在跨越整个胰淀素序列的位置上进行了同位素标记。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，表明其结构由交叉β结构基序中的两个分子层组成。固体核磁共振光谱显示了一组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。二级结构由化学位移和主链15N-15N距离的定量测量确定，表明存在两个β -链片段，由允许这些片段相互接触的弯曲分开。另外的固态核磁共振数据表明，β -片具有寄存器内平行组织。因此，整体结构是由两个分子层组成的四层β片。这与阿尔茨海默氏症β -淀粉样蛋白原纤维的结构非常相似，具有相似的形态，我们之前已经描述过（Petkova et al., Biochemistry 2006）。然而，β -淀粉样蛋白原纤维完全通过β -片之间的疏水接触来稳定，而胰淀素原纤维似乎通过疏水和极性相互作用的组合来稳定。

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.