Structural Studies of Amylin Fibrils Associated with Type 2 Diabetes

在2007财政年度，我们从固态NMR和电子显微镜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。 这一进展是相当大的努力，以开发协议的有效化学合成的全长胰淀素与特定残基的同位素标记，以及协议的制备结构均匀的胰淀素原纤维的结果。 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-protected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。 结构均匀的原纤维的制备是由在原纤维形成之前通过凝胶过滤色谱分离单体胰淀素级分以及使用原纤维种子自繁殖所需形态产生的。 从这些协议产生的形态是一个“条纹带”，由多个5 nm宽的原丝，横向关联在一个严格的平行的方式。 以这种方式制备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处同位素标记。 通过扫描透射电子显微镜测量单位长度的质量（与R. D. Leapman，DBEPS，NIBIB）表明每长度的原丝质量约为20 kD/nm，这意味着结构由交叉β结构基序中的两个分子层组成。 固态NMR光谱揭示了一组13 C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。 从化学位移和从主链15 N-15 N距离的定量测量确定的二级结构表明存在两个β链区段，其被允许这些区段彼此接触的弯曲分开。 另外的固态NMR数据表明β-折叠具有配准平行组织。 因此，整体结构是由两个分子层组成的四层β-折叠。 这非常类似于具有类似形态的阿尔茨海默氏β-淀粉样蛋白原纤维的结构，我们先前已经表征了这一点（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稳定。 这项工作是在一个完整的长度手稿，目前正在审查的生物化学描述。 目前正在进行的其他工作包括：（1）胰淀素微晶的制备，我们偶然发现其在与我们用于制备胰淀素原纤维的条件密切相关的条件下形成。 这些微晶的尺寸太小，无法进行传统的X射线晶体学，但我们计划在我们自己的实验室尝试电子衍射测量，并在阿贡国家实验室的一个新的微聚焦X射线源上进行X射线衍射测量。 如果成功的话，胰淀素的晶体结构将提供大量关于稳定胰淀素原纤维的相互作用的新信息;（2）通过细菌表达制备均匀的15 N，13 C标记的胰淀素。 在我们的合成的选择性标记的胰淀素原纤维的固态NMR谱中观察到的尖锐的13 C NMR线表明，对均匀标记的样品的测量可以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，这些约束将确定高分辨率胰淀素原纤维结构。

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.