Human islet amyloid polypeptide (hIAPP) aggregates into fibrils through oligomers that have been postulated to contain α-helices as well as β-sheets. We employ a site-specific isotope labeling strategy that is capable of detecting changes in dihedral angles when used in conjunction with 2D IR spectroscopy. The method is analogous to the chemical shift index used in NMR spectroscopy for assigning protein secondary structure. We introduce isotope labels at two neighbouring residues, which results in an increased intensity and positive frequency shift if those residues are α-helical versus a negative frequency shift in β-sheets and turns. The 2D IR dihedral index approach is demonstrated for hIAPP in micelles for which the polypeptide structure is known, using pairs of ¹³C¹⁸O isotope labels L12A13 and L16V17, along with single labeled control experiments. Applying the approach to aggregation experiments performed in buffer, we show that about 27–38% of hIAPP peptides adopt an α-helix secondary structure in the monomeric state at L12A13, prior to aggregation, but not at L16V17 residues. At L16V17, the kinetics are described solely by the monomer and fiber conformations, but at L12A13 the kinetics exhibit a third state that is created by an oligomeric intermediate. Control experiments performed with a single isotope label at A13 exhibit two-state kinetics, indicating that a previously unknown change in dihedral angle occurs at L12A13 as hIAPP transitions from the intermediate to fiber structures. We propose a mechanism for aggregation, in which helices seed oligomer formation via structures analogous to leucine rich repeat proteins.

中文翻译：

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使用二维红外二面体索引技术进行蛋白质二级结构的位点特异性检测：拟议的寡聚hIAPP组装机制

人类胰岛淀粉样多肽（hIAPP）通过低聚物聚集成原纤维，该低聚物被认为含有α-螺旋和β-折叠。我们采用了特定于站点的同位素标记策略，当与2D红外光谱法结合使用时，该策略能够检测二面角的变化。该方法类似于NMR光谱中用于指定蛋白质二级结构的化学位移指数。我们引入同位素在两个相邻的残基的标签，这导致增加的强度和阳性频移如果这些残基是α螺旋与在β片层和匝的负频移。使用¹³ C ¹⁸对，证明了已知多肽结构的胶束中hIAPP的2D IR二面体指数方法O同位素标记L12A13和L16V17，以及单个标记的对照实验。将这种方法应用于在缓冲液中进行的聚集实验，我们发现，大约27–38％的hIAPP肽在聚集之前在L12A13处处于单体状态，但在L16V17残基处未处于单体状态的α-螺旋二级结构。在L16V17，动力学仅由单体和纤维构象描述，但在L12A13，动力学表现出由低聚物中间体产生的第三态。用单一同位素标记在A13进行的对照实验显示出两种状态的动力学，表明当hIAPP从中间结构转变为纤维结构时，L12A13发生了先前未知的二面角变化。我们提出了一种聚集机制，其中螺旋通过以下方式形成种子低聚物 结构类似于富含亮氨酸的重复蛋白。