Maj Michał, Lomont Justin P, Rich Kacie L, Alperstein Ariel M, Zanni Martin T
Department of Chemistry , University of Wisconsin-Madison , Madison , Wisconsin 53706-1396 , USA . Email:
Chem Sci. 2017 Nov 3;9(2):463-474. doi: 10.1039/c7sc03789a. eCollection 2018 Jan 14.
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 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 CO 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 structures analogous to leucine rich repeat proteins.
人胰岛淀粉样多肽（hIAPP）通过低聚物聚集成纤维，据推测这些低聚物包含α-螺旋以及β-折叠。我们采用一种位点特异性同位素标记策略，当与二维红外光谱结合使用时，该策略能够检测二面角的变化。该方法类似于核磁共振光谱中用于确定蛋白质二级结构的化学位移指数。我们在两个相邻残基处引入同位素标记，如果这些残基是α-螺旋，则会导致强度增加和正频率偏移；在β-折叠和转角处则会出现负频率偏移。利用CO同位素标记对L12A13和L16V17以及单标记对照实验，对已知多肽结构的胶束中的hIAPP进行了二维红外二面角指数方法的验证。将该方法应用于在缓冲液中进行的聚集实验，我们发现，在聚集之前，约27 - 38%的hIAPP肽在L12A13处的单体状态下采用α-螺旋二级结构，但在L16V17残基处则没有。在L16V17处，动力学仅由单体和纤维构象描述，但在L12A13处，动力学表现出由低聚中间体产生的第三种状态。在A13处使用单个同位素标记进行的对照实验显示出二态动力学，表明当hIAPP从中间体转变为纤维结构时，L12A13处发生了先前未知的二面角变化。我们提出了一种聚集机制，其中螺旋引发低聚物形成，其结构类似于富含亮氨酸重复蛋白。
