Jenko Sasa, Skarabot Miha, Kenig Manca, Guncar Gregor, Musevic Igor, Turk Dusan, Zerovnik Eva
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Proteins. 2004 May 1;55(2):417-25. doi: 10.1002/prot.20041.
By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.
通过使用硫黄素T荧光、X射线衍射和原子力显微镜（AFM），已证明人丝氨酸蛋白酶抑制剂A和B（半胱氨酸蛋白酶抑制剂亚家族A）会形成淀粉样纤维。两种蛋白质纤维均显示出交叉β结构特有的4.7 Å和10 Å反射。通过AFM观察到两种蛋白质纤维的高度相似，约为3 nm，纵向重复距离为25 - 27 nm。仅在丝氨酸蛋白酶抑制剂A中观察到高度为5.6 nm的双高纤维。通过透射电子显微镜（TEM）观察，丝氨酸蛋白酶抑制剂A纤维的宽度与先前报道的丝氨酸蛋白酶抑制剂B的宽度范围相同（Zerovnik等人，《生物化学与生物物理学报》2002年；1594：1 - 5）。不过，形成纤维所需的条件有所不同。丝氨酸蛋白酶抑制剂B在pH 5时开始形成淀粉样纤维，而在丝氨酸蛋白酶抑制剂A中，预热后的样品必须酸化至pH < 2.5。在这两种情况下，添加三氟乙醇、接种和在强磁场中排列都能加速纤维生长。对单体和结构域交换二聚体的三维结构进行可视化分析表明，两种同源物稳定性的主要差异源于特定盐桥的排列，这些盐桥将丝氨酸蛋白酶抑制剂A单体和二聚体形式中的α螺旋（以及α环）固定到β折叠上。
