Flexibility of the Ure2 prion domain is important for amyloid fibril formation.
Flexibility of the Ure2 prion domain is important for amyloid fibril formation.
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DOI：
10.1042/bj20101895
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发表时间：
2011-02
期刊：
影响因子：
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通讯作者：
Yong Yu;Hong Wang;Ming Bai;S. Perrett
中科院分区：
文献类型：
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作者：
Yong Yu;Hong Wang;Ming Bai;S. Perrett
Ure 2是酿酒酵母朊病毒[URE 3]的蛋白决定簇，具有天然无序的N-末端结构域，其对于体内朊病毒形成和体外淀粉样蛋白形成很重要;球状C-结构域具有谷胱甘肽转移酶样折叠。在本研究中，我们交换了N-和C-末端区域的位置，有或没有插入肽接头，以分别产生Ure 2变体CLN-Ure 2和CN-Ure 2。的天然结构的内容和稳定性的变体是相同的野生型尿素2，所示的酶活性，远紫外CD分析和平衡变性。CLN-Ure 2能够形成淀粉样原纤维，但比野生型Ure 2具有显著更长的滞后时间;并且这两种蛋白质不能交叉接种。在相同的条件下，CN-尿素2显示出有限的能力，形成原纤维，但这是改善后，加入0.03 M的盐酸胍。对于野生型Ure 2，在CLN-Ure 2和CN-Ure 2的原纤维中观察到变构酶活性，这与原纤维内C结构域的天然样二聚体结构的保留一致。CLN-Ure 2和CN-Ure 2的蛋白水解消化的原纤维显示出与野生型Ure 2相同的残留原纤维核心形态。结果表明，朊病毒结构域的位置影响的能力，尿素形成原纤维主要是由于其灵活性的影响。
Ure2, the protein determinant of the Saccharomyces cerevisiae prion [URE3], has a natively disordered N-terminal domain that is important for prion formation in vivo and amyloid formation in vitro; the globular C-domain has a glutathione transferase-like fold. In the present study, we swapped the position of the N- and C-terminal regions, with or without an intervening peptide linker, to create the Ure2 variants CLN-Ure2 and CN-Ure2 respectively. The native structural content and stability of the variants were the same as wild-type Ure2, as indicated by enzymatic activity, far-UV CD analysis and equilibrium denaturation. CLN-Ure2 was able to form amyloid-like fibrils, but with a significantly longer lag time than wild-type Ure2; and the two proteins were unable to cross-seed. Under the same conditions, CN-Ure2 showed limited ability to form fibrils, but this was improved after addition of 0.03 M guanidinium chloride. As for wild-type Ure2, allosteric enzyme activity was observed in fibrils of CLN-Ure2 and CN-Ure2, consistent with retention of the native-like dimeric structure of the C-domains within the fibrils. Proteolytically digested fibrils of CLN-Ure2 and CN-Ure2 showed the same residual fibril core morphology as wild-type Ure2. The results suggest that the position of the prion domain affects the ability of Ure2 to form fibrils primarily due to effects on its flexibility.