Kenig Manca, Jenko-Kokalj Sasa, Tusek-Znidaric Magda, Pompe-Novak Marusa, Guncar Gregor, Turk Dusan, Waltho Jonathan P, Staniforth Rosemarie A, Avbelj Franc, Zerovnik Eva
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Proteins. 2006 Mar 1;62(4):918-27. doi: 10.1002/prot.20812.
To study the influence of whole secondary structure elements to the process of folding and amyloid-fibril formation, chimeras of stefins have been prepared. GdnHCl denaturation curves and folding rates (chevron plots) have been analyzed based on a two-state mechanism. The order of stability is: stefin A > aAbbbb > bAbbbb > stefin B = aBaaaa > bBaaaa, where the make up of chimeric proteins is designated by small letters representing the source of individual strands (a for stefin A, b for stefin B) and a capital letter representing the source of the helix (A for stefin A and B for stefin B). Only the fast folding reactions were included in the analysis and it has been found that stefin B folds the fastest (657 s(-1)). Similarly, fast folders are the chimeric proteins aBaaaa and bBaaaa, both of which contain the alpha-helix of stefin B. Unfolding rates correlate very well with protein stability, with the slowest rate for the most stable protein, stefin A. Amyloid-fibril growth was measured for each protein by monitoring thioflavin T fluorescence and was visualized using electron microscopy. The propensity to form amyloid-fibrils is in the order: stefin B > bAbbbb > aAbbbb > bBaaaa > aBaaaa > stefin A. This order does not correlate with stability, or with the folding or unfolding rates. Instead, the propensity to fibrillize is related to selected parts of structure, such as the beta-sheet of stefin B, and can be predicted reasonably well by calculating the beta-strand propensity of the denatured states.
为了研究整个二级结构元件对折叠过程和淀粉样纤维形成的影响，制备了丝氨酸蛋白酶抑制剂的嵌合体。基于两态机制分析了盐酸胍变性曲线和折叠速率（V形图）。稳定性顺序为：丝氨酸蛋白酶抑制剂A > aAbbbb > bAbbbb > 丝氨酸蛋白酶抑制剂B = aBaaaa > bBaaaa，其中嵌合蛋白的组成由代表单链来源的小写字母（a代表丝氨酸蛋白酶抑制剂A，b代表丝氨酸蛋白酶抑制剂B）和代表螺旋来源的大写字母（A代表丝氨酸蛋白酶抑制剂A，B代表丝氨酸蛋白酶抑制剂B）表示。分析中仅包括快速折叠反应，并且发现丝氨酸蛋白酶抑制剂B折叠最快（657 s⁻¹）。同样，快速折叠的是嵌合蛋白aBaaaa和bBaaaa，它们都含有丝氨酸蛋白酶抑制剂B的α-螺旋。解折叠速率与蛋白质稳定性非常相关，最稳定的蛋白质丝氨酸蛋白酶抑制剂A的解折叠速率最慢。通过监测硫黄素T荧光测量每种蛋白质的淀粉样纤维生长，并使用电子显微镜进行可视化。形成淀粉样纤维的倾向顺序为：丝氨酸蛋白酶抑制剂B > bAbbbb > aAbbbb > bBaaaa > aBaaaa > 丝氨酸蛋白酶抑制剂A。这个顺序与稳定性、折叠或解折叠速率均不相关。相反，纤维化倾向与结构的特定部分有关，例如丝氨酸蛋白酶抑制剂B的β-折叠，并且可以通过计算变性状态的β-链倾向来较好地预测。
