Skerget Katja, Vilfan Andrej, Pompe-Novak Marusa, Turk Vito, Waltho Jonathan P, Turk Dusan, Zerovnik Eva
Department of Biochemistry, Molecular and Structural Biology, JoZef Stefan Institute, 1000 Ljubljana, Slovenia.
Proteins. 2009 Feb 1;74(2):425-36. doi: 10.1002/prot.22156.
Cystatins, a family of structurally related cysteine proteinase inhibitors, have proved to be useful model system to study amyloidogenesis. We have extended previous studies of the kinetics of amyloid-fibril formation by human stefin B (cystatin B) and some of its mutants, and proposed an improved model for the reaction. Overall, the observed kinetics follow the nucleation and growth behavior observed for many other amyloidogenic proteins. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N(I) irreversible transitions with equivalent rates (k(I)), which fitted with N(I) = 64), fibril growth and nonproductive oligomerization, best explained by an off-pathway state with a rate-limiting escape rate. Three energies of activation were derived from global fitting to the minimal kinetic scheme, and independently through the fitting of the individual component rates. Nucleation was found to be a first-order process within an oligomeric species with an enthalpy of activation of 55 +/- 4 kcal mol(-1). Fibril growth was a second-order process with an enthalpy of activation (27 +/- 5 kcal mol(-1)), which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The highest enthalpy of activation (95 +/- 5 kcal mol(-1) at 35 degrees C), characteristic of a substantial degree of unfolding as observed prior to domain-swapping reactions, equated with the escape rate of the off-pathway oligomeric state.
胱抑素是一类结构相关的半胱氨酸蛋白酶抑制剂，已被证明是研究淀粉样蛋白生成的有用模型系统。我们扩展了之前关于人stefin B（胱抑素B）及其一些突变体形成淀粉样纤维动力学的研究，并提出了一个改进的反应模型。总体而言，观察到的动力学遵循许多其他淀粉样蛋白生成蛋白所观察到的成核和生长行为。最能拟合作为蛋白质浓度和温度函数的圆二色性（CD）和硫黄素T荧光变化测量值的最小动力学方案包括成核（建模为具有等效速率（k(I)）的N(I)不可逆转变，拟合得到N(I) = 64）、纤维生长和非生产性寡聚化，最好由具有限速逃逸速率的非途径状态来解释。通过对最小动力学方案的全局拟合，并独立地通过对各个组分速率的拟合，得出了三种活化能。发现成核是寡聚物种内的一级过程，活化焓为55±4 kcal mol(-1)。纤维生长是二级过程，活化焓为（27±5 kcal mol(-1)），这与胱抑素形成四聚体的情况无法区分，胱抑素形成四聚体涉及有限的构象变化，包括脯氨酸从反式到顺式的异构化。最高的活化焓（35℃时为95±5 kcal mol(-1)），这是在结构域交换反应之前观察到的大量解折叠的特征，等同于非途径寡聚状态的逃逸速率。
