Structure of the serpin-/proteinase complex and basis for metastable folding

Serpins最不寻常的两个方面是：(1)当作为蛋白酶抑制剂时，它们通过动力学捕获共价反应中间体而不是通过形成热力学稳定的非共价复合体来发挥作用；(Ii)与几乎所有其他已知的蛋白质不同，它们折叠成亚稳态构象，代表活性状态，而不是最稳定的状态，即非活性状态。在目前的提案中，我们建议测试三个与长期目标相关的假设，即了解动力学捕获的结构基础和亚稳态折叠的基础。(I)抑制机制涉及巨大的构象变化，蛋白酶移动超过70埃，并插入丝氨酸的裂解反应中心环作为蛋白质主要β片断之一的中心链。(Ii)这会产生刚性的、不可逆的共价络合物。(3)提出了蛇类的亚稳态折叠由四个具体目标驱动。AIM 1将使用荧光、核磁共振和EPR来确定总的能量转移将映射每个蛋白质上荧光团之间的分离。核磁共振将在15N标记的丝氨酸中使用顺磁加宽共振。EPR将使用不同的偶极-偶极相关分析绘制丝氨酸和蛋白酶之间的界面，利用顺磁物种的访问来区分来自丝氨酸的荧光、EPR和核磁共振可观察到的信号，以确定荧光探针中丝氨酸的运动，以确定导致亚稳态的折叠路径。这将专门测试早期形成的Beta-Sheet是否是正确折叠的关键。这将通过检测β-折叠C和反应中心环内的稳定和不稳定突变对折叠途径、亚稳态的稳定性和向潜在构象的转化率的影响来进一步检验。

Two of the most unusual aspects of serpins are(1) when acting as proteinase inhibitors, they function by kinetically trapping a covalent reaction intermediate rather than by forming a thermodynamically- stabilized non-covalent complex and (ii) that, unlike almost all other known proteins, they fold into a metastable conformation that represents the active state, rather than the most stable state, which is an inactive state. In the present proposal we propose to test three hypotheses related to the long term goals of understanding both the structural basis for kinetic trapping and the basis for metastable folding. (i) That the inhibition mechanisms involves a massive conformational change, with movement of the proteinase by over 70 angstroms and insertion of the cleaved reactive center loop of the serpin as a central strand of one of the main beta sheets of the protein. (ii) That this produces a rigid, irreversible covalent complex. (iii) That metastable folding of serpins is driven by Four Specific aims are proposed. Aim 1 will use fluorescence, NMR and EPR to determine the overall energy transfer will map the separation between fluorophores on each protein. NMR will use paramagnetic broadening of resonances in 15N-labeled serpin. EPR will use distinct dependent analysis of dipole-dipole map the interface between serpin and proteinase, using access of paramagnetic species to distinguish between fluorescence-, EPR- and NMR-observable signals from the serpin to determine the motion of the serpin in the fluorescent probes, to determine the folding pathway that leads to the metastable state. This will specifically test whether early formation of beta-sheet is key to correct folding. This will be further tested by examining the effect of stabilizing and destabilizing mutations within beta-sheet C and the reactive center loop on the folding pathway, stability of the metastable state and rate of conversion to the latent conformation.