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

蛇蛋白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物来起作用；（ii）与几乎所有其他已知蛋白质不同，它们折叠成代表活性状态的亚稳构象，而不是最稳定的状态，即失活状态。在目前的提议中，我们提出测试三个假设，这些假设与理解动力学俘获的结构基础和亚稳折叠的基础的长期目标有关。(i)抑制机制涉及大量构象变化，蛋白酶移动超过70埃，蛇形蛋白的裂解反应性中心环作为蛋白质主要β片的中心链插入。（ii）这产生刚性的，不可逆的共价复合物。（iii）提出了四个特定目标驱动蛇蛋白亚稳态折叠。目的1将使用荧光，核磁共振和EPR来确定整体能量转移，并绘制每个蛋白质上荧光团之间的分离图。核磁共振将利用顺磁增宽15n标记丝氨酸丝氨酸的共振。EPR将使用不同的偶极-偶极依赖性分析来绘制丝线蛋白和蛋白酶之间的界面，使用顺磁物质来区分丝线蛋白的荧光、EPR和核磁共振可观察信号，以确定丝线蛋白在荧光探针中的运动，确定导致亚稳态的折叠途径。这将专门测试β -薄片的早期形成是否是正确折叠的关键。这将通过检查β -sheet C和反应性中心环中稳定和不稳定突变对折叠途径、亚稳态稳定性和转化为潜在构象的速率的影响来进一步验证。

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.