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

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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.