ESR STUDY ON FOLDING AND INTERACTION WITH MEMBRANES OF ACETYLCHOLINESTERASE

Cornell University, Ithaca NY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have been using at the Weizmann Institute acetylcholinesterase (AChE), in particular Torpedo californica (Tc) AChE as a model system for studying folding, unfolding and stabilization of large proteins, taking advantage of knowledge of the 3D structure of the protein, which was solved at the Weizmann in 1991. Initially, we were able to generate a long-lived molten globule (MG) state making use of the disulfide-containing stable nitroxyl probe, biradical. We were further able to show, using CW ESR, that the transition from the MG to the fully unfolded state is a 2-state transition. We will use the methodologies and equipment of ACERT to study the following topics: 1. Use of stop-flow ESR to monitor the kinetics of the transition of native TcAChE to the quasi-native and MG states;2. Use of the same approach to study the effects of chemical and pharmacological chaperones on these processes; 3.Production of mutants of AChE bearing cysteine residues at appropriate locations, together with site-directed spin-labeling (SDSL) to further clarify the kinetics of the unfolding process;4. To make use of spin-labelled TcAChE and of liposomes containing spin-labels at different positions on the phospholipid moieties to study the detailed geometry of the interaction of the MG protein with the bilayer.