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Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones

¹ Department of Neurobiology and
² Chemical Services, Weizmann Institute of Science, Rehovoth 76100, Israel
³ Departmento de Bioquímica y Biología Molecular, Universidad de Salamanca, Salamanca 37007, Spain

Reprint requests to: Lev Weiner, Chemical Services, Weizmann Institute of Science, Rehovoth 76100, Israel; e-mail: Lev.Weiner{at}weizmann.ac.il; fax: +972-8-934-4142.

Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t_1/2 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca²⁺, Mg²⁺, and Mn²⁺ can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg²⁺ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.

Keywords: Acetylcholinesterase; calorimetry; chemical chaperone; conformational change; forces and stability; molten globule; protein folding

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