Studies on Partially Unfolded States of Torpedo californica Acetylcholinesterase

Abstract

Chemical modification, by a repertoire of thiol reagents, of the non-conserved Cys²³¹ residue of Torpedo californica acetylcholinesterase (AChE), results in inactivation, even though Cys²³¹ is not involved in catalysis (Steinberg et al., 1990; Dolginova et al., 1992; Silman et al., 1992; Salih et al., 1993). Modification by disulfides and alkylating agents produces partial unfolding of native AChE (N) to a compact state resembling a molten globule (MG). The MG is a collapsed structure possessing much of the secondary structure of the fully folded native protein, but devoid of tertiary structure (Kuwajima, 1989; Ptitsyn, 1992); it is currently believed to serve as a folding intermediate en route from the nascent polypeptide chain, generated on the ribosome, to the fully folded native protein (Kim and Baldwin, 1990; Gething and Sambrook, 1992). This structural assignment for the species produced by chemical modification of Torpedo AChE was based upon spectroscopic evidence, including CD, intrinsic fluorescence and binding of ANS, upon hydrodynamic measurements, including sucrose gradient centrifugation and quasielastic light scattering, and upon enhanced sensitivity to proteolysis (Dolginova et al., 1992). Although modification by disulfides can be rapidly reversed by exposure to reduced glutathione (GSH), the native (N) conformation is not restored, and no catalytic activity is recovered. AChE so demodified is a partially unfolded species whose physicochemical characteristics are virtually identical to those of the modified enzyme, and sucrose gradient centrifugation reveals it to be stable for many hours without aggregating.