Irreversible thermal denaturation ofTorpedo californicaacetylcholinesterase

Abstract

Thermal denaturation of Torpedo culifornicu acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the twostate kinetic scheme N 5 D, with activation energy 131 * 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 t 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.Keywords: acetylcholinesterase; differential scanning calorimetry; irreversible denaturation; molten globule; thioldisulfide exchange; two-state kinetic model Folding of many small globular proteins is a cooperative process in the course of which only two states, the fully unfolded state, U, and the native state, N, are significantly populated. In the case of large proteins, which are generally believed to contain several domains (Jaenicke, 1991;Garel, 1992), folding has generally been considered to be cooperative within each domain, the only species populated in the course of either folding or unfolding being combinations of completely folded and completely unfolded domains (Privalov, 1982;Brandts et al., 1989;Garel, 1992). Evidence is accumulating, however, that another state, intermediate between N and U, can exist. This is a compact state that lacks the unique tertiary structure of the native protein but possesses substantial secondary structure. This state has been named the molten globule (MG) state (Kuwajima, 1989;Kim & Baldwin, 1990;Ptitsyn, 1992). The MG state is considered to serve as an intermediate on the pathway from the nascent polypeptide chain to the fully folded native protein in vivo (Gething & Sambrook, 1992). For most proteins, the MG state is unstable under physiological conditions and readily converts to the N state in vitro (Ptitsyn, 1992 Because the definition of the MG state is controversial (see, for example, Griko et al., 1994; Ewbank et al., 1995;Okazaki et al., 1995), in the present paper this term is used to refer to compact states of acetylcholinesterase (AChE) that preserve substantial secondary structure and lack most of the tertiary structure of the native enzyme. We have shown recently that exposure of a native dimeric form of Torpedo AChE to various treatments generates long-lived partially unfolded species displaying many of the characteristics of the MG...

Two partially unfolded states oftorpedo californicaacetylcholinesterase

“…MG,,,, has a tendency to aggregate at the temperature of thermal denaturation of the N state, as could be seen by sucrose gradient centrifugation. Thus, the only conclusion that we can draw from the DSC traces obtained is that the native domain structure does not appear to be preserved in MG,,,, (Kreimer et al, 1995).…”

“…It was claimed recently that a native domain was preserved within a partially unfolded fragment of staphylococcal nuclease, despite the small size of this protein, and that this native domain was responsible for the twostate character of the transition of this species to a fully unfolded state as observed by DSC (Griko et al, 1994b). Thermal unfolding of native AChE reveals a single peak with the maximum at 45 "C (Kreimer et al, 1995). It is unlikely that MG,,,, retains any native domain structure, because it lacks a calorimetric peak in the temperature range of the N + MG,,,, transition (Fig.…”

“…To avoid possible complications arising from spontaneous thiol-disulfide exchange (Eichler et al, 1994b;Kreimer et al, 1995), MG,,, was prepared from chemically modified AChE. No thermally induced cooperative transition is observed for MG,,,,, whether produced by chemical modification with DTP (Fig.…”

“…No pronounced aggregation of MG,,,, occurs within this period, but more prolonged incubation at room temperature results in the appearance of aggregated species (Kreimer et al,3994b). The rate of aggregation is increased at higher temperatures (Kreimer et al, 1995), and this hampers observation of the MG,,,, -+ MG,,, transition. With the addition of 0.4 M Gdn.HC1 to MG,,,, produced by DTP, however, collapse of the band in the near UV occurs within 3 h at room temperature, resulting in a weak positive band characteristic of MG,,, (Fig.…”

“…1-2 M guanidinium hydrochloride (Gdn-HCI), whereas at 5-6 M Gdn-HCI, it is in a U state (Eichler et al, 1994b;Kreimer et al, 1994b;Weiner et al, 1994). MG states of Torpedo AChE are also produced by chemical modification of a single buried nonconserved cysteine, Cys2", by various thiol-specific reagents (Dolginova et al, 1992;Kreimer et al, 1994a) and by thermal denaturation (Kreimer et al, 1995). An important property of the MG states so generated is that they are maintained even when the perturbing agent is removed, e.g., if Gdn-HC1 is removed by dialysis or by dilution (Eichler et al, 1994b), or if chemical modification is reversed (Dolginova et al, 1992;Kreimer et al, 1994a).…”