Chemical modification of Torpedo californica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercury derivatives, is metastable. At room temperature, it converts spontaneously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of acetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, with concomitant recovery of most of the enzymic activity of the native enzyme. In contrast, similar demodification of acetylcholinesterase modified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluorescence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produced initially by mercurials is "native-like", whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurial-modified enzyme, is partially unfolded and displays many of the features of the "molten globule" state. Arrhenius plots show that the quasi-native state produced by organomercurials is separated by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D structures of native acetylcholinesterase and of a heavy-atom derivative obtained with HgAc2 suggests that the mercurial-modified enzyme may be stabilized by additional interactions of the mercury atom attached to the free thiol group of Cys231, specifically with Ser228O gamma with the main-chain nitrogen and carbonyl oxygen of the same serine residue.

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...

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Acetylcholinesterase from Torpedo californica partially unfolds to a state with the physicochemical characteristics of a ''molten globule'' upon mild thermal denaturation or upon chemical modification of a single nonconserved buried cysteine residue, Cys 231 . The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys 231 by mercuric chloride produces initially a quasinative state of Torpedo acetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1-2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteins in situ.

The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex. We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically. The possible basis of this asymmetry is discussed. We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy. Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation. The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.