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. 1997 Apr 1;94(7):2848-52.
doi: 10.1073/pnas.94.7.2848.

Membrane-promoted unfolding of acetylcholinesterase: a possible mechanism for insertion into the lipid bilayer

I Shin  1 , D Kreimer, I Silman, L Weiner
Affiliations

Membrane-promoted unfolding of acetylcholinesterase: a possible mechanism for insertion into the lipid bilayer

I Shin et al. Proc Natl Acad Sci U S A. .

Abstract

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 non-conserved buried cysteine residue, Cys231. 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 Cys231 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.

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Figures

Figure 1
Figure 1
Effect of liposomes on thermal inactivation of T. californica AcChoEase. Samples of AcChoEase (4 × 10−7 M) in buffer 1 were incubated at 28°C. Aliquots were withdrawn at appropriate times for assay of enzymic activity. (•), Control; (⋄), in the presence of DMPC liposomes (5 mg/ml). (Inset) Arrhenius plots of the temperature dependence of the rate of thermal inactivation of native AcChoEase in the presence (○) and absence (▪) of DMPC liposomes.
Figure 2
Figure 2
Interaction of thermally inactivated AcChoEase with DMPC liposomes as revealed by gel filtration. Samples of Torpedo AcChoEase (4 × 10−7 M) in buffer 1 were incubated at 34°C in the presence of DMPC liposomes (5 mg/ml). After 10 min, when AcChoEase activity was <0.5%, the reaction mixture was applied to a Sephacryl S-300 column; protein and lipid in the effluent were monitored by measuring, respectively, fluorescence and absorbance at 340 nm. (•), Fluorescence at 340 nm; (○), OD at 340 nm. Arrow marks the position of thermally inactivated AcChoEase in the absence of liposomes.
Figure 3
Figure 3
CD spectra in the far and near UV of native and unfolded preparations of Torpedo AcChoEase. (♦), Native AcChoEase; (Δ), AcChoEase in 5 M Gdn·HCl; (⋄), AcChoEase after thermal inactivation in the presence of DMPC liposomes; (○), AcChoEase after thermal inactivation in the presence of DMPC liposomes followed by removal of the liposomes (see Materials and Methods). Thermal inactivation was for 30 min at 37°C. Protein concentrations were 3–4.5 μM in buffer 1.
Figure 4
Figure 4
Kinetics of chemical modification and irreversible inactivation of T. californica AcChoEase by HgCl2 in the presence and absence of liposomes. Samples of Torpedo AcChoEase (4 × 10−7 M) in buffer 1 were incubated at 28°C with 0.1 mM HgCl2 in the presence and absence of DMPC liposomes (5 mg/ml). Aliquots were withdrawn at appropriate times and either assayed directly for enzymic activity or diluted into 1 mM GSH in 0.01 M Tris buffer (pH 8.0) prior to the assay, so as to monitor the decrease in recoverable enzymic activity (see Materials and Methods). (♦), Decrease in AcChoEase activity in the absence of liposomes; (⋄), decrease in AcChoEase activity in the presence of liposomes; (•), decrease in recoverable AcChoEase activity in control aliquots exposed to reduced GSH prior to assay; (Δ), decrease in recoverable AcChoEase activity in liposome-containing aliquots exposed to reduced GSH prior to assay. (Inset) Arrhenius plots of the temperature-dependence of the rate of the N* → MG transition in the presence (○) and absence (▪) of DMPC liposomes.
Figure 5
Figure 5
Interaction of HgCl2-modified Torpedo AcChoEase with DMPC liposomes and release by GSH. Samples of AcChoEase (3 × 10−6 M) in buffer 1 were incubated with 0.3 mM HgCl2 at 10°C. After 40 min, when AcChoEase activity was <0.5%, exposure to GSH resulted in ≈90% reactivation. (A) A sample of unreactivated enzyme (30 μl) was added to 270 μl of DMPC liposomes (35 mg/ml) in buffer 1. Within 1 min, the reaction mixture was applied to a Sephacryl S-300 column. Protein and lipid in the effluent were monitored as in the legend to Fig. 2. (•), Fluorescence at 340 nm; (⋄), absorbance at 340 nm. (B) Experimental conditions and symbols as in A, except that within 1 min of mixing modified AcChoEase and liposomes GSH was added to a final concentration of 5 mM prior to gel filtration.
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