Esther Roth

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

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 t1/2 ≃ 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.

Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4‐trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first‐order rate constant for thermal inactivation at 45°C, of single molecules entrapped within the reverse micelles (0.031 min−1), was higher than in aqueous solution (0.007 min−1) or in the presence of normal micelles (0.020 min−1). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first‐order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53°C, were 0.317 and 0.342 min−1, respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.

Targeted cross-linking of a molten globule form of acetylcholinesterase by the virucidal agent hypericin.

The natural product hypericin is a photosensitive polycyclic aromatic dione compound, which has been widely investigated because of its virucidal and antitumor properties. Although it has been suggested that singlet oxygen or a radical species might be responsible for its biological action, its mechanism of action remains unknown. Due to its amphiphilic characteristics, we considered the possibility that it might interact preferentially with partially unfolded proteins which exhibit exposed hydrophobic surfaces. We here demonstrate that hypericin binds to a molten globule species generated from Torpedo acetylcholinesterase, but not to the corresponding native enzyme. Irradiation with visible light, under aerobic conditions, causes chemical cross-linking of the catalytic subunits, to dimers and heavier species, under conditions where no cross-linking is observed for the native enzyme. Both anaerobiosis and sodium azide greatly reduce the extent of cross-linking, suggesting that singlet oxygen is responsible for the phenomenon. This agrees with our observation, using spin traps, that mainly singlet oxygen is produced by the complex of hypericin with the molten globule of acetylcholinesterase. Cross-linking is enhanced in the presence of liposomes to which the molten globule of acetylcholinesterase is quantitatively adsorbed. This may be due to high local concentrations of both hypericin and the protein resulting in close proximity, and hence in a high yield of cross-linking. Molten globule species are believed to be intermediates in both protein folding and translocation through biological membranes. Thus, hypericin may serve as a valuable tool for trapping such intermediates. This might also explain its therapeutic effectiveness toward virus-infected or tumor cells.

Stabilization of Torpedo californica Acetylcholinesterase by Reversible Inhibitors

The dimeric form of Torpedo californica acetylcholinesterase provides a valuable experimental system for studying transitions between native, partially unfolded, and unfolded states since long-lived partially unfolded states can be generated by chemical modification of a nonconserved buried cysteine residue, Cys 231, by denaturing agents, by oxidative stress, and by thermal inactivation. Elucidation of the 3D structures of complexes of Torpedo californica acetylcholinesterase with a repertoire of reversible inhibitors permits their classification into three categories: (a) active-site directed inhibitors, which interact with the catalytic anionic subsite, at the bottom of the active-site gorge, such as edrophonium and tacrine; (b) peripheral anionic site inhibitors, which interact with a site at the entrance to the gorge, such as propidium and d-tubocurarine; and (c) elongated gorge-spanning inhibitors, which bridge the two sites, such as BW284c51 and decamethonium. The effects of these three categories of reversible inhibitors on the stability of Torpedo californica acetylcholinesterase were investigated using spectroscopic techniques and differential scanning calorimetry. Thermodynamic parameters obtained calorimetrically permitted quantitative comparison of the effects of the inhibitors on the enzymes thermal stability. Peripheral site inhibitors had a relatively small effect, while gorge-spanning ligands and those binding at the catalytic anionic site, had a much larger stabilizing effect. The strongest effect was, however, observed with the polypeptide toxin, fasciculin II (FasII), even though, in terms of its binding site, it belongs to the category of peripheral site ligands. The stabilizing effect of the ligands binding at the anionic subsite of the active site, like that of the gorge-spanning ligands, may be ascribed to their capacity to stabilize the interaction between the two subdomains of the enzyme. The effect of fasciculin II may be ascribed to the large surface area of interaction (>2000 A(2)) between the two proteins. Stabilization of Torpedo californica acetylcholinesterase by both divalent cations and chemical chaperones was earlier shown to be due to a shift in equilibrium between the native state and a partially unfolded state ( Millard et al. ( 2003 ) Protein Sci. 12 , 2337 - 2347 ). The low molecular weight inhibitors used in the present study may act similarly and can thus be considered as pharmacological chaperones for stabilizing the fully folded native form of the enzyme.

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic “anionic” subsite (CAS), the peripheral “anionic” subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both “anionic” subsites, but a single site is involved in inhibition. MB also quenches TcAChEs intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active‐site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

Targeted Oxidation of Torpedo californica Acetylcholinesterase by Singlet Oxygen

The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen (1O2) trap, 9,10‐dimethylanthracene, was retarded in the presence of AChE, and the rate of photo‐inactivation was increased in the presence of D2O, indicating that inactivation was due to 1O2 generated by the photosensitizer. CD revealed slightly reduced far‐UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo‐oxidized AChE. However, both near‐UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo‐oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N′‐formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo‐oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross‐linked dimers, whereas irradiation of a partially unfolded state generated higher‐order oligomers. The active‐site gorge of AChE contains Trp in inhibitor‐binding sites that might be targets for photo‐oxidation. Indeed, reversible inhibitors retard photo‐inactivation, and photo‐inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo‐inactivation by sequestering the photosensitizer. Affinity photo‐oxidation of AChE by MB thus provides a valuable model for studying site‐specific photo‐inactivation of enzymes in both fundamental and clinical contexts.