title: Thermal denaturation of wild type and mutant recombinant acetylcholinesterase from amphioxus: effects of the temperature of in vitro expression and of reversible inhibitors.
abstract: We have studied the thermal inactivation at 37 degrees C of wild type and mutant ChE2 (C310A, F312I, C466A, C310A/F312I, and C310A/C466A) from amphioxus (Branchiostoma floridae) expressed in vitro in COS-7 monkey cells under three sets of conditions: 30 degrees C for 48 h, 30 degrees C for 24 h and 37 degrees C for 24 h, and 37 degrees C for 48 h. We found biphasic denaturation curves for all enzymes and conditions, except wild type and C310A ChE2 expressed at 30 degrees C for 48 h. Generally, single mutants are more unstable than wild type, and the double mutants are even more unstable. We propose a model involving stable and unstable conformations of the enzymes to explain these results, and we discuss the implications of the model. We also found a correlation between the melting temperature of the ChEs and the rates at which they denature at 37 degrees C, with the denaturation of the unstable conformation dominating the relationship. Reversible cholinergic inhibitors protect the ChEs from thermal denaturation, and in some cases produce monophasic denaturation curves; we also propose a model to explain this stabilization.
authors.affiliation: Department of Biology, Birmingham-Southern College, Birmingham, AL 35254, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Perrin
authors.forename: Melissa
authors.initials: M
authors.lastname: Rowland
authors.forename: Matthew
authors.initials: M
authors.lastname: Wolfe
authors.forename: Igor
authors.initials: I
authors.lastname: Tsigelny
authors.forename: Leo
authors.initials: L
authors.lastname: Pezzementi
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002800:Cholinesterase Inhibitors / Q000737:chemistry; D002802:Cholinesterases / Q000737:chemistry* / Q000235:genetics; D002816:Chordata, Nonvertebrate; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011995:Recombination, Genetic; D013696:Temperature
chemical_list: D002800:Cholinesterase Inhibitors; C116697:cholinesterase 2; D002802:Cholinesterases