title: Liposomes as chaperone mimics with controllable affinity toward heat-denatured formate dehydrogenase from Candida boidinii.
abstract: Chaperone machinery in living systems can catch denatured enzymes and induce their reactivation. Chaperone mimics are beneficial for applying enzymatic reactions in vitro. In this work, the affinity between liposomes and thermally denatured enzymes was controlled to stabilize the enzyme activity. The model enzyme is formate dehydrogenase from Candida boidinii (CbFDH) which is a homodimer and negatively charged in the phosphate buffer solution (pH 7.2) used. The activity of free CbFDH readily decreased at 58 °C following the first-order kinetics with the half-life t1/2 of 27 min. The turbidity measurements showed that the denatured enzyme molecules formed aggregates. The liposomes composed of zwitterionic phosphatidylcholines (PCs) stabilized the CbFDH activity at 58 °C, as revealed with six different PCs. The PC liposomes were indicated to bind to the aggregate-prone enzyme molecules, allowing reactivation at 25 °C. The cofactor β-reduced nicotinamide adenine dinucleotide (NADH) also stabilized the enzyme activity. The affinity between liposomes and denatured CbFDH could be modulated by incorporating cationic 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) in PC membranes. The t1/2 values significantly increased in the presence of liposomes ([lipid] = 1.5 mM) composed of PC and DOTAP at the mole fraction f(D) of 0.1. On the other hand, the DOTAP-rich liposomes (f(D) ≥ 0.7) showed strong affinity toward denatured CbFDH, accelerating its deactivation. The liposomes with low charge density function as chaperone mimics that can efficiently catch the denatured enzymes without interfering with their intramolecular interaction for reactivation.
authors.affiliation: Department of Applied Molecular Bioscience, Yamaguchi University , 2-16-1 Tokiwadai, Ube 755-8611, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Yoshimoto
authors.forename: Ryohei
authors.initials: R
authors.lastname: Kozono
authors.forename: Naoki
authors.initials: N
authors.lastname: Tsubomura
mesh_terms: D002175:Candida / Q000201:enzymology*; D005560:Formate Dehydrogenases / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D009243:NAD / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry
chemical_list: D008081:Liposomes; D010713:Phosphatidylcholines; D009243:NAD; D005560:Formate Dehydrogenases