Expression of the wild-type and the Cys-/Trp-less α3β3γ complex of thermophilic F1-ATPase in Escherichia coli

Abstract

The alpha, beta and gamma subunits of F1-ATPase from thermophilic Bacillus PS3 were expressed in Escherichia coli cells simultaneously in large amounts. Most of the expressed subunits assembled into a form of alpha 3 beta 3 gamma complex in E. coli cells and this complex was easily purified to homogeneity. The recombinant alpha 3 beta 3 gamma complex thus obtained showed similar enzymatic properties to the alpha 3 beta 3 gamma complex obtained by in vitro reconstitution from individual subunits (Yokoyama, K. et al. (1989) J. Biol. Chem. 264, 21837-21841) except that the former had several-fold higher ATPase activity than the latter. Using this expression system, a mutant alpha 3 beta 3 gamma complex with no Trp and Cys was generated by replacing alpha Cys193 and alpha Trp463 with Ser and Phe, respectively. This mutant complex was functionally intact, indicating both residues are not essential for catalysis. The Cys-/Trp-less complex is a convenient 'second wild type' enzyme from which one can generate mutants with Trp (as a fluorescent probe) or Cys (as an acceptor of a variety of probes) at desired positions without concern for 'background' Trp and Cys residues.

Thermophilic F1-ATPase Is Activated without Dissociation of an Endogenous Inhibitor, ε Subunit

“…Other chemicals were the highest grade commercially available. ␣ 3 ␤ 3 ␥ complex of TF 1 was prepared as described previously (7). Purified ␣ 3 ␤ 3 ␥ complex contained less than 0.1 mol of adenine nucleotides/mol of enzyme when analyzed by HPLC (41).…”

Biological application of multi-component nanowires in hybrid devices powered by F1-ATPase motors

“…Preparation of the F 1 -ATPase F 1 -ATPase, whose coding sequence was isolated from thermophilic bacterium, Bacillus PS3, was a kindly gift from Dr. Montemagno. Thermophilic bacterium, Bacillus PS3, with ten histidine on β subunits were expressed and purified as follows (Matsui and Yoshida, 1995). The expressed strain was cultured in 2 × YT medium (AMP + ) at 37 • C for 4 h. When the value of A 660 is between 0.6 and 0.8, the expression of the F 1 -ATPase was induced by addition of 1 mmol/L isoprophylthio-β-D-galactoside for 3 h. Then, the products were collected after centrifuged at 4000 × g for 15 min.…”

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1‐ATPase β subunit determined by 13C‐NMR

“…In this experiment, we used a complex containing ~(C193S/W463F) to avoid the carboxymethylation of Cys-193 of the ~ subunit. This complex was reported to have the same level of ATPase activity as that of the wild-type 0 [3[33] t complex at 25°C [8]. The number of free sulfhydryl groups of the complex was estimated to be 3.2 by DTNB titration.…”

“…I3(E190C) and offCI93S/W463F) correspond to mutant [3 in which Glu-190 was substituted by cysteine (Cys), and mutant ~ in which Cys-193 and Trp-463 were substituted by serine and phenylalanine, respectively. For expression of the mutant cc:d3:~ 7 complex, E. coli strain JMIO3A(uncB-D) was transformed with the constructed plasmid [8]. [3(E190C) and a3fJ( E190C)37 [3(E190C) was purified as described previously [7].…”

“…Completion of carboxymethylation was confirmed by quantitative analysis of sulfhydryl groups using DTNB [10]. The overproduction and purification of o~313(E190C)37 were carried out as reported by Matsui et al [8]. Urea was added to a solution containing 250 mg of the complex at more than 8 M, followed by the addition of 5 mole equivalents of an iodo[1-ISC]acetic acid solution.…”