Abstract
Previously, we generated genetically fused dimers and trimers of subunit c of the Escherichia coli ATP synthase based upon the precedent of naturally occurring dimers in V-type H(+)-transporting ATPases. The c(2) and c(3) oligomers have proven useful in testing hypothesis regarding the mechanism of energy coupling. In the first part of this paper, the uncoupling Q42E substitution has been introduced into the second loop of the c(2) dimer or the third loop of the c(3) trimer. Both mutant proteins proved to be as functional as the wild type c(2) dimer or wild type c(3) trimer. The results argue against an obligatory movement of the epsilon subunit between loops of monomeric subunit c in the c(12) oligomer during rotary catalysis. Rather, the results support the hypothesis that the c-epsilon connection remains fixed as the c-oligomer rotates. In the second section of this paper, we report on the effect of substitution of the proton translocating Asp(61) in every second helical hairpin of the c(2) dimer, or in every third hairpin of the c(3) trimer. Based upon the precedent of V-type ATPases, where the c(2) dimer occurs naturally with a single proton translocating carboxyl in every second hairpin, these modified versions of the E. coli c(2) and c(3) fused proteins were predicted to have a functional H(+)-transporting ATPase activity, with a reduced H(+)/ATP stoichiometry, but to be inactive as ATP synthases. A variety of Asp(61)-substituted proteins proved to lack either activity indicating that the switch in function in V-type ATPases is a consequence of more than a single substitution.
Highlights
Hϩ-transporting F1F0 ATP synthases utilize the energy of a transmembrane electrochemical Hϩ gradient to catalyze formation of ATP
Effect on Function of the Q42E Substitution in the Last Loop of Subunits c2 and c3—A Q42E mutation was introduced into the last polar loop in the c2 dimer, c3 trimer, and c4 tetramer to generate subunits c2(Q42ЈE), c3(Q42ЈЈE), and c4(Q42ЈЈЈE), respectively
The anti-c serum used had previously been shown to be directed to residues in the polar loop [41], and the lighter band for c1Q42E membranes may reflect reduced antibody binding to the substituted polar loop
Summary
Strains and Plasmids—The plasmids used in this study are derivatives of plasmid pDF163, which contains the wild type uncBEFH genes (bases 870 –32161 coding subunits a, c, b, and ␦) cloned between the HindIII and SphI sites of plasmid pBR322 [36]. The plasmids carrying the wild type c1 monomer (pNOC) and fused c2 dimer (pPJC2), c3 trimer (pPJC3), and c4 tetramer (pPJC4) were derived from plasmid pNOC, which has a C21S substitution in subunit b, a mutation which has no effect on function [4, 24]. These plasmids and the derivatives described below were expressed in the recA chromosomal ⌬uncBEFH deletion strain JJ001 (pyrE41, entA403, argHI, rspsL109, supE44, ⌬uncBEFH), recA56, srl::Tn10) [4]. The substitutions generated by this method were c2(D61N), c2(D61G), and c2(D61S). Plasmids bearing the c3(D61ЈN), c3(D61ЈG), and c3(D61ЈS)
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