Structural Stability of Chloroplast Coupling Factor 1 as Determined by Differential Scanning Calorimetry and Cold Inactivation

Abstract

At least part of the γ subunit of the catalytic portion of the chloroplast ATP synthase (CF₁) is present in the middle of the α₃β₃ heterohexamer. Interactions of the α/β subunits with the γ subunit stabilize the hexameric structure. Surprisingly, neither reduction of the γ disulfide nor selective proteolysis of α, β, and γ affects the thermal stability of EDTA-treated CF₁ preparations, as determined by differential scanning calorimetry. Dissociation of the enzyme in the cold may be monitored by loss of the ATPase activity of CF₁ depleted of its ε subunit [CF₁(−ε)]. The rate of cold inactivation of ATPase activity of reduced and alkylated CF₁(−ε) treated with trypsin in solution was much faster than that of CF₁(−ε) (8.1 versus 38.7 min, respectively, for 50% loss of activity). The increased cold lability of the trypsin-treated enzyme was not a consequence of the cleavage of the γ. CF₁ incubated with trypsin under conditions in which γ is not cleaved was as cold labile as CF₁ with cleaved γ. Instead, loss of the δ subunit and a few residues from the C-termini of the β subunits were responsible for the increased cold lability of the enzyme.