Thiol modification as a probe of conformational forms of the F 1 ATPase of Escherichia coli and of the structural asymmetry of its β subunits

The sulfhydryl groups of soluble and membrane‐bound F 1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5‐iodoacetamidofluorescein, 2‐[(4′‐iodoacetamido)anilino]naphthalene‐6‐sulfonic acid, 4‐[ N ‐(iodoacetoxy)ethyl‐ N ‐methyl]amino‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole and 2‐[(4′‐maleimidyl)anilino]naphthalene‐6‐sulfonic acid. Whereas γ and δ subunits were always labeled by these reagents, the β subunit reacted preferentially in the soluble enzmye, and the α subunit in the membrane‐bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three β subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One β subunit was cross‐linked to the ɛ subunit on treatment of the ATPase with 1‐ethyl‐3‐[3‐(dimethyl‐amino)propyl]carbodiimide, as observed previously by Lötscher et al. [ Biochemistry (1984) 23 , 4134–4140]. A second β subunit, which did not cross‐link to the ɛ subunit, was modified preferentially by the fluorescent thiol reagents and by 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole. The third β subunit was less chemically reactive than the others. Both α and β subunits of the soluble 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.