TFPACD, a novel bifunctional reagent for reacting with DCCD sites in proteins: studies using Escherichia coli ATP synthase.

A novel cross-linker, 1-[6-(4-azido-2,3,5,6-tetrafluorobenzamido)hexyl]-3-cyclohexylc arbodiimide (TFPACD), has been synthesized and tested by reaction with the Escherichia coli ATP Synthase (ECF1F0). The reagent has a carbodiimide as one reactive group, which is shown to react with ECF1F0 in a similar way to 1,3-dicyclohexylcarbodiimide (DCCD) and modify the beta subunit of the ECF1 part and the c subunit of the F0 part. Reaction with both the ECF1 and F0 parts of the complex inhibited ATPase activity. The second reactive group in the reagent is the photoactivatable tetrafluorophenylazide moiety. Subsequent UV photolysis of TFPACD--modified ECF1 and ECF1F0 led to generation of cross-linked products in significant yields, one between beta and alpha subunits; the second, dimers of the c subunit of the F0 part.