Insights into ATP Synthase Structure and Function Using Affinity and Site-Specific Spin Labeling

A variety of different approaches has been used during the last couple of decades to investigatestructure and function relationships within the catalytic portion of the F₀F₁-ATP synthase andof its interactions with the proton-translocator F₀. In our group, we employ ESR spectroscopywith the use of stable organic radicals, so-called spin labels, as reporter groups. The radicalsare either attached to substrates/ligands or specifically inserted into the protein structure bysite-specific spin labeling. Both approaches bear intrinsic advantages for their special usesand result in the specific information that is available through ESR, e.g., structural changesdue to binding of effector molecules (e.g., Mg²⁺ ions), conformational transitions duringcatalytic turnover, distance information on radicals bound at 20 Å or less, and information onthe binding characteristics of labeled substrates. This review summarizes the results of a varietyof different approaches we have used during the last years to study, with the help of ESRspectroscopy, the structure of the nucleotide binding sites of F₁-ATPases of different originsas well as interactions with F₀ subunits.