Sulfhydryl-reactive, cleavable, and radioiodinatable benzophenone photoprobes for study of protein-protein interaction.

The major task in proteomics is to understand how proteins interact with their partners. The photo-cross-linking technique enables direct probing of protein-protein interaction. Here we report the development of three novel sulfhydryl-reactive benzophenone photoprobes of short "arm" length, each with a substitution of either amino, iodo, or nitro at the para-position, rendering the benzophenone moiety directly radioiodinatable. Their potential for study of protein-protein interaction was assessed using the inhibitory subunit of rod cGMP phosphodiesterase (PDEgamma) and the activated transducin alphasubunit (G alpha t-GTPgammaS) as a model system. These photoprobes proved to be stable at neutral pH and dithiothreitol-cleavable in addition. The PDEgamma constructs derivatized at the C-terminal positions with these probes could be readily purified, had unaltered PDEgamma functional activity, and were shown to photo-cross-link to G alpha t-GTPgammaS with an efficiency as high as 40%. Additionally, the amino benzophenone probe was radioiodinated, facilitating sensitive detection of label transfer. The uniquely combined features of these benzophenone photoprobes promise robust and flexible methods for characterization of protein-protein interaction, either by mass spectrometry when a nonradioactive label is available or by autoradiography when using radioiodinated derivatives.