title: Sulfhydryl-reactive, cleavable, and radioiodinatable benzophenone photoprobes for study of protein-protein interaction.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA. Lianwangguo@wisc.edu
authors.forename: Lian-Wang
authors.initials: LW
authors.lastname: Guo
authors.forename: Abdol R
authors.initials: AR
authors.lastname: Hajipour
authors.forename: Monica L
authors.initials: ML
authors.lastname: Gavala
authors.forename: Marty
authors.initials: M
authors.lastname: Arbabian
authors.forename: Kirill A
authors.initials: KA
authors.lastname: Martemyanov
authors.forename: Vadim Y
authors.initials: VY
authors.lastname: Arshavsky
authors.forename: Arnold E
authors.initials: AE
authors.lastname: Ruoho
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D006900:Hydroxylation; D007457:Iodine Radioisotopes; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry*; D015394:Molecular Structure; D010777:Photochemistry; D011485:Protein Binding; D013431:Sulfates / Q000737:chemistry*
chemical_list: D001577:Benzophenones; D007457:Iodine Radioisotopes; D015335:Molecular Probes; D013431:Sulfates; C047723:benzophenone; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6