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.
Guo
Lian-Wang
LW
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA. Lianwangguo@wisc.edu
Hajipour
Abdol R
AR
Gavala
Monica L
ML
Arbabian
Marty
M
Martemyanov
Kirill A
KA
Arshavsky
Vadim Y
VY
Ruoho
Arnold E
AE
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*
D001577:Benzophenones; D007457:Iodine Radioisotopes; D015335:Molecular Probes; D013431:Sulfates; C047723:benzophenone; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6