Instability of the G-protein beta(5) Subunit in detergent

Heterotrimeric guanine nucleotide-binding proteins are important mediators in signal transduction and function by transmitting information from membra ne-bound receptors to effecters. Because these proteins are membrane bound and contain covalent lipid modifications, detergents are required for solub ilization and purification. It was discovered that the interaction between the beta(5) subunit and the gamma(2) subunit was disrupted in two detergent s, cholate and Chaps (3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfo nate). A beta(5)gamma(2) column was constructed in which recombinant py dim ers were immobilized on a FLAG antibody column via a hexahistidine-FLAG-tag ged gamma(2) subunit, gamma(2HF). Greater than 95% of the beta(5) subunit w as specifically eluted from the immobilized gamma(2HF) subunit using a chol ate gradient from 0.05 to 1.0% and greater than 40% of the beta(5) subunit was eluted using a Chaps gradient from 0.05 to 1.0%. In contrast, the beta( 1), beta(2),and beta(3) subunits remained bound to the gamma(2HF) subunit i n all concentrations of Chaps and cholate, Genapol C-100, Triton X-100, and polyoxyethylene-10-lauryl ether did not interfere with any of the four bet a subunits' ability to interact with the gamma(2) subunit. These data sugge st that the beta(5) subunit is not stable in bile acid or Chaps-type deterg ents (i.e,, Chapso, glycocholate, deoxycholate). Therefore, alternative det ergents should be used to extract dimers containing the beta(5) subunit. (C ) 1999 Academic Press.