Instability of the G-protein beta5 subunit in detergent.
Heterotrimeric guanine nucleotide-binding proteins are important mediators in signal transduction and function by transmitting information from membrane-bound receptors to effectors. Because these proteins are membrane bound and contain covalent lipid modifications, detergents are required for solubilization and purification. It was discovered that the interaction between the beta5 subunit and the gamma2 subunit was disrupted in two detergents, cholate and Chaps (3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate). A beta5gamma2 column was constructed in which recombinant betagamma dimers were immobilized on a FLAG antibody column via a hexahistidine-FLAG-tagged gamma2 subunit, gamma2HF. Greater than 95% of the beta5 subunit was specifically eluted from the immobilized gamma2HF subunit using a cholate gradient from 0.05 to 1.0% and greater than 40% of the beta5 subunit was eluted using a Chaps gradient from 0.05 to 1.0%. In contrast, the beta1, beta2, and beta3 subunits remained bound to the gamma2HF subunit in 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 beta subunits' ability to interact with the gamma2 subunit. These data suggest that the beta5 subunit is not stable in bile acid or Chaps-type detergents (i.e., Chapso, glycocholate, deoxycholate). Therefore, alternative detergents should be used to extract dimers containing the beta5 subunit.
Department of Pharmacology, Health Sciences Center, Charlottesville, Virginia, 22908, USA.
M B
MB
Jones
J C
JC
Garrison
D016367:Baculoviridae; D020355:Cholates / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D044387:GTP-Binding Protein beta Subunits; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005822:Genetic Vectors; D020962:Heterotrimeric GTP-Binding Proteins; D015151:Immunoblotting; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D020355:Cholates; D003902:Detergents; D044387:GTP-Binding Protein beta Subunits; C094668:Gnb5 protein, mouse; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins