A, reaction of selenocystamine with cysteine. Selenocystamine is known to react with protein cysteines to form mixed sulfide-selenides and selenocysteamine. B, titration of selenocystamine in the ELISA leaving the compound in the reaction. The experiment was carried out as described under Materials and Methods, Competition ELISA, except that 5 × 10⁹ SIGK-displaying phage particles per well were used instead of 0.1 × 10¹⁰. Data are representative of five independent experiments, shown as mean ± S.E.M. of duplicate determinations, and are fit with a sigmoid dose-response curve using the Prism 5 software (GraphPad Software, San Diego, CA). C, titration of selenocystamine in the ELISA, removing the compound from the reaction after pretreating Gβγ with it for 30 min at 4°C. The experiment was carried out as described under Materials and Methods. Deactivation ELISA. Data are representative of five independent experiments, shown as mean ± S.E.M. of duplicate determinations, and are fit with a sigmoid dose-response curve using the GraphPad Prism 5 software. D, prevention of the action of selenocystamine by DTT and GSH. The experiment was carried out as described under Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁γ₂ was immobilized in the wells. Five millimolar DTT or GSH (final concentration) was added together with 1 μM selenocystamine and incubated for 30 min at 4°C. The wells were washed six times with TBS plus 0.5% Tween 20, and then phage was added. Data are representative of five independent experiments and are shown as mean ± S.E.M. of duplicate determinations. E, reversal of the effect of selenocystamine by DTT and GSH. The experiment was carried out as described under Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁γ₂ was pretreated with 1 μM selenocystamine for 30 min at 4°C and then immobilized in the wells. After six washes with TBS plus 0.5% Tween 20, 5 mM DTT or GSH (final concentration) was added and incubated for 1 h at 4°C. Data are representative of five independent experiments and are shown as mean ± S.E.M. of duplicate determinations.

Effect of selenocystamine on the interaction between Gβγ and three physiological binding partners. A, pull-down assay against Gα_i. Ten nanomolar biotinylated-Gβ₁γ₂ was pretreated with 1 μM selenocystamine, then incubated with 10 nM Gα_i, and finally immobilized on streptavidin-agarose beads. Associated Gα_i was detected with antibody to Gα_i, and Gβγ was detected with antibody specific to the C terminus of Gβ (B600). Data are representative of two independent experiments. B, flow cytometry GRK2 binding assay. 20 nM biotinylated-Gβ₁γ₂ was immobilized on streptavidin-polystyrene particles, treated with selenocystamine at the shown concentrations, and finally incubated with 10 nM Alexa Fluor-532-labeled GRK2. The bead-associated fluorescence was recorded in a BD FACSCanto flow cytometer. Data are representative of two independent experiments, are corrected for background, are shown as mean ± S.E.M. of duplicate determinations, and are fit with a sigmoid dose-response curve using the Prism 5 software. C, PLCβ2 activity assay. Purified PLCβ2 (0.5 ng) was assayed with 1.5 mM CaCl₂ and selenocystamine at the shown concentrations, in the presence or absence of 200 nM purified Gβ₁γ₂. The reactions were incubated for 30 min at 30°C. [³H]inositol-1,4,5-trisphosphate released from sonicated phospholipid micelles containing 50 μM PIP₂, 200 μM phosphatidylethanolamine, and [³H]PIP₂ (3.0 × 10⁻³ μCi/assay) was measured by liquid scintillation counting. Data are representative of two independent experiments, are shown as mean ± S.E.M. of duplicate determinations, and are fit with a sigmoid dose-response curve using the Graph Pad Prism 5 software.

A, SIGK bound to the Gβγ “hot spot.” Electrostatic surface representation prepared with the Molsoft ICM-Pro software from Davis et al., 2005 (PDB code 1XHM). Positively charged areas are in blue, negatively charged in red, and hydrophobic in white. The hydrophobic core in SIGK interacts with a hydrophobic pocket in the Gβγ “hot spot,” whereas Lys4 interacts with a negative pocket. B, Gβγ “hot spot” without SIGK bound to it. Electrostatic surface representation of the Gβγ “hot spot” (large circle) without SIGK bound. Trp332, Lys57, Tyr59, Trp99, Val100, Met101, Leu117, Tyr145, and Met188 comprise a hydrophobic pocket (oval) that interacts with the hydrophobic core of SIGK. Asp228, Asn230, and Asp246 delimit a negative pocket (small circle) that interacts with the first lysine of SIGK. C, SIGK, SIGC, and SIGC(-cysteamine). Stick and ball representations prepared with the Molsoft ICM-Browser software from the structure published by Davis et al., 2005 (PDB code 1XHM). When SIGK is bound to Gβγ, the side chain of Lys4 is near Cys204.

A, mass spectrum of SIGC(-cysteamine)-treated Gβγ. Top left, Gβ₁6hisγ₂C68S (2.5 μM) in 20 mM ammonium acetate, pH 7.0, was analyzed in a Waters “Synapt” nano ESI QToF Mass Spectrometer designed for intact protein analysis. +12, +13, and +14 charged species were observed. Only the +13 charged species is shown for simplicity. Bottom left, Gβ₁6hisγ₂C68S (2.5 μM) in 20 mM ammonium acetate, pH 7.0, was incubated with 20 μM SIGC(-cysteamine) for 15 min and then analyzed as described above. Right, tandem MS dissociation of Gβ and Gγ by increasing the collision cell voltage results in release of free Gγ, whose mass is unchanged by SIGC(-cysteamine) (data not shown), and free Gβ₁ (+8 ion shown for simplicity). (It is highly unlikely that a noncovalent complex between SIGC(-cysteamine) and Gβ survive these collision conditions necessary to dissociate Gβ from Gγ.) B, action of SIGC(-cystamine) on the Gβ(C204A)γ. No wash, ELISA leaving 100 μM SIGK, 100 μΜ SIGC(-cysteamine), or 10 mM cystamine in the reaction. The experiment was carried out as described under Deactivation ELISA in Materials and Methods, except that 5 × 10⁹ SIGK-displaying phage particles per well were used instead of 1 × 10⁹. Wash, ELISA removing SIGC(-cysteamine) from the reaction after pretreating Gβγ with it for 30 min at 4°C, as described under Deactivation ELISA in Materials and Methods. Data are representative of two independent experiments and are shown as mean ± S.E.M. of duplicate determinations.

A, action of selenocystamine on the GβC204A-mutant Gβγ. The experiment was carried out as described Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁(C204A)γ₂ was pretreated with 1 μM selenocystamine for 30 min at 4°C, and then immobilized in the wells. After six washes with TBS plus 0.5% Tween 20, 5 × 10⁹ SIGK-displaying phage particles were added per well. Data are representative of five independent experiments and are shown as mean ± S.E.M. of duplicate determinations. B, mass spectrum of selenocystamine-treated Gβγ. Top left, Gβ₁6hisγ₂C68S (2.5 μM) in 20 mM ammonium acetate, pH 7.0, was analyzed in a Waters “Synapt” nano ESI QToF Mass Spectrometer. +12, +13, and +14 charged species were observed. Only the +13 charged species is shown for simplicity. The major peak at 3542.66 (46,042 Da) represents the Gβγ heterodimer where Gβ has the N terminal Met cleaved and Gγ subunits that have not been processed. Other peaks represent either differential N terminal processing of Gγ or ion adducts. Bottom left, Gβ₁6hisγ₂C68S (2.5 μM) in 20 mM ammonium acetate, pH 7.0, was incubated with 20 μM selenocystamine for 15 min and then analyzed as described above. Right, tandem MS dissociation of Gβ and Gγ by increasing the collision cell voltage results in release of free Gγ, whose mass is unchanged by selenocystamine (not shown), and free Gβ₁ (+8 ion shown for simplicity). (It is highly unlikely that a noncovalent complex between selenocystamine and Gβ survive these collision conditions necessary to dissociate Gβ from Gγ.)

A, titrations of cystamine, l-cystine, and HEDS in the ELISA. Titrations were carried out as described under Deactivation ELISA in Materials and Methods. The compounds were removed from the reaction after pretreating Gβγ with them for 30 min at 4°C. Data are representative of three independent experiments, are shown as mean ± S.E.M. of duplicate determinations, and are fit with a sigmoid dose-response curve using Prism 5 software. B, prevention of the action of cystamine by DTT and GSH. The ELISA was carried out as described under Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁γ₂ was immobilized in the wells. Five millimolar DTT or GSH (final concentration) was added together with 1 mM cystamine and incubated for 30 min at 4°C. The wells were next washed six times with TBS plus 0.5% Tween 20, and then the phage was added. Data are representative of three independent experiments and are shown as mean ± S.E.M. of duplicate determinations. C, reversal of the effect of cystamine by DTT and GSH. The ELISA was carried out as described under Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁γ₂ was pretreated with 1 mM cystamine for 30 min at 4°C, and then immobilized in the wells. After six washes with TBS plus 0.5% Tween 20, 5 mM DTT or GSH (final concentration) was added and incubated for 1 h at 4°C. Data are representative of three independent experiments and are shown as mean ± S.E.M. of duplicate determinations. D, action of cystamine on the GβC204A-mutant Gβγ. The ELISA was carried out as described under Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁γ₂ was pretreated with 10 mM cystamine for 30 min at 4°C, and then immobilized in the wells. After six washes with TBS plus 0.5% Tween 20, 5 × 10⁹ SIGK-displaying phage particles were added per well. Data are representative of five independent experiments and are shown as mean ± S.E.M. of duplicate determinations.

A, titration of SIGC(-cysteamine) in the ELISA. Left, titration of SIGC(-cysteamine) in the ELISA leaving the peptide in the reaction. The experiment was carried out as described under Deactivation ELISA in Materials and Methods, except that 5 × 10⁹ SIGK-displaying phage particles per well were used instead of 10⁹. Right, titration of SIGC(-cysteamine) in the ELISA removing the peptide from the reaction after pretreating Gβγ with it for 30 min at 4°C, as described under Deactivation ELISA in Materials and Methods. Data are representative of two independent experiments, are shown as mean ± S.E.M. of duplicate determinations, and are fit with a sigmoid dose-response curve using GraphPad Prism 5. B, prevention of the action of SIGC(-cysteamine) by DTT and GSH. The ELISA was carried out as described under Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁γ₂ was immobilized in the wells. Five millimolar DTT or GSH (final concentration) was added together with 10 μM SIGC(-cysteamine), 1 mM cystamine, or 1 μM selenocystamine and incubated for 30 min at 4°C. The wells were next washed six times with TBS plus 0.5% Tween 20, and then the phage was added. Data are representative of two independent experiments and are shown as mean ± S.E.M. of duplicate determinations. C, reversal of the effect of SIGC(-cysteamine) by DTT and GSH. The ELISA was carried out as described under Deactivation ELISA in Materials and Methods. Biotinylated-Gβ₁γ₂ was pretreated with 10 μM SIGC(-cysteamine), 1 mM cystamine, or 1 μM selenocystamine for 30 min at 4°C, and then immobilized in the wells. After six washes with TBS plus 0.5% Tween 20, 5 mM DTT or GSH (final concentration) was added and incubated for 1 h at 4°C. Data are representative of two independent experiments and are shown as mean ± S.E.M. of duplicate determinations.