G-proteins
G protein-coupled receptors couple to heterotrimeric guanine nucleotide binding proteins (G proteins), which are composed of three subunits: α, β and γ. There are at least 6 different β, 12 γ and 21 α-subunit types, which can give rise to over a thousand different G protein heterotrimers. In our group we have comprised the following collection of G proteins:
Mutants: αi2, αslong, αq, γ2
Wild-types: αi1, αi2, αi3, αslong, αq, β1
We express both wild-type and mutant G proteins using Sf9 cells as a host. To get a functional βγ-dimer we express a strep-tagged γ-subunit and a β-subunit under a double promoter system. In the purification step, the strep-tagged γ-subunits are attached to a streptactin affinity column which now serves as an affinity matrix for purification of all subunits (wild type or mutants). Additionally, we have introduced a tetracysteine tag into α-subunits and γ-subunits, which allows us to purify them without a need to use the abovementioned βγ-dimer as an affinity matrix for α-subunit. Using a custom-synthesized FlAsh-affinity column, which is highly specific for tetracysteine-containing mutants, we get α-subunits in a highly pure form and use it in various in vitro assays. To avoid G protein degradation during and after purification we can add GDP. However, using the Streptactin column we can easily remove endogenous nucleotides from the sample, if necessary. By removing endogenous Mg2+ using EDTA, we can reduce the affinity between guanine nucleotides and α-subunits without the risk of the sample losing its affinity for the column (as would be the case if Ni-columns were used when purifying His-tagged proteins).
We have synthesized F2FlAsh, which is better than the commercial FlAsh – brighter, less bleachable, less sensitive to BSA (Tõntson et al. 2012). Also, we have been using a commercial ReAsh dye. F2FlAsh and ReAsh bind covalently to tetracysteine tags, but ReAsh with different affinity depending on the specific tetracysteine tag. Using F2FlAsh and ReAsh dyes we are able to detect FRET between α- and γ-subunits. These biarsenical ligands bind to endogenous G protein α-subunits and enable allosteric sensing of nucleotide binding (Tõntson et al. 2013). Since FlAsh and ReAsh are cell-penetrating dyes it is easy to apply them extracellularly on live cells. With TIRF microscopy we can conveniently eliminate possible signal interference that might result from the dye interacting with the nuclei which is suggested to be an issue when using ReAsh and FlAsh dyes.
We have two fluorescently labelled GTPγS ligands (Bodipy-TR-GTPγS and Bodipy-FL-GTPγS), which can bind to Gα-subunits. GTPγS can’t enter the cells, therefore this approach is limited mainly to in vitro applications. Bodipy-labelled α-subunits can be used to measure the effects of various nucleotides on GTPγS binding by detecting change in anisotropy signal (Tõntson et al. 2012).
Our experiments have shown that co-expression of 5-HT1A and αi2+βγ is possible in a budded baculovirus system and hence we are able to use this system to study the effects of Mn2+ and guanine nucleotides on high affinity ligand binding to these signaling complexes (Tõntson et al. 2014).
Mutants: αi2, αslong, αq, γ2
Wild-types: αi1, αi2, αi3, αslong, αq, β1
We express both wild-type and mutant G proteins using Sf9 cells as a host. To get a functional βγ-dimer we express a strep-tagged γ-subunit and a β-subunit under a double promoter system. In the purification step, the strep-tagged γ-subunits are attached to a streptactin affinity column which now serves as an affinity matrix for purification of all subunits (wild type or mutants). Additionally, we have introduced a tetracysteine tag into α-subunits and γ-subunits, which allows us to purify them without a need to use the abovementioned βγ-dimer as an affinity matrix for α-subunit. Using a custom-synthesized FlAsh-affinity column, which is highly specific for tetracysteine-containing mutants, we get α-subunits in a highly pure form and use it in various in vitro assays. To avoid G protein degradation during and after purification we can add GDP. However, using the Streptactin column we can easily remove endogenous nucleotides from the sample, if necessary. By removing endogenous Mg2+ using EDTA, we can reduce the affinity between guanine nucleotides and α-subunits without the risk of the sample losing its affinity for the column (as would be the case if Ni-columns were used when purifying His-tagged proteins).
We have synthesized F2FlAsh, which is better than the commercial FlAsh – brighter, less bleachable, less sensitive to BSA (Tõntson et al. 2012). Also, we have been using a commercial ReAsh dye. F2FlAsh and ReAsh bind covalently to tetracysteine tags, but ReAsh with different affinity depending on the specific tetracysteine tag. Using F2FlAsh and ReAsh dyes we are able to detect FRET between α- and γ-subunits. These biarsenical ligands bind to endogenous G protein α-subunits and enable allosteric sensing of nucleotide binding (Tõntson et al. 2013). Since FlAsh and ReAsh are cell-penetrating dyes it is easy to apply them extracellularly on live cells. With TIRF microscopy we can conveniently eliminate possible signal interference that might result from the dye interacting with the nuclei which is suggested to be an issue when using ReAsh and FlAsh dyes.
We have two fluorescently labelled GTPγS ligands (Bodipy-TR-GTPγS and Bodipy-FL-GTPγS), which can bind to Gα-subunits. GTPγS can’t enter the cells, therefore this approach is limited mainly to in vitro applications. Bodipy-labelled α-subunits can be used to measure the effects of various nucleotides on GTPγS binding by detecting change in anisotropy signal (Tõntson et al. 2012).
Our experiments have shown that co-expression of 5-HT1A and αi2+βγ is possible in a budded baculovirus system and hence we are able to use this system to study the effects of Mn2+ and guanine nucleotides on high affinity ligand binding to these signaling complexes (Tõntson et al. 2014).