Galpha subunits of heterotrimeric G proteins (Gi, Go, Gs). The G-Protein antagonist peptide is a hydrophobic, truncated substance P-related peptide that binds directly to Galpha subunits (or the receptor-G protein interface), thereby competing with activated GPCRs for G protein binding. Unlike many GPCR antagonists that act at the receptor orthosteric site, this peptide targets the G protein itself. By binding to Galpha subunits, it prevents the receptor-catalyzed exchange of GTP for GDP, effectively blocking G protein activation. This antagonism is receptor-independent and applies to multiple G protein subtypes, including Gi, Go, and Gs, as demonstrated by the peptide's ability to block M2 mAChR-mediated activation of Gi/Go and beta-adrenergic receptor-mediated activation of Gs in reconstituted phospholipid vesicles. The peptide does not discriminate among Galpha subtypes. Its hydrophobic nature may facilitate insertion into the membrane bilayer to access the G protein binding interface. The TFA salt is used for solubility. This peptide is a research tool for dissecting GPCR-G protein interactions.

In vitro, G-Protein antagonist peptide TFA (pGlu-Gln-Trp-Phe-Trp-Trp-Met-NH2) inhibits the activation of heterotrimeric G proteins (Gi, Go, Gs) by GPCRs in a non-competitive manner. In reconstituted phospholipid vesicle systems containing purified M2 muscarinic cholinergic receptor (M2 mAChR) and Gi or Go, the peptide (10-500 uM) prevents agonist-induced GTPgammaS binding and GTP hydrolysis, with an IC50 in the 10-100 uM range. Specifically, the peptide blocks M2 mAChR-mediated activation of Gi and Go, as assayed by receptor-promoted GTP hydrolysis. Similarly, the peptide inhibits beta-adrenergic receptor-mediated activation of Gs in reconstituted vesicles. The peptide does not require the presence of the receptor to bind to G proteins; it can directly interact with purified Galpha subunits in vitro. The inhibition is competitive with respect to G protein binding but is not overcome by increasing receptor concentration, indicating that the peptide acts directly on Galpha. The peptide's mechanism is believed to involve binding to the C-terminal region of Galpha or the switch regions that interact with the receptor. The peptide is not active in intact cells without permeabilization or microinjection due to its hydrophilicity and size. It is primarily used in cell-free systems (purified proteins or membrane preparations) to study G protein coupling. The TFA salt does not affect peptide activity. The sequence is sometimes referred to as "G-protein antagonist peptide" or "M35".

Not applicable (used in cell-free systems). The G-Protein antagonist peptide TFA is not intended for in vivo administration; it is used exclusively in cell-free biochemical assays or in permeabilized cells. It does not cross cell membranes due to its size (MW ~1.2 kDa) and hydrophilicity. Therefore, no in vivo activity data are available, and there are no studies of this peptide in animal models of disease. For ex vivo studies, the peptide may be added to membrane preparations from tissues (e.g., brain, heart, lung) to block receptor-G protein coupling. For example, in brain membranes expressing M2 mAChR or beta-adrenergic receptors, the peptide (10-200 uM) inhibits agonist-stimulated GTPgammaS binding. However, such applications are considered in vitro/ex vivo, not in vivo. For cell-based experiments, the peptide must be delivered by microinjection or electroporation, or cell-permeable versions must be developed. This product is a research reagent for studying G protein signaling mechanisms, not a therapeutic drug.

For direct binding assays (non-cellular), a fluorescence polarization (FP) assay or surface plasmon resonance (SPR) can be used to measure binding of the peptide to purified Galpha subunits. For FP: Purified recombinant Galphai1, Galphao, or Galphas protein (10-50 nM) is incubated with fluorescently labeled G-protein antagonist peptide (FITC-peptide, 10-50 nM) in binding buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% NP-40, 0.1 mg/mL BSA) in black 384-well plates. After 30-60 min at room temperature, fluorescence polarization (excitation 485 nm, emission 520 nm) is measured. The Kd is determined from direct binding curves using a one-site binding hyperbola. For competitive FP assays, unlabeled G-protein antagonist peptide is added to compete with FITC-peptide for binding to Galpha; IC50 is determined, and Ki is calculated. For SPR: Galpha protein is immobilized on a CM5 sensor chip. Increasing concentrations of the peptide (0.1-1000 uM) are flowed over the surface, and sensorgrams are recorded. KD is calculated. For functional assays (receptor-promoted GTP hydrolysis): M2 mAChR or beta2-adrenergic receptor is reconstituted into phospholipid vesicles with Gi/Go or Gs protein, respectively. The vesicles are incubated with varying concentrations of G-protein antagonist peptide (0-1000 uM) for 10 min at 30degC. Then, agonist (carbachol for M2, 100 uM; or isoproterenol for beta2, 1 uM) and [gamma-32P]GTP (or unlabeled GTP) are added to initiate the reaction. After incubation (5-15 min), the reaction is terminated, and the amount of hydrolyzed Pi (32P) is measured. Percent inhibition is calculated relative to no-peptide control. IC50 values for inhibition of agonist-stimulated GTPase activity typically range from 10-100 uM. For GTPgammaS binding assays: Agonist-stimulated [35S]GTPgammaS binding to membranes or reconstituted vesicles is measured in the presence and absence of peptide. For example, rat brain membranes (expressing M2 mAChR or beta2-AR) are incubated with 0.1-0.5 nM [35S]GTPgammaS, 10 uM GDP, agonist (carbachol or isoproterenol), and varying peptide concentrations in assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT). After 60 min at 30degC, bound [35S]GTPgammaS is separated by vacuum filtration (GF/B filters) and quantified by liquid scintillation counting. Percent inhibition is calculated. The peptide is effective in the 1-500 uM range. All assays should be performed in triplicate with at least 3 independent experiments.

For cell-based assays, the peptide is not cell-permeable; therefore, it must be delivered by permeabilization, microinjection, or using a cell-penetrating peptide conjugate. A typical method is to use digitonin-permeabilized cells. For example, HEK293 cells expressing M2 mAChR or beta2-AR are cultured in 6-well plates. Cells are washed with cold PBS, then permeabilized with 20-50 ug/mL digitonin in intracellular buffer (20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2, 2 mM EGTA, 0.5 mg/mL BSA, 1 mM DTT, 1 mM ATP, 0.1 mM GTP) for 10 min on ice. After removing the permeabilization buffer, cells are incubated with varying concentrations of G-protein antagonist peptide (0-1000 uM) in intracellular buffer for 30 min at 30degC. Then, agonist (carbachol or isoproterenol) is added, and after 10-15 min, cells are lysed, and GTPase activity or GTPgammaS binding is measured. Alternatively, cells can be microinjected with the peptide using a micromanipulator (1-100 uM in injection buffer). Due to the complexity and low throughput of these methods, most studies use membrane preparations rather than intact cells. Therefore, a detailed cell-based protocol for intact, non-permeabilized cells is not standard. For researchers requiring intracellular delivery, conjugation to a cell-penetrating peptide (e.g., TAT, penetratin) is recommended. However, such modified peptides are not represented by this product. The TFA salt may be used to prepare stock solutions in water or 10% DMSO. For electroporation: cells are suspended in electroporation buffer with 10-100 uM peptide, and electroporation is performed using a Gene Pulser (Bio-Rad). After recovery, cells are plated and used for signaling assays. These methods are feasible but not routine. The peptide is not typically used in standard cell-based assays without permeabilization.

Not applicable (cell-free tool). The G-Protein antagonist peptide TFA is not used in in vivo animal studies because it does not cross cell membranes and is rapidly cleared from circulation. There are no published in vivo efficacy or toxicity studies in animals. For researchers wishing to test the peptide in animal models of disease, a cell-penetrating version (e.g., TAT-conjugated G-protein antagonist peptide) would be required. However, such modified peptides are not covered by this product. Standard in vivo protocols are not available for this specific peptide. The peptide is strictly a biochemical research tool for in vitro and ex vivo membrane-based assays. It is not intended for use in living animals. If an investigator desires to test the peptide in animals despite the limitations, a typical protocol would involve conjugation to a cell-penetrating peptide (CPP) and then intravenous (i.v.) or intraperitoneal (i.p.) administration at 1-20 mg/kg, but this is outside the scope of this product. No such studies are reported in the literature. Therefore, it is recommended that users treat the peptide as an in vitro reagent only.

Not applicable. The G-Protein antagonist peptide is not used in vivo, and no pharmacokinetic data are available. As a 7-amino acid peptide (MW ~1.2 kDa), if administered intravenously, it would be rapidly cleared by the kidneys (glomerular filtration) and degraded by proteases (t1/2 < 5 min). It is not orally bioavailable. The TFA salt does not affect PK properties. The peptide is designed for in vitro use only. For research involving cell-free systems or membrane preparations, PK is irrelevant. Some researchers may conjugate the peptide to a carrier (e.g., PEGylation or fusion to a cell-penetrating peptide) to improve stability, but such derivatives are not represented by this product. Therefore, no detailed PK parameters (absorption, distribution, metabolism, excretion, half-life, bioavailability) are reported or applicable. The product is for research use only, not for drug development.

No specific toxicity data are available for the G-Protein antagonist peptide TFA. As a short peptide composed of naturally occurring L-amino acids (except for the N-terminal pyroglutamate), it is generally considered to have low toxicity. In vitro, the peptide is not cytotoxic to cells at concentrations up to 500 uM when added extracellularly, but due to its low cell permeability, it does not enter cells and thus does not affect viability. In permeabilized cell preparations, concentrations up to 500 uM may cause some disruption of cellular integrity due to the permeabilization process itself (digitonin), but this is not attributable to the peptide. No genotoxicity, organ toxicity, carcinogenicity, or reproductive toxicity studies have been conducted. The TFA salt (trifluoroacetate) is present in low amounts and is considered non-toxic. The peptide is for research use only and is not approved for human or animal use. Standard laboratory safety precautions (gloves, lab coat, eye protection) are sufficient. The peptide is not intended for in vivo use due to its poor bioavailability and lack of cell permeability.

[1]. G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding. J Biol Chem. 1992;267(23):16237-16243.

GPCRs (G protein-coupled receptors) are the largest family of membrane receptors and signal through heterotrimeric G proteins (Galpha, Gbetagamma). G protein antagonist peptides, such as the one described, have been used to probe the molecular details of receptor-G protein coupling. This specific peptide (pGlu-Gln-Trp-Phe-Trp-Trp-Met-NH2) was discovered by Mukai et al. (1992) and is also known as "G-Protein antagonist peptide" or "M35". It is derived from substance P (a neuropeptide) by deletion of N-terminal residues and amidation at the C-terminus. The peptide is often used in combination with reconstituted receptor-G protein systems to study the specificity and mechanism of G protein activation. This product is supplied as a TFA salt to enhance solubility and stability. The peptide is for research use only and is not a drug. It is not approved for clinical or therapeutic use. The peptide can be stored as a lyophilized powder at -20degC and reconstituted in water or DMSO.

C59H65F3N12O11S

1207.28

G-Protein antagonist peptide;143675-79-0

White to off-white solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~100 mg/mL (~82.83 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.07 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)