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Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase.

Fres JM, Müller S, Praefcke GJ - J. Lipid Res. (2010)

Bottom Line: The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins.We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase.Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne, Institute for Genetics, Zülpicher Strasse 47, 50674 Köln, Germany.

ABSTRACT
Over a hundred proteins in eukaryotic cells carry a C-terminal CaaX box sequence, which targets them for posttranslational isoprenylation of the cysteine residue. This modification, catalyzed by either farnesyl or geranylgeranyl transferase, converts them into peripheral membrane proteins. Isoprenylation is usually followed by proteolytic cleavage of the aaX tripeptide and methylation of the carboxyl group of the newly exposed isoprenylcysteine. The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins. We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase. Our system is based on the coexpression of hGBP1 with the two subunits of human farnesyltransferase in Escherichia coli and a chromatographic separation of farnesylated and unmodified protein. Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

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Related in: MedlinePlus

Concentration-dependent GTPase activity of hGBP1. Different concentrations of (A) unmodified and (B) farnesylated hGBP1 were incubated with 1 mM GTP at 37°C in the presence (closed circles) or in the absence (open circles) of liposomes. The specific activities obtained from linear fits were plotted against the protein concentration. Maximal activity and dissociation equilibrium constant of the hGBP1 dimer were calculated by quadratic fits. C: Comparison of maximal activity, dimer dissociation equilibrium constant (Kd), and produced GMP for unmodified and farnesylated hGBP1.
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fig4: Concentration-dependent GTPase activity of hGBP1. Different concentrations of (A) unmodified and (B) farnesylated hGBP1 were incubated with 1 mM GTP at 37°C in the presence (closed circles) or in the absence (open circles) of liposomes. The specific activities obtained from linear fits were plotted against the protein concentration. Maximal activity and dissociation equilibrium constant of the hGBP1 dimer were calculated by quadratic fits. C: Comparison of maximal activity, dimer dissociation equilibrium constant (Kd), and produced GMP for unmodified and farnesylated hGBP1.

Mentions: The effect of farnesylation and liposome binding on the GTPase activity of hGBP1 was measured by HPLC (Fig. 4). At 37°C, both farnesylated and unmodified hGBP1 showed a cooperative behavior, explained by self-association and self-activation of hGBP1. The fit of a quadratic equation to the data for the unmodified form displayed a maximum specific activity of 85 min−1 and a dimer dissociation constant of 4 µM. For the farnesylated hGBP1, the dimer dissociation constant was 2-fold higher, while the maximum activity of farnesylated hGBP1 was identical to the unmodified form. The major product of GTP hydrolysis by the unmodified form at 37°C was GMP (85% of total) but the attachment of the farnesyl moiety resulted in a change of the product ratio to 30% GMP. The presence of folch liposomes had no significant impact on the GTPase activity of any modified form of hGBP1 (Fig. 4, filled circles).


Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase.

Fres JM, Müller S, Praefcke GJ - J. Lipid Res. (2010)

Concentration-dependent GTPase activity of hGBP1. Different concentrations of (A) unmodified and (B) farnesylated hGBP1 were incubated with 1 mM GTP at 37°C in the presence (closed circles) or in the absence (open circles) of liposomes. The specific activities obtained from linear fits were plotted against the protein concentration. Maximal activity and dissociation equilibrium constant of the hGBP1 dimer were calculated by quadratic fits. C: Comparison of maximal activity, dimer dissociation equilibrium constant (Kd), and produced GMP for unmodified and farnesylated hGBP1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903825&req=5

fig4: Concentration-dependent GTPase activity of hGBP1. Different concentrations of (A) unmodified and (B) farnesylated hGBP1 were incubated with 1 mM GTP at 37°C in the presence (closed circles) or in the absence (open circles) of liposomes. The specific activities obtained from linear fits were plotted against the protein concentration. Maximal activity and dissociation equilibrium constant of the hGBP1 dimer were calculated by quadratic fits. C: Comparison of maximal activity, dimer dissociation equilibrium constant (Kd), and produced GMP for unmodified and farnesylated hGBP1.
Mentions: The effect of farnesylation and liposome binding on the GTPase activity of hGBP1 was measured by HPLC (Fig. 4). At 37°C, both farnesylated and unmodified hGBP1 showed a cooperative behavior, explained by self-association and self-activation of hGBP1. The fit of a quadratic equation to the data for the unmodified form displayed a maximum specific activity of 85 min−1 and a dimer dissociation constant of 4 µM. For the farnesylated hGBP1, the dimer dissociation constant was 2-fold higher, while the maximum activity of farnesylated hGBP1 was identical to the unmodified form. The major product of GTP hydrolysis by the unmodified form at 37°C was GMP (85% of total) but the attachment of the farnesyl moiety resulted in a change of the product ratio to 30% GMP. The presence of folch liposomes had no significant impact on the GTPase activity of any modified form of hGBP1 (Fig. 4, filled circles).

Bottom Line: The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins.We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase.Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne, Institute for Genetics, Zülpicher Strasse 47, 50674 Köln, Germany.

ABSTRACT
Over a hundred proteins in eukaryotic cells carry a C-terminal CaaX box sequence, which targets them for posttranslational isoprenylation of the cysteine residue. This modification, catalyzed by either farnesyl or geranylgeranyl transferase, converts them into peripheral membrane proteins. Isoprenylation is usually followed by proteolytic cleavage of the aaX tripeptide and methylation of the carboxyl group of the newly exposed isoprenylcysteine. The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins. We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase. Our system is based on the coexpression of hGBP1 with the two subunits of human farnesyltransferase in Escherichia coli and a chromatographic separation of farnesylated and unmodified protein. Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

Show MeSH
Related in: MedlinePlus