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Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length.

Fahrer J, Kranaster R, Altmeyer M, Marx A, Bürkle A - Nucleic Acids Res. (2007)

Bottom Line: In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer).In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA.Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Molecular Toxicology Group, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany.

ABSTRACT
Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR-protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.

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SPR real-time binding studies with PAR 14-mer (A, C and E) and 63-mer (B, D and F). Experimental data are depicted in black and fitted curves in red. (A) Sensorgram of binding of antibody 10H (various concentrations from 0.01 to 10 nM) to PAR 14-mer using bivalent binding model for data evaluation. (B) Sensorgram for antibody 10H (various concentrations from 0.01 to 10 nM) binding to PAR 63-mer and bivalent binding model for data evaluation. (C) Sensorgram for XPA (50 and 100 nM) injected over immobilized PAR 14-mer. Even at 500 nM XPA no binding was observed. (D) Sensorgram for XPA binding to PAR 63-mer, with data fitted using a conformational change binding model. (E) Kinetic titration sensorgram for p53 binding to PAR 14-mer using a 1:1 binding model for data fitting. (F) Kinetic titration sensorgram for p53 binding to PAR 63-mer. Due to complex binding behavior (up to three different complexes) no satisfactory data fit was possible.
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Figure 4: SPR real-time binding studies with PAR 14-mer (A, C and E) and 63-mer (B, D and F). Experimental data are depicted in black and fitted curves in red. (A) Sensorgram of binding of antibody 10H (various concentrations from 0.01 to 10 nM) to PAR 14-mer using bivalent binding model for data evaluation. (B) Sensorgram for antibody 10H (various concentrations from 0.01 to 10 nM) binding to PAR 63-mer and bivalent binding model for data evaluation. (C) Sensorgram for XPA (50 and 100 nM) injected over immobilized PAR 14-mer. Even at 500 nM XPA no binding was observed. (D) Sensorgram for XPA binding to PAR 63-mer, with data fitted using a conformational change binding model. (E) Kinetic titration sensorgram for p53 binding to PAR 14-mer using a 1:1 binding model for data fitting. (F) Kinetic titration sensorgram for p53 binding to PAR 63-mer. Due to complex binding behavior (up to three different complexes) no satisfactory data fit was possible.

Mentions: Antibody 10H showed very high affinity and fast association to both short and long PAR chains. Curves were fitted with a bivalent binding model (Figure 4A and B). The binding equilibrium of the first binding event was calculated to be 2.8 nM for the short and 0.35 nM for the long PAR chain (Table 1; Supplementary Table S1).Figure 4.


Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length.

Fahrer J, Kranaster R, Altmeyer M, Marx A, Bürkle A - Nucleic Acids Res. (2007)

SPR real-time binding studies with PAR 14-mer (A, C and E) and 63-mer (B, D and F). Experimental data are depicted in black and fitted curves in red. (A) Sensorgram of binding of antibody 10H (various concentrations from 0.01 to 10 nM) to PAR 14-mer using bivalent binding model for data evaluation. (B) Sensorgram for antibody 10H (various concentrations from 0.01 to 10 nM) binding to PAR 63-mer and bivalent binding model for data evaluation. (C) Sensorgram for XPA (50 and 100 nM) injected over immobilized PAR 14-mer. Even at 500 nM XPA no binding was observed. (D) Sensorgram for XPA binding to PAR 63-mer, with data fitted using a conformational change binding model. (E) Kinetic titration sensorgram for p53 binding to PAR 14-mer using a 1:1 binding model for data fitting. (F) Kinetic titration sensorgram for p53 binding to PAR 63-mer. Due to complex binding behavior (up to three different complexes) no satisfactory data fit was possible.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175335&req=5

Figure 4: SPR real-time binding studies with PAR 14-mer (A, C and E) and 63-mer (B, D and F). Experimental data are depicted in black and fitted curves in red. (A) Sensorgram of binding of antibody 10H (various concentrations from 0.01 to 10 nM) to PAR 14-mer using bivalent binding model for data evaluation. (B) Sensorgram for antibody 10H (various concentrations from 0.01 to 10 nM) binding to PAR 63-mer and bivalent binding model for data evaluation. (C) Sensorgram for XPA (50 and 100 nM) injected over immobilized PAR 14-mer. Even at 500 nM XPA no binding was observed. (D) Sensorgram for XPA binding to PAR 63-mer, with data fitted using a conformational change binding model. (E) Kinetic titration sensorgram for p53 binding to PAR 14-mer using a 1:1 binding model for data fitting. (F) Kinetic titration sensorgram for p53 binding to PAR 63-mer. Due to complex binding behavior (up to three different complexes) no satisfactory data fit was possible.
Mentions: Antibody 10H showed very high affinity and fast association to both short and long PAR chains. Curves were fitted with a bivalent binding model (Figure 4A and B). The binding equilibrium of the first binding event was calculated to be 2.8 nM for the short and 0.35 nM for the long PAR chain (Table 1; Supplementary Table S1).Figure 4.

Bottom Line: In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer).In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA.Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Molecular Toxicology Group, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany.

ABSTRACT
Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR-protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.

Show MeSH
Related in: MedlinePlus