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Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation.

Janssen BM, van Ommeren SP, Merkx M - Int J Mol Sci (2015)

Bottom Line: The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR).Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates.The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5600 MB Eindhoven, The Netherlands. b.m.g.janssen@tue.nl.

ABSTRACT
The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py-Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py-Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py-Im polyamides. The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates. The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

No MeSH data available.


Related in: MedlinePlus

Native chemical ligation of peptides and proteins with a C-terminal thioester and Cys-Py–Im-polyamide. (a) RP-HPLC and m/z-spectrum of the purified N-acyl-benzimidazolinone (Nbz) anti-HIV epitope peptide; (b) RP-HPLC and m/z-spectrum of the purified NCL-reaction between Cys-Py–Im-polyamide and Nbz-aHIV-epitope in 200 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP·HCl, 6 mM Guanidine·HCl at pH 6.8. The # and * indicate different diastereomers with the same ESI-MS spectrum; (c,e,g) RP-HPLC and m/z-spectra of the purified EYFP-MESNA (Mwcalc = 27,845 Da, Mwexp = 27,852 Da) (c), ECFP-MESNA (Mwcalc = 27,763 Da, Mwexp = 27,765 Da) (e) and CNA35-MESNA (Mwcalc = 36,028 Da, Mwexp = 36,025 Da) (g); (d,f,h) RP-HPLC and m/z-spectra of protein-Py–Im-polyamide conjugates of EYFP (Mwcalc = 29,254 Da, Mwexp = 29,252 Da) (d), ECFP (Mwcalc = 29,167 Da, Mwexp = 29,165 Da) (f) and CNA35 (Mwcalc = 37,427 Da, Mwexp = 37,425 Da) (h); The additional peak at 27,637 Da corresponds to ECFP (f) in which the MESNA-thioester was hydrolysed to a carboxylic acid. All reactions with recombinant proteins were performed at room temperature in 200 mM Na2HPO4, 100 mM MPAA, 20 mM TCEP·HCl at pH 6.8. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.
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ijms-16-12631-f002: Native chemical ligation of peptides and proteins with a C-terminal thioester and Cys-Py–Im-polyamide. (a) RP-HPLC and m/z-spectrum of the purified N-acyl-benzimidazolinone (Nbz) anti-HIV epitope peptide; (b) RP-HPLC and m/z-spectrum of the purified NCL-reaction between Cys-Py–Im-polyamide and Nbz-aHIV-epitope in 200 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP·HCl, 6 mM Guanidine·HCl at pH 6.8. The # and * indicate different diastereomers with the same ESI-MS spectrum; (c,e,g) RP-HPLC and m/z-spectra of the purified EYFP-MESNA (Mwcalc = 27,845 Da, Mwexp = 27,852 Da) (c), ECFP-MESNA (Mwcalc = 27,763 Da, Mwexp = 27,765 Da) (e) and CNA35-MESNA (Mwcalc = 36,028 Da, Mwexp = 36,025 Da) (g); (d,f,h) RP-HPLC and m/z-spectra of protein-Py–Im-polyamide conjugates of EYFP (Mwcalc = 29,254 Da, Mwexp = 29,252 Da) (d), ECFP (Mwcalc = 29,167 Da, Mwexp = 29,165 Da) (f) and CNA35 (Mwcalc = 37,427 Da, Mwexp = 37,425 Da) (h); The additional peak at 27,637 Da corresponds to ECFP (f) in which the MESNA-thioester was hydrolysed to a carboxylic acid. All reactions with recombinant proteins were performed at room temperature in 200 mM Na2HPO4, 100 mM MPAA, 20 mM TCEP·HCl at pH 6.8. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.

Mentions: To test the efficiency of the native chemical ligation (NCL) to Cys-Py–Im-polyamide, a C-terminal thioester functionalized peptide epitope (ELDRWEKIRLRP) was synthesized that specifically binds to an anti-HIV p17 antibody [42,43]. The C-terminal thioester peptide was synthesized according to the method of Dawson and coworkers and involved Fmoc-solid phase synthesis using a Rink amide aminomethyl (AM) resin containing a diaminobenzyl linker [44]. The resin-bound N-acyl-benzimidazolinone (Nbz) peptide was deprotected and cleaved from the resin using trifluoroacetic acid. The desired peptide AELDRWEKIRLRPAA-Nbz was obtained in high purity and a final yield of 15% after RP-HPLC purification (Figure 2a). Since the Nbz-moiety is a poor leaving group, the NCL-reaction with Cys-Py–Im-polyamide was performed using 4-mercaptophenylacetic acid (MPAA) as a catalyst [45]. Cys-Py–Im-polyamide was mixed with a slight excess of peptide-Nbz to yield final concentrations of 2 and 2.2 mM, respectively. Analysis by ESI-MS showed full conversion of the Cys-Py–Im-polyamide to the epitope-Py–Im-polyamide conjugate after overnight incubation at room temperature (Figure 2b). The HPLC-trace actually showed two peaks with the expected mass for the epitope-Py–Im-polyamide product, which we attribute to the formation of a small amount of a diastereomer.


Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation.

Janssen BM, van Ommeren SP, Merkx M - Int J Mol Sci (2015)

Native chemical ligation of peptides and proteins with a C-terminal thioester and Cys-Py–Im-polyamide. (a) RP-HPLC and m/z-spectrum of the purified N-acyl-benzimidazolinone (Nbz) anti-HIV epitope peptide; (b) RP-HPLC and m/z-spectrum of the purified NCL-reaction between Cys-Py–Im-polyamide and Nbz-aHIV-epitope in 200 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP·HCl, 6 mM Guanidine·HCl at pH 6.8. The # and * indicate different diastereomers with the same ESI-MS spectrum; (c,e,g) RP-HPLC and m/z-spectra of the purified EYFP-MESNA (Mwcalc = 27,845 Da, Mwexp = 27,852 Da) (c), ECFP-MESNA (Mwcalc = 27,763 Da, Mwexp = 27,765 Da) (e) and CNA35-MESNA (Mwcalc = 36,028 Da, Mwexp = 36,025 Da) (g); (d,f,h) RP-HPLC and m/z-spectra of protein-Py–Im-polyamide conjugates of EYFP (Mwcalc = 29,254 Da, Mwexp = 29,252 Da) (d), ECFP (Mwcalc = 29,167 Da, Mwexp = 29,165 Da) (f) and CNA35 (Mwcalc = 37,427 Da, Mwexp = 37,425 Da) (h); The additional peak at 27,637 Da corresponds to ECFP (f) in which the MESNA-thioester was hydrolysed to a carboxylic acid. All reactions with recombinant proteins were performed at room temperature in 200 mM Na2HPO4, 100 mM MPAA, 20 mM TCEP·HCl at pH 6.8. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490465&req=5

ijms-16-12631-f002: Native chemical ligation of peptides and proteins with a C-terminal thioester and Cys-Py–Im-polyamide. (a) RP-HPLC and m/z-spectrum of the purified N-acyl-benzimidazolinone (Nbz) anti-HIV epitope peptide; (b) RP-HPLC and m/z-spectrum of the purified NCL-reaction between Cys-Py–Im-polyamide and Nbz-aHIV-epitope in 200 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP·HCl, 6 mM Guanidine·HCl at pH 6.8. The # and * indicate different diastereomers with the same ESI-MS spectrum; (c,e,g) RP-HPLC and m/z-spectra of the purified EYFP-MESNA (Mwcalc = 27,845 Da, Mwexp = 27,852 Da) (c), ECFP-MESNA (Mwcalc = 27,763 Da, Mwexp = 27,765 Da) (e) and CNA35-MESNA (Mwcalc = 36,028 Da, Mwexp = 36,025 Da) (g); (d,f,h) RP-HPLC and m/z-spectra of protein-Py–Im-polyamide conjugates of EYFP (Mwcalc = 29,254 Da, Mwexp = 29,252 Da) (d), ECFP (Mwcalc = 29,167 Da, Mwexp = 29,165 Da) (f) and CNA35 (Mwcalc = 37,427 Da, Mwexp = 37,425 Da) (h); The additional peak at 27,637 Da corresponds to ECFP (f) in which the MESNA-thioester was hydrolysed to a carboxylic acid. All reactions with recombinant proteins were performed at room temperature in 200 mM Na2HPO4, 100 mM MPAA, 20 mM TCEP·HCl at pH 6.8. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.
Mentions: To test the efficiency of the native chemical ligation (NCL) to Cys-Py–Im-polyamide, a C-terminal thioester functionalized peptide epitope (ELDRWEKIRLRP) was synthesized that specifically binds to an anti-HIV p17 antibody [42,43]. The C-terminal thioester peptide was synthesized according to the method of Dawson and coworkers and involved Fmoc-solid phase synthesis using a Rink amide aminomethyl (AM) resin containing a diaminobenzyl linker [44]. The resin-bound N-acyl-benzimidazolinone (Nbz) peptide was deprotected and cleaved from the resin using trifluoroacetic acid. The desired peptide AELDRWEKIRLRPAA-Nbz was obtained in high purity and a final yield of 15% after RP-HPLC purification (Figure 2a). Since the Nbz-moiety is a poor leaving group, the NCL-reaction with Cys-Py–Im-polyamide was performed using 4-mercaptophenylacetic acid (MPAA) as a catalyst [45]. Cys-Py–Im-polyamide was mixed with a slight excess of peptide-Nbz to yield final concentrations of 2 and 2.2 mM, respectively. Analysis by ESI-MS showed full conversion of the Cys-Py–Im-polyamide to the epitope-Py–Im-polyamide conjugate after overnight incubation at room temperature (Figure 2b). The HPLC-trace actually showed two peaks with the expected mass for the epitope-Py–Im-polyamide product, which we attribute to the formation of a small amount of a diastereomer.

Bottom Line: The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR).Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates.The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5600 MB Eindhoven, The Netherlands. b.m.g.janssen@tue.nl.

ABSTRACT
The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py-Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py-Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py-Im polyamides. The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates. The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

No MeSH data available.


Related in: MedlinePlus