Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions
Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations....
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Bonhoure, Anna [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2020transfer abstract |
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| Schlagwörter: |
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| Übergeordnetes Werk: |
Enthalten in: Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems - Kroon, Frederieke J. ELSEVIER, 2014, methods in the biological sciences, San Diego, Calif |
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| Übergeordnetes Werk: |
volume:603 ; year:2020 ; day:15 ; month:08 ; pages:0 |
| Links: |
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| DOI / URN: |
10.1016/j.ab.2020.113772 |
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ELV050821091 |
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| 520 | |a Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. | ||
| 520 | |a Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. | ||
| 650 | 7 | |a Papillomavirus |2 Elsevier | |
| 650 | 7 | |a Protein-protein interaction |2 Elsevier | |
| 650 | 7 | |a Linear motif |2 Elsevier | |
| 650 | 7 | |a Oncoprotein |2 Elsevier | |
| 650 | 7 | |a Peptide array |2 Elsevier | |
| 650 | 7 | |a Conservative replacement |2 Elsevier | |
| 700 | 1 | |a Forster, Anne |4 oth | |
| 700 | 1 | |a Babah, Khaled Ould |4 oth | |
| 700 | 1 | |a Gógl, Gergő |4 oth | |
| 700 | 1 | |a Eberling, Pascal |4 oth | |
| 700 | 1 | |a Kostmann, Camille |4 oth | |
| 700 | 1 | |a Volkmer, Rudolf |4 oth | |
| 700 | 1 | |a Tapia Mancilla, Victor |4 oth | |
| 700 | 1 | |a Travé, Gilles |4 oth | |
| 700 | 1 | |a Nominé, Yves |4 oth | |
| 773 | 0 | 8 | |i Enthalten in |n Elsevier |a Kroon, Frederieke J. ELSEVIER |t Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems |d 2014 |d methods in the biological sciences |g San Diego, Calif |w (DE-627)ELV018040411 |
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10.1016/j.ab.2020.113772 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000001084.pica (DE-627)ELV050821091 (ELSEVIER)S0003-2697(20)30304-3 DE-627 ger DE-627 rakwb eng 550 VZ 333.7 VZ 610 VZ 15,3 ssgn PHARM DE-84 fid 44.40 bkl Bonhoure, Anna verfasserin aut Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions 2020transfer abstract nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Papillomavirus Elsevier Protein-protein interaction Elsevier Linear motif Elsevier Oncoprotein Elsevier Peptide array Elsevier Conservative replacement Elsevier Forster, Anne oth Babah, Khaled Ould oth Gógl, Gergő oth Eberling, Pascal oth Kostmann, Camille oth Volkmer, Rudolf oth Tapia Mancilla, Victor oth Travé, Gilles oth Nominé, Yves oth Enthalten in Elsevier Kroon, Frederieke J. ELSEVIER Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems 2014 methods in the biological sciences San Diego, Calif (DE-627)ELV018040411 volume:603 year:2020 day:15 month:08 pages:0 https://doi.org/10.1016/j.ab.2020.113772 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U FID-PHARM SSG-OLC-PHA SSG-OPC-PHA 44.40 Pharmazie Pharmazeutika VZ AR 603 2020 15 0815 0 |
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10.1016/j.ab.2020.113772 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000001084.pica (DE-627)ELV050821091 (ELSEVIER)S0003-2697(20)30304-3 DE-627 ger DE-627 rakwb eng 550 VZ 333.7 VZ 610 VZ 15,3 ssgn PHARM DE-84 fid 44.40 bkl Bonhoure, Anna verfasserin aut Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions 2020transfer abstract nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Papillomavirus Elsevier Protein-protein interaction Elsevier Linear motif Elsevier Oncoprotein Elsevier Peptide array Elsevier Conservative replacement Elsevier Forster, Anne oth Babah, Khaled Ould oth Gógl, Gergő oth Eberling, Pascal oth Kostmann, Camille oth Volkmer, Rudolf oth Tapia Mancilla, Victor oth Travé, Gilles oth Nominé, Yves oth Enthalten in Elsevier Kroon, Frederieke J. ELSEVIER Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems 2014 methods in the biological sciences San Diego, Calif (DE-627)ELV018040411 volume:603 year:2020 day:15 month:08 pages:0 https://doi.org/10.1016/j.ab.2020.113772 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U FID-PHARM SSG-OLC-PHA SSG-OPC-PHA 44.40 Pharmazie Pharmazeutika VZ AR 603 2020 15 0815 0 |
| allfields_unstemmed |
10.1016/j.ab.2020.113772 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000001084.pica (DE-627)ELV050821091 (ELSEVIER)S0003-2697(20)30304-3 DE-627 ger DE-627 rakwb eng 550 VZ 333.7 VZ 610 VZ 15,3 ssgn PHARM DE-84 fid 44.40 bkl Bonhoure, Anna verfasserin aut Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions 2020transfer abstract nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Papillomavirus Elsevier Protein-protein interaction Elsevier Linear motif Elsevier Oncoprotein Elsevier Peptide array Elsevier Conservative replacement Elsevier Forster, Anne oth Babah, Khaled Ould oth Gógl, Gergő oth Eberling, Pascal oth Kostmann, Camille oth Volkmer, Rudolf oth Tapia Mancilla, Victor oth Travé, Gilles oth Nominé, Yves oth Enthalten in Elsevier Kroon, Frederieke J. ELSEVIER Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems 2014 methods in the biological sciences San Diego, Calif (DE-627)ELV018040411 volume:603 year:2020 day:15 month:08 pages:0 https://doi.org/10.1016/j.ab.2020.113772 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U FID-PHARM SSG-OLC-PHA SSG-OPC-PHA 44.40 Pharmazie Pharmazeutika VZ AR 603 2020 15 0815 0 |
| allfieldsGer |
10.1016/j.ab.2020.113772 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000001084.pica (DE-627)ELV050821091 (ELSEVIER)S0003-2697(20)30304-3 DE-627 ger DE-627 rakwb eng 550 VZ 333.7 VZ 610 VZ 15,3 ssgn PHARM DE-84 fid 44.40 bkl Bonhoure, Anna verfasserin aut Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions 2020transfer abstract nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Papillomavirus Elsevier Protein-protein interaction Elsevier Linear motif Elsevier Oncoprotein Elsevier Peptide array Elsevier Conservative replacement Elsevier Forster, Anne oth Babah, Khaled Ould oth Gógl, Gergő oth Eberling, Pascal oth Kostmann, Camille oth Volkmer, Rudolf oth Tapia Mancilla, Victor oth Travé, Gilles oth Nominé, Yves oth Enthalten in Elsevier Kroon, Frederieke J. ELSEVIER Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems 2014 methods in the biological sciences San Diego, Calif (DE-627)ELV018040411 volume:603 year:2020 day:15 month:08 pages:0 https://doi.org/10.1016/j.ab.2020.113772 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U FID-PHARM SSG-OLC-PHA SSG-OPC-PHA 44.40 Pharmazie Pharmazeutika VZ AR 603 2020 15 0815 0 |
| allfieldsSound |
10.1016/j.ab.2020.113772 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000001084.pica (DE-627)ELV050821091 (ELSEVIER)S0003-2697(20)30304-3 DE-627 ger DE-627 rakwb eng 550 VZ 333.7 VZ 610 VZ 15,3 ssgn PHARM DE-84 fid 44.40 bkl Bonhoure, Anna verfasserin aut Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions 2020transfer abstract nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. Papillomavirus Elsevier Protein-protein interaction Elsevier Linear motif Elsevier Oncoprotein Elsevier Peptide array Elsevier Conservative replacement Elsevier Forster, Anne oth Babah, Khaled Ould oth Gógl, Gergő oth Eberling, Pascal oth Kostmann, Camille oth Volkmer, Rudolf oth Tapia Mancilla, Victor oth Travé, Gilles oth Nominé, Yves oth Enthalten in Elsevier Kroon, Frederieke J. ELSEVIER Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems 2014 methods in the biological sciences San Diego, Calif (DE-627)ELV018040411 volume:603 year:2020 day:15 month:08 pages:0 https://doi.org/10.1016/j.ab.2020.113772 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U FID-PHARM SSG-OLC-PHA SSG-OPC-PHA 44.40 Pharmazie Pharmazeutika VZ AR 603 2020 15 0815 0 |
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Informing policy to protect coastal coral reefs: Insight from a global review of reducing agricultural pollution to coastal ecosystems |
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benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions |
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Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions |
| abstract |
Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. |
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Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. |
| abstract_unstemmed |
Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities. |
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Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions |
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The assay is based on the “holdup” principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. 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