Details of experiments between the fragments O00499~513-593 and P50570~846-860
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.06
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.06.29.
Number of measurements: 4
Measured pKd: 3.53
Experiment series 2
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: T532M
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.03
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.06.29.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 3
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: D537V
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 4
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: F584S
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: -0
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 5
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: P551L
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 6
Experiment method: HU (DAPF)
Details of affinity fitting: Bait concentration was adjusted to fit the profile to WT
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 6
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Q540H
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.07
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 4
Measured pKd: 3.63
Experiment series 7
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: T532M
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 8
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V566M
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.02
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 9
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V583I
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.02
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 9
Experiment method: HU (DAPF)
Details of affinity fitting: Bait concentration was adjusted to fit the profile to WT
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 10
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.05
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 4
Measured pKd: 3.5
Experiment series 11
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Y531S
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 12
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: R581C
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: DNM2
Protein B fragment: 846-860
Protein B site: PxxP_7
Protein B sequence: VPSRRPPAAPSRPTI
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 6
Experiment method: HU (DAPF)
Details of affinity fitting: Bait concentration was adjusted to fit the profile to WT
Date of measurement: 2022.10.24.
Number of measurements: 4
The affinity was below the detection threshold of the assay.