Details of experiments between the fragments O00499~513-593 and P30260~396-410
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.81
Holdup BI standard deviation: 0.01
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: 5.47
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.77
Holdup BI standard deviation: 0.01
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: 5.35
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.21
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: 4.2
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: -0.07
Holdup BI standard deviation: 0.05
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.5
Holdup BI standard deviation: 0.01
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
Measured pKd: 5.32
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.72
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: 5.22
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.72
Holdup BI standard deviation: 0
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: 5.22
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.69
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: 5.16
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.61
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
Measured pKd: 5.37
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.74
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: 5.29
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.19
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: 4.14
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: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.54
Holdup BI standard deviation: 0.02
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
Measured pKd: 5.41
Experiment series 13
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.65
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/16
Number of measurements: 1
Measured pKd: 5.08
Experiment series 14
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: T532M
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.67
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/16
Number of measurements: 1
Measured pKd: 5.13
Experiment series 15
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Q540H
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.69
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 1
Measured pKd: 5.15
Experiment series 16
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: D537V
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.18
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 1
Measured pKd: 4.09
Experiment series 17
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V583I
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.53
Immobilized partner concentration in holdup experiment (10-6M): 13
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 1
Measured pKd: 5.02
Experiment series 18
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: P551L
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.47
Immobilized partner concentration in holdup experiment (10-6M): 8
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 1
Measured pKd: 5.12
Experiment series 19
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: F584S
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 1
The affinity was below the detection threshold of the assay.
Experiment series 20
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Y531S
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.16
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/18
Number of measurements: 1
Measured pKd: 4.05
Experiment series 21
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: R581C
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.52
Immobilized partner concentration in holdup experiment (10-6M): 10
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/18
Number of measurements: 1
Measured pKd: 5.12
Experiment series 22
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V566M
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: CDC27
Protein B fragment: 396-410
Protein B site: PxxP_1
Protein B sequence: LKMKFPPKIPNRKTK
Protein B construct: Bio E E Ado Ado-LKMKFPPKIPNRKTK-CONH2
Average holdup BI: 0.67
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/18
Number of measurements: 1
Measured pKd: 5.13