Details of experiments between the fragments O14974~493-507 and O00499~513-593
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.21
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.06.29.
Number of measurements: 2
Measured pKd: 4.18
Experiment series 2
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: T532M
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.2
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.06.29.
Number of measurements: 2
Measured pKd: 4.15
Experiment series 3
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: D537V
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.04
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: 2
The affinity was below the detection threshold of the assay.
Experiment series 4
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: F584S
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.01
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: 2
The affinity was below the detection threshold of the assay.
Experiment series 5
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: P551L
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.05
Holdup BI standard deviation: 0.06
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: 2
Measured pKd: 3.96
Experiment series 6
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: Q540H
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.19
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: 2
Measured pKd: 4.13
Experiment series 7
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: T532M
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.15
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: 2
Measured pKd: 4
Experiment series 8
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: V566M
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.15
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: 2
Measured pKd: 3.99
Experiment series 9
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: V583I
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.09
Holdup BI standard deviation: 0
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: 2
Measured pKd: 4.03
Experiment series 10
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.17
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: 2
Measured pKd: 4.07
Experiment series 11
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: Y531S
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.02
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: 2
The affinity was below the detection threshold of the assay.
Experiment series 12
Protein A protein: PPP1R12A
Protein A fragment: 493-507
Protein A site: PxxP_2
Protein A sequence: RLAYVAPTIPRRLAS
Protein A construct: Biotinylated 15-mer peptide (crude)
Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: R581C
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3
Average holdup BI: 0.09
Holdup BI standard deviation: 0.03
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: 2
Measured pKd: 4.21