Details of experiments between the fragments P46013~686-700 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: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.16
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: 2
Measured pKd: 4.05


Experiment series 2

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.13
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: 2
Measured pKd: 3.92


Experiment series 3

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.01
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: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: -0.06
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: 2
The affinity was below the detection threshold of the assay.


Experiment series 5

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.03
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: 2
The affinity was below the detection threshold of the assay.


Experiment series 6

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.13
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: 3.94


Experiment series 7

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.08
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: 3.72


Experiment series 8

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.08
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: 3.69


Experiment series 9

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.01
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: 2
The affinity was below the detection threshold of the assay.


Experiment series 10

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.07
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: 2
Measured pKd: 3.65


Experiment series 11

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.01
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 12

Protein A protein: MKI67
Protein A fragment: 686-700
Protein A site: PxxP_14
Protein A sequence: NKRQRRPATPKKPVG
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: 0.04
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
The affinity was below the detection threshold of the assay.