2. The Ns group is removed by adding 1,8-diazabicyclo[5.4.0]
undec-7-ene (DBU, 0.5 mmol) in DMF (2 mL) and
β-mercaptoethanol (0.5 mmol) in DMF (2 mL) and shake it
for 30 min (see Fig. 4, step 2.1).
3. Wash with DMF (5 times, each of 10 mL) and repeat the
treatment with β-mercaptoethanol/DBU. Wash the resin
again consecutively with DMF, DCM, MeOH, and DCM
(5 times each, each of 10 mL). For Tat- (AVLX-144) and
ReTat-N-dimer (AVLX-147) the first amino acid from the
sequence (L- or D-Arg, respectively) was coupled to the linker
nitrogen by six consecutive couplings of Fmoc-L/D-Arg(Pbf)OH.
4. For each coupling, activate the Fmoc-L/D-Arg(Pbf)-OH
(0.5 mmol) with HATU in DMF (2 mL, 0.244 M) and collidine (132 μL) (see Fig. 4, step 2.2).
5. Remove the Fmoc protection group with 20% (v/v) piperidine
in DMF and add the remaining Tat- or ReTat sequence following the same procedures as described in Subheading 3.1.1 (see
Fig. 4, step 3).
3.3 Affinity
and Selectivity
Experiments
3.3.1 Fluorescence
Polarization Assays
The expressed PDZ domains can be used directly in any in vitro
assay for detection of protein–protein interactions. In this case, we
describe the use of an FP assay to assess the binding affinity of the
target protein with the synthesized peptide ligands. The protocol
involves a fluorescence-labeled peptide probe (in this case Cy5),
which is at a constant concentration. To the probe the protein is
titrated with increasing concentration to obtain a saturation curve.
The dissociation constant, K d , indicates the strength of the binding
interaction between protein and ligand is interpolated from the
saturation curve.
1. Prepare a series of 12 twofold dilutions of the desired PDZ
domain, diluting from a starting concentration of 200 μM (see
Note 13) with FP buffer. For triplicate measurements, transfer
three times 15 μL of each dilution onto a 384-well black flatbottom plate.
2. In an Eppendorf tube, prepare BSA blank by diluting 10x BSA
stock solution into FP buffer. For triplicate measurements of
BSA blank and FP buffer controls, transfer three times 30 μL
BSA blank and 15 μL FP buffer, respectively, into empty wells
of the 384-well plate.
3. In an Eppendorf tube, prepare 2Â probe stock by diluting 10Â
BSA stock and a Cy5 conjugated peptide ligand to a final assay
concentration of 200 nM with FP buffer (see Note 14). Add
15 μL of the 2Â probe stock to each of the protein and buffer
control wells of the 384-well plate. Incubate plate at room
temperature for 15 min. Protect the plate from light.
PDZ Domain Peptide Inhibitors
169
undec-7-ene (DBU, 0.5 mmol) in DMF (2 mL) and
β-mercaptoethanol (0.5 mmol) in DMF (2 mL) and shake it
for 30 min (see Fig. 4, step 2.1).
3. Wash with DMF (5 times, each of 10 mL) and repeat the
treatment with β-mercaptoethanol/DBU. Wash the resin
again consecutively with DMF, DCM, MeOH, and DCM
(5 times each, each of 10 mL). For Tat- (AVLX-144) and
ReTat-N-dimer (AVLX-147) the first amino acid from the
sequence (L- or D-Arg, respectively) was coupled to the linker
nitrogen by six consecutive couplings of Fmoc-L/D-Arg(Pbf)OH.
4. For each coupling, activate the Fmoc-L/D-Arg(Pbf)-OH
(0.5 mmol) with HATU in DMF (2 mL, 0.244 M) and collidine (132 μL) (see Fig. 4, step 2.2).
5. Remove the Fmoc protection group with 20% (v/v) piperidine
in DMF and add the remaining Tat- or ReTat sequence following the same procedures as described in Subheading 3.1.1 (see
Fig. 4, step 3).
3.3 Affinity
and Selectivity
Experiments
3.3.1 Fluorescence
Polarization Assays
The expressed PDZ domains can be used directly in any in vitro
assay for detection of protein–protein interactions. In this case, we
describe the use of an FP assay to assess the binding affinity of the
target protein with the synthesized peptide ligands. The protocol
involves a fluorescence-labeled peptide probe (in this case Cy5),
which is at a constant concentration. To the probe the protein is
titrated with increasing concentration to obtain a saturation curve.
The dissociation constant, K d , indicates the strength of the binding
interaction between protein and ligand is interpolated from the
saturation curve.
1. Prepare a series of 12 twofold dilutions of the desired PDZ
domain, diluting from a starting concentration of 200 μM (see
Note 13) with FP buffer. For triplicate measurements, transfer
three times 15 μL of each dilution onto a 384-well black flatbottom plate.
2. In an Eppendorf tube, prepare BSA blank by diluting 10x BSA
stock solution into FP buffer. For triplicate measurements of
BSA blank and FP buffer controls, transfer three times 30 μL
BSA blank and 15 μL FP buffer, respectively, into empty wells
of the 384-well plate.
3. In an Eppendorf tube, prepare 2Â probe stock by diluting 10Â
BSA stock and a Cy5 conjugated peptide ligand to a final assay
concentration of 200 nM with FP buffer (see Note 14). Add
15 μL of the 2Â probe stock to each of the protein and buffer
control wells of the 384-well plate. Incubate plate at room
temperature for 15 min. Protect the plate from light.
PDZ Domain Peptide Inhibitors
169