3. Top up the concentrator with phosphate buffer and centrifuge
for 20 min at 4000 Â g.
4. After the first centrifugation, discard the flow through and top
up the concentrator again with fresh phosphate buffer and spin
again for up to 20 min at 4000 Â g.
5. Repeat step 4 for another 4 times.
6. Concentrate the protein sample (now in phosphate buffer) to a
final concentration of 0.3 mg/mL and use approximately
300 μL for CD experiment (see Note 1).
3.2 Interactions
of Polarity Protein PDZ
Domains with Ligands
Techniques used to analyze protein interactions such as surface
plasmon resonance (SPR) can be hampered by the requirement
for immobilization of proteins before the interaction can be
observed. Isothermal titration calorimetry (ITC) provides a quantitative in-solution method for measuring protein interactions
without immobilization. In a single measurement, ITC allows for
accurate measurements of the binding constant (K D ), reaction
stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) of a given
biomolecular binding event whereby heat is either released or
absorbed [23]. ITC is highly sensitive to small differences in concentration of buffer components and pH, thus making control
measurements to establish buffer effects advisable. In addition to
buffer control measurements, control measurements to gauge PDZ
domain protein quality interactions are useful. For this purpose, we
use a synthetic peptide that binds to a vast range of PDZ domains
with high affinity termed superpeptide (RSWFETWV), which was
identified via phage display experiments [24].
1. Ensure reference cell contains water.
2. Rinse sample cell and syringe with dH 2 O and storage buffer
three times each (see Note 4).
3. Titrations are performed at 25
C with a stirring speed of
750 rpm.
4. A total of 20 injections with 2 μL of the peptide solution each
and a spacing of 180 s were titrated into the 200 μL protein
sample, except for the first injection which was only 0.4 μL (see
Note 5).
5. Process the raw thermograms with Origin
® to obtain the binding parameters of each interaction. Typical examples are shown
in Fig. 1.
3.3 Preparation
of Cell-Polarity
Protein–Peptide
Complexes
for Crystallization
Scribble and Dlg PDZ domains interact with a diverse range of
known partners, and preparation of complexes of these domains
with the interacting partners is a key step to understand their multifunctionality and promiscuous behavior, and ultimately their role in
disease mechanisms. In this example we demonstrate how to
Biophysics of Scribble Module PDZ Interactions
131
for 20 min at 4000 Â g.
4. After the first centrifugation, discard the flow through and top
up the concentrator again with fresh phosphate buffer and spin
again for up to 20 min at 4000 Â g.
5. Repeat step 4 for another 4 times.
6. Concentrate the protein sample (now in phosphate buffer) to a
final concentration of 0.3 mg/mL and use approximately
300 μL for CD experiment (see Note 1).
3.2 Interactions
of Polarity Protein PDZ
Domains with Ligands
Techniques used to analyze protein interactions such as surface
plasmon resonance (SPR) can be hampered by the requirement
for immobilization of proteins before the interaction can be
observed. Isothermal titration calorimetry (ITC) provides a quantitative in-solution method for measuring protein interactions
without immobilization. In a single measurement, ITC allows for
accurate measurements of the binding constant (K D ), reaction
stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) of a given
biomolecular binding event whereby heat is either released or
absorbed [23]. ITC is highly sensitive to small differences in concentration of buffer components and pH, thus making control
measurements to establish buffer effects advisable. In addition to
buffer control measurements, control measurements to gauge PDZ
domain protein quality interactions are useful. For this purpose, we
use a synthetic peptide that binds to a vast range of PDZ domains
with high affinity termed superpeptide (RSWFETWV), which was
identified via phage display experiments [24].
1. Ensure reference cell contains water.
2. Rinse sample cell and syringe with dH 2 O and storage buffer
three times each (see Note 4).
3. Titrations are performed at 25
C with a stirring speed of
750 rpm.
4. A total of 20 injections with 2 μL of the peptide solution each
and a spacing of 180 s were titrated into the 200 μL protein
sample, except for the first injection which was only 0.4 μL (see
Note 5).
5. Process the raw thermograms with Origin
® to obtain the binding parameters of each interaction. Typical examples are shown
in Fig. 1.
3.3 Preparation
of Cell-Polarity
Protein–Peptide
Complexes
for Crystallization
Scribble and Dlg PDZ domains interact with a diverse range of
known partners, and preparation of complexes of these domains
with the interacting partners is a key step to understand their multifunctionality and promiscuous behavior, and ultimately their role in
disease mechanisms. In this example we demonstrate how to
Biophysics of Scribble Module PDZ Interactions
131