1. Wash 1 mL centrifuge concentrator with PDZ storage buffer.
2. Add 1 mg of PDZ domain in final sample buffer.
3. Slowly add at least 2–5 molar excess of the target peptide to
centrifugal concentrator while stirring with pipette to avoid
local precipitation of sample (see Note 6).
4. Concentrate the complex to a final concentration of 5 mg/mL
(see Note 7).
3.4 Crystallization
of Tandem Scribble
PDZ Domains Bound
to Interacting Peptides
As Scribble and Dlg consist of multi-PDZ domains, two or more of
these PDZ domains can be connected to each other in tandem to
form PDZ supramodules, which may give rise to unique binding
properties with their interactors that are different from when they
exist as individual domains [27]. Since these tandem PDZ domains
are often connected to each other via very short linkers, these
domains act as a one structural and functional unit [27]. Preparation of complexes of tandem domains with a target peptide/peptide can be also achieved using a similar method as mentioned
above. The protein concentration and the protein–peptide ratio
may vary. A successful crystallization example is the structure determination of Scribble PDZ34 (8 mg/mL) in complex with a commercially synthesized peptide (S-W-F-Q-T-D-L) in a 5:1 peptide–
protein molar ratio (PDB Id:4WYU) [28].
4 Notes
1. Phosphate buffer is prepared by mixing Na 2 HPO 4 and
NaH 2 PO 4 to achieve the desired pH in order to avoid the use
of HCl.
2. Suitable peptide length for ITC is eight or more amino acids.
However, peptides of four amino acids in length have been
successfully characterized.
3. The synthetic peptide stocks are stored in dH 2 O at a concentration of 5 mM.
4. It is crucial to have an identical pH for sample and peptide
buffers when performing ITC to prevent spurious heat peaks
due to the heat of dilution of protons.
5. Usually PDZ domain proteins yield high-quality thermograms
at concentrations of 75 μM for protein and 900 μM for peptide;
however, some concentration optimization may be required.
6. Peptide concentration in crystallization trials should be at least
at a twofold molar excess.
7. Final concentration of the sample for crystallization experiment
may vary with each sample and should be confirmed using a
spectrophotometer to measure UV absorbance at 280 nm
wavelength.
Biophysics of Scribble Module PDZ Interactions
133
2. Add 1 mg of PDZ domain in final sample buffer.
3. Slowly add at least 2–5 molar excess of the target peptide to
centrifugal concentrator while stirring with pipette to avoid
local precipitation of sample (see Note 6).
4. Concentrate the complex to a final concentration of 5 mg/mL
(see Note 7).
3.4 Crystallization
of Tandem Scribble
PDZ Domains Bound
to Interacting Peptides
As Scribble and Dlg consist of multi-PDZ domains, two or more of
these PDZ domains can be connected to each other in tandem to
form PDZ supramodules, which may give rise to unique binding
properties with their interactors that are different from when they
exist as individual domains [27]. Since these tandem PDZ domains
are often connected to each other via very short linkers, these
domains act as a one structural and functional unit [27]. Preparation of complexes of tandem domains with a target peptide/peptide can be also achieved using a similar method as mentioned
above. The protein concentration and the protein–peptide ratio
may vary. A successful crystallization example is the structure determination of Scribble PDZ34 (8 mg/mL) in complex with a commercially synthesized peptide (S-W-F-Q-T-D-L) in a 5:1 peptide–
protein molar ratio (PDB Id:4WYU) [28].
4 Notes
1. Phosphate buffer is prepared by mixing Na 2 HPO 4 and
NaH 2 PO 4 to achieve the desired pH in order to avoid the use
of HCl.
2. Suitable peptide length for ITC is eight or more amino acids.
However, peptides of four amino acids in length have been
successfully characterized.
3. The synthetic peptide stocks are stored in dH 2 O at a concentration of 5 mM.
4. It is crucial to have an identical pH for sample and peptide
buffers when performing ITC to prevent spurious heat peaks
due to the heat of dilution of protons.
5. Usually PDZ domain proteins yield high-quality thermograms
at concentrations of 75 μM for protein and 900 μM for peptide;
however, some concentration optimization may be required.
6. Peptide concentration in crystallization trials should be at least
at a twofold molar excess.
7. Final concentration of the sample for crystallization experiment
may vary with each sample and should be confirmed using a
spectrophotometer to measure UV absorbance at 280 nm
wavelength.
Biophysics of Scribble Module PDZ Interactions
133