cholesterol [12]. Lipid interactions are important for the organization, segregation and trafficking of PDZ protein complexes to
specific cell compartments. For example, crystal structures and
complementary biophysical, biochemical, and cell biological
approaches demonstrate that tripartite PDZ domain–peptide–
phosphatidylinositol 4,5-bisphosphate (PIP 2 ) interactions function
in membrane compartmentalization and dynamics to control noncanonical Wnt signaling [6]. To better understand the biology of
PDZ proteins, it is thus important to clarify the crosstalk between
PDZ domains, cognate peptides, and lipids. Here we describe
protocols for such interaction studies using BIAcore. We explain
how to measure the binding of a PDZ domain/protein to a biotinylated peptide (Figs. 1, 2, and 3), specific lipids embedded in
liposomes (Figs. 4 and 5) as well as tripartite interactions (Fig. 6).
Further information and the description of complementary biochemical and cell biological approaches can be found in manuscripts from Zimmermann et al.
2 Materials
2.1 Lyophilized
Synthetic Biotinylated
Peptides
1. Peptide. The use of N-terminally biotinylated peptides, ideally
15 mer long or longer, purity ~95% is recommended.
2. Storage of lyophilized peptides. Peptides should be stored at
À80
C. Most peptides stored at this temperature will remain
stable for several years.
3. Solubility. Try to dissolve a small amount of peptide in deionized water. If the peptide is insoluble, sonication may help.
The solubility of basic peptides can be increased by the addition
of 10% (v/v) acetic acid. In case of acidic peptides it is recommended to add 1% NH 4 OH. For hydrophobic peptides it
might be necessary to add organic solvents such as DMSO or
Acetonitrile (from 5 to 50%). Stock solutions at 1 mg/ml are
usually stable for several months when conserved at À80
C.
2.2 Liposomes: We
Recommend to Use
Liposomes Mimicking
the Lipid Composition
of the Biological
Membranes of Interest
1. Lipid solvent. Chloroform–methanol solution (1:1). Store at
room temperature in a bottle wrapped with aluminum foil and
sealed with Teflon caps (see Note 1).
2. Preparation of lipid stocks (from powder). Use chloroform–
methanol (1:1) to solubilize and aliquot into glass vials sealed
with Teflon caps. The stock of lipids can be stored at À20
C
for months.
2.3 BIAcore
All buffers and solutions for BIAcore should be filtered through a
0.22 μm filter. Degassing before use is also essential.
76
Pascale Zimmermann and Antonio Luis Egea-Jimenez
specific cell compartments. For example, crystal structures and
complementary biophysical, biochemical, and cell biological
approaches demonstrate that tripartite PDZ domain–peptide–
phosphatidylinositol 4,5-bisphosphate (PIP 2 ) interactions function
in membrane compartmentalization and dynamics to control noncanonical Wnt signaling [6]. To better understand the biology of
PDZ proteins, it is thus important to clarify the crosstalk between
PDZ domains, cognate peptides, and lipids. Here we describe
protocols for such interaction studies using BIAcore. We explain
how to measure the binding of a PDZ domain/protein to a biotinylated peptide (Figs. 1, 2, and 3), specific lipids embedded in
liposomes (Figs. 4 and 5) as well as tripartite interactions (Fig. 6).
Further information and the description of complementary biochemical and cell biological approaches can be found in manuscripts from Zimmermann et al.
2 Materials
2.1 Lyophilized
Synthetic Biotinylated
Peptides
1. Peptide. The use of N-terminally biotinylated peptides, ideally
15 mer long or longer, purity ~95% is recommended.
2. Storage of lyophilized peptides. Peptides should be stored at
À80
C. Most peptides stored at this temperature will remain
stable for several years.
3. Solubility. Try to dissolve a small amount of peptide in deionized water. If the peptide is insoluble, sonication may help.
The solubility of basic peptides can be increased by the addition
of 10% (v/v) acetic acid. In case of acidic peptides it is recommended to add 1% NH 4 OH. For hydrophobic peptides it
might be necessary to add organic solvents such as DMSO or
Acetonitrile (from 5 to 50%). Stock solutions at 1 mg/ml are
usually stable for several months when conserved at À80
C.
2.2 Liposomes: We
Recommend to Use
Liposomes Mimicking
the Lipid Composition
of the Biological
Membranes of Interest
1. Lipid solvent. Chloroform–methanol solution (1:1). Store at
room temperature in a bottle wrapped with aluminum foil and
sealed with Teflon caps (see Note 1).
2. Preparation of lipid stocks (from powder). Use chloroform–
methanol (1:1) to solubilize and aliquot into glass vials sealed
with Teflon caps. The stock of lipids can be stored at À20
C
for months.
2.3 BIAcore
All buffers and solutions for BIAcore should be filtered through a
0.22 μm filter. Degassing before use is also essential.
76
Pascale Zimmermann and Antonio Luis Egea-Jimenez