selectivity and specificity of PDZ domain protein–protein interactions will be instrumental in providing novel insights into disease
mechanisms.
Human Scribble and Dlg PDZ domains have been shown to
interact with a wide range of proteins involved in various cellular
functions such as signaling, trafficking, and polarity, many of which
are implicated in disease progression [3]. A number of structures of
Dlg and Scribble PDZ-PBM protein complexes have been solved
using either X-ray crystallography or NMR and deposited in the
protein data bank (PDB) as listed in Table 1. However, for the
Scribble module, the vast majority of its interactions and their
molecular basis remain to be characterized in humans and other
relevant model organisms. Here we will focus on methods and
strategies related to PDZ-related structure determination of Scribble module components using X-ray crystallography. Furthermore,
we discuss some of the biophysical analyses such as circular dichroism spectroscopy (CD) and isothermal titration calorimetry (ITC)
which complement efforts to determine X-ray crystallographic
structures.
Circular Dichroism (CD) is a spectroscopic method that utilizes the differential absorption of right-and-left polarized light
through an optically active molecule [20]. This is an invaluable
tool for the determination of secondary structure of proteins,
with CD spectra in the far UV region between 180 nm and
260 nm revealing different secondary structure features of sample
proteins: alpha helix, parallel and antiparallel beta sheet, turn, and
other. Thus, CD spectroscopy can determine the percentage of
secondary structure components a protein contains. CD spectroscopy can also determine the folding state of expressed and purified
recombinant proteins, and reveal how mutations affect protein
structure and stability. Variations of the secondary structure of
proteins upon ligand bindings and protein–protein interactions in
different conditions such as temperature, pH, and salts, can also be
determined using CD spectroscopy [21]. While CD spectroscopy is
not a substitute for high-resolution structure determination by
X-ray crystallography, NMR, or cryo-EM, CD analysis significantly
improves the sample preparation for the higher resolution
techniques.
2 Materials
2.1 Sample
Preparation for CD
Analysis
1. Recombinant cell polarity protein (e.g., Scribble PDZ and Dlg
PDZ domains) purified to homogeneity.
2. PDZ storage buffer: 25 mM HEPES–NaOH pH 7.2,
150 mM NaCl.
3. Phosphate buffer: 50 mM sodium phosphate pH 7.0 (see Note 1).
4. Centrifugal concentrator (MWCO 3 kDa).
Biophysics of Scribble Module PDZ Interactions
127
mechanisms.
Human Scribble and Dlg PDZ domains have been shown to
interact with a wide range of proteins involved in various cellular
functions such as signaling, trafficking, and polarity, many of which
are implicated in disease progression [3]. A number of structures of
Dlg and Scribble PDZ-PBM protein complexes have been solved
using either X-ray crystallography or NMR and deposited in the
protein data bank (PDB) as listed in Table 1. However, for the
Scribble module, the vast majority of its interactions and their
molecular basis remain to be characterized in humans and other
relevant model organisms. Here we will focus on methods and
strategies related to PDZ-related structure determination of Scribble module components using X-ray crystallography. Furthermore,
we discuss some of the biophysical analyses such as circular dichroism spectroscopy (CD) and isothermal titration calorimetry (ITC)
which complement efforts to determine X-ray crystallographic
structures.
Circular Dichroism (CD) is a spectroscopic method that utilizes the differential absorption of right-and-left polarized light
through an optically active molecule [20]. This is an invaluable
tool for the determination of secondary structure of proteins,
with CD spectra in the far UV region between 180 nm and
260 nm revealing different secondary structure features of sample
proteins: alpha helix, parallel and antiparallel beta sheet, turn, and
other. Thus, CD spectroscopy can determine the percentage of
secondary structure components a protein contains. CD spectroscopy can also determine the folding state of expressed and purified
recombinant proteins, and reveal how mutations affect protein
structure and stability. Variations of the secondary structure of
proteins upon ligand bindings and protein–protein interactions in
different conditions such as temperature, pH, and salts, can also be
determined using CD spectroscopy [21]. While CD spectroscopy is
not a substitute for high-resolution structure determination by
X-ray crystallography, NMR, or cryo-EM, CD analysis significantly
improves the sample preparation for the higher resolution
techniques.
2 Materials
2.1 Sample
Preparation for CD
Analysis
1. Recombinant cell polarity protein (e.g., Scribble PDZ and Dlg
PDZ domains) purified to homogeneity.
2. PDZ storage buffer: 25 mM HEPES–NaOH pH 7.2,
150 mM NaCl.
3. Phosphate buffer: 50 mM sodium phosphate pH 7.0 (see Note 1).
4. Centrifugal concentrator (MWCO 3 kDa).
Biophysics of Scribble Module PDZ Interactions
127