Although PDZ–PBM interactions have been extensively characterized, there remains inadequate understanding of the general
molecular mechanisms that determine PDZ–PBM specificity, particularly for internal PBMs. This is the result of the low sequence
identity among PDZ domain homologs, promiscuous binding
profiles, and context-dependent interaction mechanisms. Physiological PDZ–PBM interactions have relatively weak binding affinities, with a dissociation constant (K d ) ranging from μM to low
mM [2–4]. To thoroughly characterize PDZ–PBM interactions it is
necessary to determine the binding energetics (i.e., ΔG b , Gibbs free
energy of binding) of PDZ–PBM interactions. The binding energetics coupled with high-resolution structural information and
mutagenesis can provide deep insights into the binding mechanism
and specificity of PDZ–PBM interactions [5–14]. Importantly, this
information can be used to design potential PDZ–PBM protein–
ligand inhibitors. Indeed, over the past ~10 years PDZ–PBM interactions have been identified as potential therapeutic targets
(reviewed in [1]). Here, we describe a general protocol for determining the binding energetics of PDZ–PBM interactions using a
robust and simple fluorescence anisotropy-based assay sensitive to
interactions with dissociation constants in the 1 to ~500 μM range
[15–17].
2 Materials
2.1 Equipment
1. A spectrofluorometer equipped with excitation and emission
polarizers and a magnetic stirrer is used to collect fluorescence
anisotropy data [15]. Here, we use a Fluorolog-3 (Jobin Yvon,
Horiba, NJ) controlled by the FluorEssence V3.8 software
program (Jobin Yvon, Horiba, NJ). The spectrofluorometer is
set to an excitation wavelength at 340 nm and an emission
wavelength at 550 nm, specific for the dansyl [5-(dimethyl
amino)naphthalene-1-sulfonyl] chloride fluorophore (see
Note 1), with constant stirring at 25
C. The instrument
light slit widths are adjusted in the range of 3–9 nm to optimize
the signal-to-noise ratio and maximum output intensity—aiming for ~one million counts per second on the detector (see
Note 2).
2. A quartz cuvette containing 4 polished windows, compatible
with a magnetic stirring platform is used. We use a
2 mL,10 mm length path cuvette equipped with a stopper
and stir bar (Hellma, NY; catalog #119F-10-40).
2.2 Constructs,
Medium, and Reagents
for PDZ Domain
Purification
1. PDZ domains cloned into bacterial expression plasmids are
used. Here we use the CASK PDZ domain cloned into
pET28a (Novagen) and the Scribble PDZ1 cloned into a modified pET21a (Novagen) [18].
138
Young Joo Sun and Ernesto J. Fuentes
molecular mechanisms that determine PDZ–PBM specificity, particularly for internal PBMs. This is the result of the low sequence
identity among PDZ domain homologs, promiscuous binding
profiles, and context-dependent interaction mechanisms. Physiological PDZ–PBM interactions have relatively weak binding affinities, with a dissociation constant (K d ) ranging from μM to low
mM [2–4]. To thoroughly characterize PDZ–PBM interactions it is
necessary to determine the binding energetics (i.e., ΔG b , Gibbs free
energy of binding) of PDZ–PBM interactions. The binding energetics coupled with high-resolution structural information and
mutagenesis can provide deep insights into the binding mechanism
and specificity of PDZ–PBM interactions [5–14]. Importantly, this
information can be used to design potential PDZ–PBM protein–
ligand inhibitors. Indeed, over the past ~10 years PDZ–PBM interactions have been identified as potential therapeutic targets
(reviewed in [1]). Here, we describe a general protocol for determining the binding energetics of PDZ–PBM interactions using a
robust and simple fluorescence anisotropy-based assay sensitive to
interactions with dissociation constants in the 1 to ~500 μM range
[15–17].
2 Materials
2.1 Equipment
1. A spectrofluorometer equipped with excitation and emission
polarizers and a magnetic stirrer is used to collect fluorescence
anisotropy data [15]. Here, we use a Fluorolog-3 (Jobin Yvon,
Horiba, NJ) controlled by the FluorEssence V3.8 software
program (Jobin Yvon, Horiba, NJ). The spectrofluorometer is
set to an excitation wavelength at 340 nm and an emission
wavelength at 550 nm, specific for the dansyl [5-(dimethyl
amino)naphthalene-1-sulfonyl] chloride fluorophore (see
Note 1), with constant stirring at 25
C. The instrument
light slit widths are adjusted in the range of 3–9 nm to optimize
the signal-to-noise ratio and maximum output intensity—aiming for ~one million counts per second on the detector (see
Note 2).
2. A quartz cuvette containing 4 polished windows, compatible
with a magnetic stirring platform is used. We use a
2 mL,10 mm length path cuvette equipped with a stopper
and stir bar (Hellma, NY; catalog #119F-10-40).
2.2 Constructs,
Medium, and Reagents
for PDZ Domain
Purification
1. PDZ domains cloned into bacterial expression plasmids are
used. Here we use the CASK PDZ domain cloned into
pET28a (Novagen) and the Scribble PDZ1 cloned into a modified pET21a (Novagen) [18].
138
Young Joo Sun and Ernesto J. Fuentes