Chapter 8
A Fluorescence-Based Assay to Determine PDZ–Ligand
Binding Thermodynamics
Young Joo Sun and Ernesto J. Fuentes
Abstract
Postsynaptic density-95, disks-large, and zonula occludens-1 (PDZ) domain interactions with cognate
linear binding motifs (i.e., PDZ-binding motifs or PBMs) are important for many biological processes and
can be pathological when disrupted. There are hundreds of PDZ–PBM interactions reported but few have
been quantitatively determined. Moreover, PDZ–PBM interactions have been identified as potential
therapeutic targets. To thoroughly understand PDZ–PBM binding energetics and their specificity, we
have developed a sensitive and quantitative equilibrium binding assay. Here, we describe a protocol for
determining PDZ–PBM binding energetics using fluorescence anisotropy-based methodology.
Key words PDZ domain, PDZ-binding motif, Fluorescence anisotropy, Protein–protein binding,
CASK, Scribble, SGEF
1 Introduction
Postsynaptic density-95, Disks-large, and Zonula occludens-1
(PDZ) proteins are ubiquitously found in many types of mammalian cells and regulate the spatial and temporal function of a diverse
set of signaling pathways. A distinguishing feature of these proteins
is the small (~90 amino acids, ~10 kDa), structurally conserved
protein–protein interaction module known as a PDZ domain that
selectively interacts with linear C-terminal and internal peptide
motifs (i.e., PDZ-binding-motifs or PBMs) [1]. PDZ–PBM interactions and their specificity are critical for many biological processes
including the maintenance of cell polarity, neuronal development,
and signal transduction. Thus, it is not surprising that genetic
mutations in PDZ proteins or perturbation of PDZ–PBM interactions can contribute to pathologies such as neuronal disorders and
complications form brain injury, cancer, cystic fibrosis, and viral
infections (reviewed in [1]).
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
137
A Fluorescence-Based Assay to Determine PDZ–Ligand
Binding Thermodynamics
Young Joo Sun and Ernesto J. Fuentes
Abstract
Postsynaptic density-95, disks-large, and zonula occludens-1 (PDZ) domain interactions with cognate
linear binding motifs (i.e., PDZ-binding motifs or PBMs) are important for many biological processes and
can be pathological when disrupted. There are hundreds of PDZ–PBM interactions reported but few have
been quantitatively determined. Moreover, PDZ–PBM interactions have been identified as potential
therapeutic targets. To thoroughly understand PDZ–PBM binding energetics and their specificity, we
have developed a sensitive and quantitative equilibrium binding assay. Here, we describe a protocol for
determining PDZ–PBM binding energetics using fluorescence anisotropy-based methodology.
Key words PDZ domain, PDZ-binding motif, Fluorescence anisotropy, Protein–protein binding,
CASK, Scribble, SGEF
1 Introduction
Postsynaptic density-95, Disks-large, and Zonula occludens-1
(PDZ) proteins are ubiquitously found in many types of mammalian cells and regulate the spatial and temporal function of a diverse
set of signaling pathways. A distinguishing feature of these proteins
is the small (~90 amino acids, ~10 kDa), structurally conserved
protein–protein interaction module known as a PDZ domain that
selectively interacts with linear C-terminal and internal peptide
motifs (i.e., PDZ-binding-motifs or PBMs) [1]. PDZ–PBM interactions and their specificity are critical for many biological processes
including the maintenance of cell polarity, neuronal development,
and signal transduction. Thus, it is not surprising that genetic
mutations in PDZ proteins or perturbation of PDZ–PBM interactions can contribute to pathologies such as neuronal disorders and
complications form brain injury, cancer, cystic fibrosis, and viral
infections (reviewed in [1]).
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
137