10. To prevent fluorophore bleaching by light exposure, it is highly
recommended to cover the cuvette (containing the
fluorophore-peptide) in the spectrofluorometer with the lid
closed. The sample is now ready for the data acquisition. If
necessary, the sample can be equilibrated for several minutes
(typically 1–5 min) or until the measured anisotropy value is
stable over time.
11. The number of data points can vary depending on the sample
and affinity of the PDZ–ligand interaction. Data collection is
complete when the anisotropy value plateaus (i.e., three consecutive data points have similar anisotropy values). However,
we typically collect two or three additional data points after
approaching the anisotropy plateau to ensure binding saturation has been reached.
12. The following assumptions are made when fitting the binding
data. First, the PDZ–PBM binding stoichiometry is 1:1, which
is true for all known PDZ–PBM interactions. Second, the
concentration of free PDZ domain is on the order of the
total PDZ domain concentration. Third, there is no significant
change in fluorescence intensity (<10%) upon PDZ binding.
We have not observed a significant change in fluorescence
intensity in our experiments. Otherwise, correction factors
should be applied [15].
Acknowledgments
The authors thank members of the Fuentes laboratory for helpful
comments. E.J.F. is supported by National Institutes of Health
grant R21-AI135305.
References
1. Liu X, Fuentes EJ (2019) Emerging themes in
PDZ domain signaling: structure, function,
and inhibition. Int Rev Cell Mol Biol
343:129–218
2. Stiffler MA, Chen JR, Grantcharova VP, Lei Y,
Fuchs D, Allen JE et al (2007) PDZ domain
binding selectivity is optimized across the
mouse proteome. Science 317:364–369
3. Chen JR, Chang BH, Allen JE, Stiffler MA,
MacBeath G (2008) Predicting PDZ domainpeptide interactions from primary sequences.
Nat Biotechnol 26:1041–1045
4. Tonikian R, Zhang Y, Sazinsky SL, Currell B,
Yeh JH, Reva B et al (2008) A specificity map
for the PDZ domain family. PLoS Biol 6:e239
5. Shepherd TR, Klaus SM, Liu X, Ramaswamy S,
DeMali KA, Fuentes EJ (2010) The Tiam1
PDZ domain couples to Syndecan1 and promotes cell-matrix adhesion. J Mol Biol
398:730–746
6. Shepherd TR, Hard RL, Murray AM, Pei D,
Fuentes EJ (2011) Distinct ligand specificity of
the Tiam1 and Tiam2 PDZ domains. Biochemistry 50:1296–1308
7. Liu X, Shepherd TR, Murray AM, Xu Z,
Fuentes EJ (2013) The structure of the
Tiam1 PDZ domain/phospho-syndecan1
complex reveals a ligand conformation that
modulates protein dynamics. Structure
21:342–354
8. Liu X, Speckhard DC, Shepherd TR, Sun YJ,
Hengel SR, Yu L et al (2016) Distinct roles for
conformational dynamics in protein-ligand
interactions. Structure 24:2053–2066
PDZ Domain Binding Thermodynamics
147
recommended to cover the cuvette (containing the
fluorophore-peptide) in the spectrofluorometer with the lid
closed. The sample is now ready for the data acquisition. If
necessary, the sample can be equilibrated for several minutes
(typically 1–5 min) or until the measured anisotropy value is
stable over time.
11. The number of data points can vary depending on the sample
and affinity of the PDZ–ligand interaction. Data collection is
complete when the anisotropy value plateaus (i.e., three consecutive data points have similar anisotropy values). However,
we typically collect two or three additional data points after
approaching the anisotropy plateau to ensure binding saturation has been reached.
12. The following assumptions are made when fitting the binding
data. First, the PDZ–PBM binding stoichiometry is 1:1, which
is true for all known PDZ–PBM interactions. Second, the
concentration of free PDZ domain is on the order of the
total PDZ domain concentration. Third, there is no significant
change in fluorescence intensity (<10%) upon PDZ binding.
We have not observed a significant change in fluorescence
intensity in our experiments. Otherwise, correction factors
should be applied [15].
Acknowledgments
The authors thank members of the Fuentes laboratory for helpful
comments. E.J.F. is supported by National Institutes of Health
grant R21-AI135305.
References
1. Liu X, Fuentes EJ (2019) Emerging themes in
PDZ domain signaling: structure, function,
and inhibition. Int Rev Cell Mol Biol
343:129–218
2. Stiffler MA, Chen JR, Grantcharova VP, Lei Y,
Fuchs D, Allen JE et al (2007) PDZ domain
binding selectivity is optimized across the
mouse proteome. Science 317:364–369
3. Chen JR, Chang BH, Allen JE, Stiffler MA,
MacBeath G (2008) Predicting PDZ domainpeptide interactions from primary sequences.
Nat Biotechnol 26:1041–1045
4. Tonikian R, Zhang Y, Sazinsky SL, Currell B,
Yeh JH, Reva B et al (2008) A specificity map
for the PDZ domain family. PLoS Biol 6:e239
5. Shepherd TR, Klaus SM, Liu X, Ramaswamy S,
DeMali KA, Fuentes EJ (2010) The Tiam1
PDZ domain couples to Syndecan1 and promotes cell-matrix adhesion. J Mol Biol
398:730–746
6. Shepherd TR, Hard RL, Murray AM, Pei D,
Fuentes EJ (2011) Distinct ligand specificity of
the Tiam1 and Tiam2 PDZ domains. Biochemistry 50:1296–1308
7. Liu X, Shepherd TR, Murray AM, Xu Z,
Fuentes EJ (2013) The structure of the
Tiam1 PDZ domain/phospho-syndecan1
complex reveals a ligand conformation that
modulates protein dynamics. Structure
21:342–354
8. Liu X, Speckhard DC, Shepherd TR, Sun YJ,
Hengel SR, Yu L et al (2016) Distinct roles for
conformational dynamics in protein-ligand
interactions. Structure 24:2053–2066
PDZ Domain Binding Thermodynamics
147