core PDZ structure. If PDZ domains are mainly robust, soluble
domains, inappropriate delimitations can lead to unstable protein.
Indeed, many studies have shown that extensions can influence the
dynamics, stability, and solubility of the PDZ domains [3] for often
unknown reasons. The extension of N- and C-terminus can entropically stabilize the domain affecting its internal dynamics. Thus,
the thermostability and the folding of PDZ domain are dependent
in many cases on short disordered extensions at their two termini,
but also of adjacent modules forming homotypic and heterotypic
PDZ supramodules [4]. These N- and C-terminal extensions could
form functional and structural units playing a crucial role of activity
modulation of the adjacent PDZ domain. Indeed, sequence context impacts the stability and solubility of constructs and can deeply
influence binding affinity and specificity, and structured and disordered extensions may affect the structure and function of the core
PDZ domain [5].
Optimal folding and solubility of PDZ domains are needed to
ensure accurate affinity measurement and structure determination.
Conformational exchange can also be an intrinsic property of PDZ
domains, for example with the tandem of PDZ domains in whirlin
[6] or of GRIP1 [7], and also the autoassociation capacity of the
unique PDZ domain of MAST2 (microtubule-associated serine and
threonine kinase 2; [8]) or of the second PDZ domain of ZO2
[9]. Notably, the frequency of PDZ-PDZ interactions in eukaryotic
proteins has been documented, and dimerization occurs in vitro in
30% of the 157 PDZ domains tested [10].
Altogether, these examples illustrate the need for a strict quality
assessment of the PDZ domains before deeply characterizing their
biochemical, biophysical, and functional properties. Appropriate
experimental approaches should be selected to overcome the
small size of such domains. Here, we report the contribution of a
series of biochemical and biophysical approaches applied to PDZ
domain constructs for sample quality evaluation. These analyses
were used to assay the purity, identity, homogeneity, stability, and
folding of PDZ domain construct samples. We use as an example
the PDZ domain of the kinase protein MAST2 and detail every step
of this workflow, the principles, the techniques used, and some
protocols in this chapter. The biochemical/biophysical characterizations are divided into four parts: purity, homogeneity, identity,
and conformational stability/folding state.
1.1 Purity
1.1.1 SDS-PAGE
Electrophoresis
SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis
containing sodium dodecyl sulfate) is a technique that separates
proteins in a gel [11]. The migration is controlled by an electric
field, enabling their separation according to their molecular weight.
The polyacrylamide gel is made by the copolymerization of
acrylamide and bisacrylamide, in the presence of polymerization
agents (e.g., TEMED, ammonium persulfate). The concentration
90
Ce ´ lia Caillet-Saguy et al.
domains, inappropriate delimitations can lead to unstable protein.
Indeed, many studies have shown that extensions can influence the
dynamics, stability, and solubility of the PDZ domains [3] for often
unknown reasons. The extension of N- and C-terminus can entropically stabilize the domain affecting its internal dynamics. Thus,
the thermostability and the folding of PDZ domain are dependent
in many cases on short disordered extensions at their two termini,
but also of adjacent modules forming homotypic and heterotypic
PDZ supramodules [4]. These N- and C-terminal extensions could
form functional and structural units playing a crucial role of activity
modulation of the adjacent PDZ domain. Indeed, sequence context impacts the stability and solubility of constructs and can deeply
influence binding affinity and specificity, and structured and disordered extensions may affect the structure and function of the core
PDZ domain [5].
Optimal folding and solubility of PDZ domains are needed to
ensure accurate affinity measurement and structure determination.
Conformational exchange can also be an intrinsic property of PDZ
domains, for example with the tandem of PDZ domains in whirlin
[6] or of GRIP1 [7], and also the autoassociation capacity of the
unique PDZ domain of MAST2 (microtubule-associated serine and
threonine kinase 2; [8]) or of the second PDZ domain of ZO2
[9]. Notably, the frequency of PDZ-PDZ interactions in eukaryotic
proteins has been documented, and dimerization occurs in vitro in
30% of the 157 PDZ domains tested [10].
Altogether, these examples illustrate the need for a strict quality
assessment of the PDZ domains before deeply characterizing their
biochemical, biophysical, and functional properties. Appropriate
experimental approaches should be selected to overcome the
small size of such domains. Here, we report the contribution of a
series of biochemical and biophysical approaches applied to PDZ
domain constructs for sample quality evaluation. These analyses
were used to assay the purity, identity, homogeneity, stability, and
folding of PDZ domain construct samples. We use as an example
the PDZ domain of the kinase protein MAST2 and detail every step
of this workflow, the principles, the techniques used, and some
protocols in this chapter. The biochemical/biophysical characterizations are divided into four parts: purity, homogeneity, identity,
and conformational stability/folding state.
1.1 Purity
1.1.1 SDS-PAGE
Electrophoresis
SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis
containing sodium dodecyl sulfate) is a technique that separates
proteins in a gel [11]. The migration is controlled by an electric
field, enabling their separation according to their molecular weight.
The polyacrylamide gel is made by the copolymerization of
acrylamide and bisacrylamide, in the presence of polymerization
agents (e.g., TEMED, ammonium persulfate). The concentration
90
Ce ´ lia Caillet-Saguy et al.