Chapter 9
Unveiling the Folding Mechanism of PDZ Domains
Candice Gautier and Stefano Gianni
Abstract
Understanding the mechanism of folding of single domain proteins demands a complete characterization of
their equilibrium and kinetic properties. By using a well-studied class of protein domain, the PDZ domain,
here we exemplify the typical procedure to address this problem.
Key words Protein Stability, Equilibrium, Kinetics, Fluorescence, Protein Engineering
1 Introduction
Understanding the mechanism of folding of single domain proteins
is a difficult task that demands the execution of several experiments.
Of particular importance is the employment of both equilibrium
and kinetic experiments to address quantitatively the simplest reaction scheme that adequately describes the observed reaction
[1]. Furthermore, by employing site directed mutagenesis it is
possible to further verify the proposed scheme, as well as to infer
the structural features of the identified folding intermediate, as well
as the intervening transition states [2].
PDZ domains represent a class of globular domains displaying a
conserved structure composed of five to six β-strands and two
α-helices [3]. Because of their small size, good expression yields,
high solubility and reversible folding, they proved as very good
candidate to perform folding studies. In fact, the folding mechanism of PDZ domains has been studied extensively and different
members of this protein family have been characterized in detail
[4–12].
Because of their complexity, the analysis of the equilibrium and
kinetic folding mechanism of PDZ domains represent a good
example to illustrate the classical approaches that are employed to
describe the folding of single domain proteins. By taking PDZ
domains as a prototypical case, here we recapitulate the standard
experiments that are generally performed.
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_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
149
Unveiling the Folding Mechanism of PDZ Domains
Candice Gautier and Stefano Gianni
Abstract
Understanding the mechanism of folding of single domain proteins demands a complete characterization of
their equilibrium and kinetic properties. By using a well-studied class of protein domain, the PDZ domain,
here we exemplify the typical procedure to address this problem.
Key words Protein Stability, Equilibrium, Kinetics, Fluorescence, Protein Engineering
1 Introduction
Understanding the mechanism of folding of single domain proteins
is a difficult task that demands the execution of several experiments.
Of particular importance is the employment of both equilibrium
and kinetic experiments to address quantitatively the simplest reaction scheme that adequately describes the observed reaction
[1]. Furthermore, by employing site directed mutagenesis it is
possible to further verify the proposed scheme, as well as to infer
the structural features of the identified folding intermediate, as well
as the intervening transition states [2].
PDZ domains represent a class of globular domains displaying a
conserved structure composed of five to six β-strands and two
α-helices [3]. Because of their small size, good expression yields,
high solubility and reversible folding, they proved as very good
candidate to perform folding studies. In fact, the folding mechanism of PDZ domains has been studied extensively and different
members of this protein family have been characterized in detail
[4–12].
Because of their complexity, the analysis of the equilibrium and
kinetic folding mechanism of PDZ domains represent a good
example to illustrate the classical approaches that are employed to
describe the folding of single domain proteins. By taking PDZ
domains as a prototypical case, here we recapitulate the standard
experiments that are generally performed.
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_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
149