Chapter 12
Chemical Synthesis of PDZ Domains
Christin Kossmann, Sana Ma, Louise S. Clemmensen,
and Kristian Strømgaard
Abstract
Developments in chemical protein synthesis have enabled the generation of tailor-made proteins including
incorporation of many types of modifications into proteins, enhancing our ability to control site-specificity
of protein posttranslational modifications (PTMs), modify protein backbones and introduce photocrosslinking probes. For PDZ (postsynaptic density protein, disks large, zonula occludens) protein domains,
expressed protein ligation (EPL) has been employed to introduce analogs of cognate amino acids, amideto-ester bond mutations, and phosphorylations in the study of PDZ domain-mediated protein-protein
interactions (PPIs). Here, we present protocols for EPL of PDZ domains focusing on phosphorylation and
amide-to-ester modifications in the PDZ domain proteins.
Key words Expressed protein ligation, Protein modification, Phosphorylation, Amide-to-ester mutation, Solid-phase peptide synthesis, Native chemical ligation
1 Introduction
The introduction of chemical modifications into proteins has broad
applicability for not only biotechnological and biomedical purposes
but also in studies of fundamental biological functions such as cell
signaling, protein-protein interactions (PPIs), and posttranslational
modifications (PTMs). Chemical synthesis of proteins via solidphase peptide synthesis (SPPS) enables veritably any modification
at any desired position [1, 2] and allows both the introduction of
noncanonical amino acids as well as peptide or protein backbone
modifications. The seemingly limitless scope of chemical space that
can be leveraged in SPPS is a major advantage over recombinant
methods to introduce protein modifications. However, the constraints in the coupling efficiency of longer peptide chains severely
restrict the size of peptides or proteins that can be generated by
SPPS. Undeniably, improvements in 9-fluorenylmethoxycarbonyl
(Fmoc)/tert-butyl (t-Bu)-SPPS and tert-butyloxycarbonyl (Boc)/
benzyl (Bzl)-SPPS, such as building blocks to minimize
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
193
Chemical Synthesis of PDZ Domains
Christin Kossmann, Sana Ma, Louise S. Clemmensen,
and Kristian Strømgaard
Abstract
Developments in chemical protein synthesis have enabled the generation of tailor-made proteins including
incorporation of many types of modifications into proteins, enhancing our ability to control site-specificity
of protein posttranslational modifications (PTMs), modify protein backbones and introduce photocrosslinking probes. For PDZ (postsynaptic density protein, disks large, zonula occludens) protein domains,
expressed protein ligation (EPL) has been employed to introduce analogs of cognate amino acids, amideto-ester bond mutations, and phosphorylations in the study of PDZ domain-mediated protein-protein
interactions (PPIs). Here, we present protocols for EPL of PDZ domains focusing on phosphorylation and
amide-to-ester modifications in the PDZ domain proteins.
Key words Expressed protein ligation, Protein modification, Phosphorylation, Amide-to-ester mutation, Solid-phase peptide synthesis, Native chemical ligation
1 Introduction
The introduction of chemical modifications into proteins has broad
applicability for not only biotechnological and biomedical purposes
but also in studies of fundamental biological functions such as cell
signaling, protein-protein interactions (PPIs), and posttranslational
modifications (PTMs). Chemical synthesis of proteins via solidphase peptide synthesis (SPPS) enables veritably any modification
at any desired position [1, 2] and allows both the introduction of
noncanonical amino acids as well as peptide or protein backbone
modifications. The seemingly limitless scope of chemical space that
can be leveraged in SPPS is a major advantage over recombinant
methods to introduce protein modifications. However, the constraints in the coupling efficiency of longer peptide chains severely
restrict the size of peptides or proteins that can be generated by
SPPS. Undeniably, improvements in 9-fluorenylmethoxycarbonyl
(Fmoc)/tert-butyl (t-Bu)-SPPS and tert-butyloxycarbonyl (Boc)/
benzyl (Bzl)-SPPS, such as building blocks to minimize
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
193