Deciphering functional roles of protein succinylation and glutarylation using genetic code expansion

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Post-translational modifications (PTMs) dynamically regulate cellular processes.
• Lysine acylations, such as succinylation (SucK) and glutarylation (GluK), are PTMs that reverse lysine's charge, impacting protein function.
• Studying these negatively charged PTMs is challenging due to limitations in existing methods.

Purpose of the Study:

• To develop a method for site-specific incorporation of succinylation and glutarylation into proteins.
• To enable biochemical studies of homogeneously modified proteins.
• To investigate the functional roles of succinylation and glutarylation in biological processes.

Main Methods:

• Site-specific incorporation of succinylation and glutarylation using thioester derivatives to mask negative charges.
• Preparation of modified bacterial and mammalian target proteins, including multidomain proteins.
• Biochemical assays to assess impacts on enzymatic activity and protein interactions.

Main Results:

• Successfully incorporated succinylation and glutarylation at specific sites in various proteins.
• Demonstrated the ability to modify non-refoldable multidomain proteins.
• Enabled the study of how these PTMs affect metabolic enzyme activity and protein interactions.

Conclusions:

• The developed method overcomes previous limitations in studying negatively charged lysine acylations.
• This approach facilitates the investigation of succinylation and glutarylation roles in diverse cellular functions, from replication to signaling.
• Provides a valuable tool for understanding the impact of PTMs on protein structure and function.