Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics.

Area of Science:

• Protein-protein interactions
• Molecular biology
• Biochemistry

Background:

• Protein complexes are crucial for biological regulation but challenging to study.
• The bacterial toxin CcdB interacts with DNA Gyrase, and its antitoxin CcdA releases CcdB via a ternary complex.

Purpose of the Study:

• To introduce Ter-Seq, a high-throughput method for stabilizing and characterizing transient protein ternary complexes.
• To investigate the kinetics of the CcdA-CcdB-GyrA14 ternary complex using this new methodology.

Main Methods:

• Yeast surface display (YSD) with saturation mutagenesis of CcdB to screen for enhanced ternary complex formation.
• Surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) for kinetic validation of identified mutants.

Main Results:

• Ter-Seq successfully identified CcdB mutants with altered ternary complex formation and rejuvenation kinetics.
• Mutations affecting CcdA binding sites slowed rejuvenation, while mutations at GyrA14-interacting sites enhanced it.
• Analysis revealed correlations between CcdA/GyrA14 affinity, surface accessibility, and evolutionary conservation with rejuvenation rates.

Conclusions:

• Ter-Seq is an effective high-throughput method for studying transient protein complexes and their kinetics.
• Understanding CcdA-CcdB-GyrA interactions provides insights into allosteric regulation and protein complex dynamics.
• The study highlights the utility of Ter-Seq for dissecting mechanisms of protein complex formation and regulation.