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

ABSTRACT: Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either

SUBMITTER: Chattopadhyay G 

PROVIDER: S-EPMC9793979 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature