Chattopadhyay, G and Ahmed, S and Srilatha, NS and Asok, A and Varadarajan, R (2023) Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics. In: Protein Science, 32 (1).
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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 resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.
| Item Type: | Journal Article |
|---|---|
| Publication: | Protein Science |
| Publisher: | John Wiley and Sons Inc |
| Additional Information: | The copyright for this article belongs to the Authors. |
| Keywords: | allosterism; article; complex formation; fluorescence resonance energy transfer; genetic conservation; high throughput technology; kinetics; molecular evolution; nonhuman; protein engineering; rejuvenation; saturation mutagenesis; surface plasmon resonance; toxin-antitoxin system; yeast; chemistry; Escherichia coli; genetics; kinetics; mutation, bacterial protein; DNA topoisomerase (ATP hydrolysing), Bacterial Proteins; DNA Gyrase; Escherichia coli; Kinetics; Mutation |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit |
| Date Deposited: | 31 Jan 2023 07:13 |
| Last Modified: | 31 Jan 2023 07:13 |
| URI: | https://eprints.iisc.ac.in/id/eprint/79624 |
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