[Functional reconstitution of the ethylene receptor ETR1-GAF and characterization of its binding with the inhibitory peptide NOP-1].

Ethylene, a pivotal gaseous plant hormone regulating plant growth and environmental responses, initiates its signaling through receptor proteins on the membrane. Ethylene response 1 (ETR1) is a key ethylene receptor in plants, and its GAF domain plays a central role in mediating the interaction with the downstream signaling protein ethylene insensitive 2 (EIN2). The research on the interactions between ethylene receptors and downstream components still lacks high-throughput and rapid detection systems, which limits in-depth understanding of the recognition mechanisms. In this study, we utilized the Pichia pastoris expression system to express and purify the transmembrane and GAF domains of ETR1. Membrane proteins were solubilized in the detergent Fos-14 and successfully purified through a two-step strategy involving the Anti-Flag antibody and Ni-NTA affinity chromatography, which yielded high-purity ETR1-GAF protein with structural integrity. Circular dichroism spectroscopy revealed that ETR1-GAF adopted a predominantly α-helical conformation in solution. Using bio-layer interferometry, we conducted kinetic binding assays between ETR1-GAF and the inhibitory peptide NOP-1 at various concentrations. The results revealed a high-affinity, concentration-dependent specific interaction, with a dissociation constant KD=(6.885×10-5±7.944×10-6) mol/L, R2=0.991 4. This study establishes a stable and quantifiable in vitro interaction analysis system for ethylene receptors, providing a crucial tool for molecular-level investigation into the dynamic recognition mechanisms between receptors and downstream elements in ethylene signaling. Moreover, it lays a methodological foundation for further exploration of the ethylene signaling network and the development of novel regulatory strategies.