Fig. 1
Detection of ephrinB2 binding to NiV G by SHG. a Crystal structure of the globular domain of NiV G bound to the globular domain of ephrinB2. NiV G conformational mutants are indicated in purple, and the dominant labeling site of NiV G is indicated in yellow. Epitopes for conformation-sensitive antibodies mAb45 and mAb213 are indicated in gray. b Prior to ligand binding, the SHG label representing protein conformational ensemble S1 has an average orientation angle θ 1 relative to the surface normal. Ligand binding elicits protein conformation ensemble S2 with an average label orientation θ 2. c The SHG signal depends on both the average label orientation θ and the width of the orientational distribution σ of the probe. Rearrangements in protein structure upon ligand binding that result in changes of either θ, σ, or both will be reflected in the SHG signal change. d Biodesy read plate setup. A NiV G ectodomain construct with an N-terminal His6-tag was oriented in a fixed manner via binding of the His6-tag to Ni-NTA groups on a supported lipid bilayer. The globular domains are represented by pink rectangles, and the stalk domain by dashed pink lines. e Δ SHG concentration-dependent dose-response of ephrinB2-Fc binding to NiV G. His6-tagged NiV G ectodomain construct was bound to Ni-NTA-containing supported lipid bilayer at 0.5 μM. EphrinB2-Fc was added to the indicated final concentrations. f Negative controls for SHG response in NiV G ectodomain constructs. NiV G was bound at 0.5 μM and ephrinB2 constructs and competitors were added at indicated amounts (μM). Mean and s.d. values for SHG data are shown from a representative experiment, n = 3
