Fig. 1

Nanobody-mediated shift in heterotrimeric G_t equilibrium. a Effect of Nb5 on the heterotrimeric state of G_t in solution. Lane 1 shows the dissociation of heterotrimeric G_t in the presence of light-activated Rh (Rh*), GTP, and Nb5. Gβ₁γ₁ was later co-purified with Nb5 by immobilized-Ni²⁺ affinity chromatography and Gα_t emerged as an unbound protein in the flow-through (lane 2). Note the decrease in Gγ₁ Coomassie staining in the presence of 300 mM imidazole during protein elution. Similar results were obtained from the reaction mixture in the absence of either Rh* alone (lanes 3 and 4) or both Rh* and GTP (lanes 5 and 6). However, Nb17 did not affect the heterotrimeric configuration of G_t (lanes 7 and 8, compare lanes marked with asterisks). Additionally, non-specific interactions between Gβ₁γ₁ and Ni²⁺-NTA resin were not seen (lanes 9 and 10). The eluate and flow-through obtained from affinity chromatography purification are denoted as E and FT, respectively. b Proteins in typical eluates obtained after immobilized-Ni²⁺ affinity chromatography were analyzed by SDS-PAGE (lane 1) and immunoblotting with antibodies specific to Gβ₁, His-tag, and Gγ₁, respectively (lanes 2–4). c Light activation of ROS membranes in the presence of GTP released Gα_t in the high salt wash (lane 1). Similar Gα_t release was obtained with Nb5 with or without GTP during the high salt wash (lanes 2 and 3). Treatment of ROS membranes with Nb17 had no effect on Gα_t release (lane 4, compare lanes marked with asterisks). Untreated ROS membranes showed no Gα_t release during the high salt wash (lane 5). d The initial G_t activation rate of Rh* was reduced upon pre-treatment of heterotrimeric G_t with Nb5 (greencyan) as compared to either untreated (red) or Nb17-treated G_t (purple). e Quantification of the initial G_t activation rates of Rh* with and without nanobody pre-treatment. Results are expressed as the mean ± SD, n = 3 replicates, ****P < 0.0001, Student’s t-test. f The decrease in G_t activation rates after Nb5 treatment were explained by Rh single turnover experiments that exhibited an exponential increase in G_t activation rates with increasing concentrations of G_t