Figure 2: Biochemical properties of TTN5 proteins suggest that TTN5 is present in a GTP-loaded active form in cells.

(A), Schematic illustration of the stopped-flow fluorescence device for monitoring the nucleotide-binding kinetics of the purified TTN5 protein heterologously expressed in bacteria (Supplementary Figure S2A–D). It consists of two motorized, thermostated syringes, a mixing chamber and a fluorescence detector. Two different reagents 1 and 2 are rapidly mixed and transferred to a fluorescence detection cell within 4 ms. One of the reagents must contain a fluorescent reporter group. Here, mdGDP and mGppNHp were used to mimic GDP and GTP. (B), Schematic illustration of the nucleotide association. Nucleotide-free TTN5 (reagent 1; preparation see Supplementary Figure S2E) was rapidly mixed with mdGDP (reagent 2). A fluorescence increase is expected upon association of mdGDP with TTN5. Similar measurements are performed with mGppNHp instead of mdGDP. (C), Schematic illustration of the intrinsic nucleotide dissociation. mdGDP-bound TTN5 (reagent 1) is mixed with a molar excess of GDP (reagent 2). A fluorescence decrease is expected upon mdGDP dissociation from TTN5 and binding of free GDP. Similar measurements are performed with mGppNHp. (D-E), Kinetics of association and dissociation of fluorescent nucleotides mdGDP (D) or mGppNHp (E) with TTN5 proteins (WT, TTN5^T30N, TTN5^Q70L) are illustrated as bar charts. The association of mdGDP (0.1 μM) or mGppNHp (0.1 μM) with increasing concentration of TTN5^WT, TTN5^T30N and TTN5^Q70L was measured using a stopped-flow device (see A, B; data see Supplementary Figure S3A–F, S4A–E). Association rate constants (k_on in μM⁻¹s⁻¹) were determined from the plot of increasing observed rate constants (k_obs in s⁻¹) against the corresponding concentrations of the TTN5 proteins. Intrinsic dissociation rates (k_off in s⁻¹) were determined by rapidly mixing 0.1 μM mdGDP-bound or mGppNHp-bound TTN5 proteins with the excess amount of unlabeled GDP (see A, C, data see Supplementary Figure S3G–I, S4F–H). The nucleotide affinity (dissociation constant or K_d in μM) of the corresponding TTN5 proteins was calculated by dividing k_off by k_on. When mixing mGppNHp with nucleotide-free TTN5^T30N, no binding was observed (n.b.o.) under these experimental conditions. (F-G), GTP hydrolysis of TTN5 proteins determined by HPLC. (F), Schematic illustration of the GTP hydrolysis measurement. (G), GTP-bound TTN5 proteins (100 μM) were incubated at room temperature at different time points before injecting them on a reversed-phase HPLC system. Evaluated data (data see Supplementary Figure S5) resulted in the determination of the GTP hydrolysis rates (k_cat in s⁻¹) illustrated as bar charts. (H), TTN5 accumulated in a GTP-loaded active form. GST-TTN5^WT (46.5 kDa) was purified from bacterial cell lysates at three different volumes in the presence of 0.1 μM unbound free GppNHp using glutathione beads. The nucleotide contents and the protein purities were determined by HPLC and Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis. The presence of much higher amounts of GppNHp-bound versus GDP-bound GST-TTN5 protein indicates that TTN5 rapidly exchanged bound nucleotide und accumulated in this state.