Mant gdp

Biochemical and molecular biological procedures were performed using commercially available enzymes, chemicals, and other materials. GTP, GDP, and guanosine 5′-(β,γ-imido)triphosphate (GMPPNP) were purchased from Sigma-Aldrich (USA). [2′-/3′-O-(N-methylanthraniloyl)guanosine-5′-O-triphosphate] (Mant-GTP) and [2′-/3′-O-(N-methylanthraniloyl)guanosine-5′-O-diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany). [α-³²P]GTP (800 Ci/mmol) and [¹⁴C] l-cystine (74 GBq/mmol) were purchased from Perkin Elmer (USA).

To obtain prenylated Rab-chaperone complexes the prenylation reaction was performed as described previously (Langemeyer et al., 2018b (link); Thomas and Fromme, 2016 (link)). Rab-GTPases were preloaded with either GDP (Sigma Aldrich, Germany) or MANT-GDP (Jena Bioscience, Germany). For Drosophila or yeast Rab-GTPases in complex with GDI, the respective purified GDI from the same organism was used. To complex Rab-GTPases with REP, the yeast REP was used.

Nucleotide binding kinetics were determined by fluorescence stopped-flow (SX18.MV; Applied Photophysics). All measurements were performed at 20°C in 50 mM tris (pH 7.5), 2 mM MgCl₂, 40 mM NH₄Cl, and 2 mM β-mercaptoethanol. EF-Tu or pEF-Tu_T382 at a concentration of 1 μM was rapidly mixed with a concentration range (5 to 25 μM) of MANT-labeled nucleotides (MANT-GDP and MANT-GTPγS; Jena Bioscience). MANT-labeled nucleotides were excited indirectly at 280 nm using resonance energy transfer (FRET) between tryptophan and the MANT-labeled nucleotide, and the change in fluorescence was monitored through a 405-nm cutoff filter. For each nucleotide concentration, the data of at least five time traces were averaged and fitted to a single exponential function, yielding the observed rate constant k_obs. The association rate constant k_on was obtained from the slope of the linear fit plotting the k_obs versus the nucleotide concentration. The dissociation rate constant k_off was obtained by mixing 200 μM unlabeled GDP with a mixture of 0.4 μM protein and 1.5 μM MANT-labeled nucleotide. The resulting time traces were fitted to a single exponential function. All measurements were performed as three independent repeats, and k_on and k_off values are given as means ± SD. The K_d values were calculated from the ratio of k_off and k_on.

The fluorescent nucleotide analog 2′/3′-O-(N-methyl-anthraniloyl)-guanosine-5′-diphosphate (Mant-GDP) (51 (link)) was obtained from Jena biosciences. Fluorescence emission spectra were recorded on Horiba FluoroMax^® 4 spectrophotometer (Jobin Yvon) at 25°C in a 10 × 10 mm quartz cuvette. The excitation wavelength was set at 360 nm and single point intensities were measured at 440 nm (I₄₄₀). For the fluorescence measurements, 0.5 μM protein in buffer (50 mM KCl, 10 mM Tris–Cl pH 8, 5 mM MgCl₂ and 1 mM DTT) was added to the cuvette and a blank spectrum was taken. Mant-GDP was added to the protein gradually with increasing concentration steps and I₄₄₀ were recorded for each concentration. Before each measurement, the protein was incubated with Mant-GDP for one minute prior to collection of the spectra. I₄₄₀ readings of Mant-GDP without protein were taken as control. The difference of I₄₄₀ in presence and absence of protein were plotted against Mant-GDP concentration.

First 10 nmol of hexahistidine-GST-Rab was loaded with 2′-(3′)-bis-O-(N-methylanthraniloyl)-GDP (Mant-GDP) (Jena Bioscience, Germany) in 20 mM HEPES, pH 6.8, 1 mg/ml BSA, 20 mM EDTA, pH 8.0, 40 mM Mant-GDP at 30°C for 30 min. After loading 25 mmol MgCl₂ was added and the sample was exchanged into reaction buffer (20 mM HEPES, pH 6.8, 1 mg/ml BSA, 150 mM NaCl, 1 mM MgCl₂) using Zeba spin columns (Fisher Scientific). This step removes the free Mant-GDP leaving only Rab bound nucleotide. Nucleotide exchange was then measured using 1 nmol of the loaded Rab and the amount of GEF specified in the figure legends in a final volume of 100 µl reaction buffer by monitoring the quenching of fluorescence after release of Mant-GDP using a Tristar LB 941 plate reader (Berthold Technologies, UK) under control of MikroWin Software. Samples were excited at 350 nm and emission monitored at 440 nm. GTP was added to a final concentration of 0.1 mM to start the exchange reaction at 30°C. Curve fitting and extraction of pseudo first order rate constants (k_obs) was carried out as described previously (Delprato et al., 2004 (link); Delprato and Lambright, 2007 (link)). Since k_obs = (k_cat/K_m)[GEF] + k_basal where k_basal is the rate constant measured in the absence of GEF, catalytic efficiency (k_cat/K_m) can be obtained.