Felixgx software

For multiple-turnover assays with either GTP or GMPPNP (Fig. 3, E and F), 2 µM cyt-DATL labeled with Cy3 as described above was mixed with 1 mM nucleotide (final concentrations after mixing) in SEC buffer at 25°C using a stopped-flow accessory mounted on a PTI QuantaMaster-400 fluorimeter (Horiba Instruments Inc.), and 570 nm fluorescence was monitored at 100-ms intervals after 540-nm excitation. Data were acquired using the FelixGX software (Horiba Instruments Inc.) and normalized using the equation (Fluorescence – Minimum fluorescence observed)/(Maximum fluorescence observed – Minimum fluorescence observed), where fluorescence is F/Fo. Single-turnover PIFE assays (Figs. 1 B, 4 B, and 5 A) were essentially the same as above except that the final concentrations after mixing were 15 µM cyt-DATL (or cyt-hATL1) and 7.5 µM GTP in SEC buffer, and data were acquired at 50-ms intervals. Data were plotted either as F/Fo or as normalized F/Fo using the equation (Fluorescence – Initial fluorescence)/(Maximum fluorescence observed – Initial fluorescence). All data shown are the average of three to five runs per condition. All data analysis was in Microsoft Excel. Each of the three to five PIFE traces was nearly identical to the others, and each result was reproduced with at least two independent protein preparations.

For GMPPNP PIFE kinetics (Fig. 3), cytoDATL labeled with Cy3 was mixed with 1 mM nucleotide (final) using a stopped flow accessory mounted on a PTI QuantaMaster-400 fluorometer (Horiba Instruments Inc.) and 570-nm fluorescence was monitored at 1-s intervals after 540-nm excitation. Data were acquired using the FelixGX software (Horiba Instruments Inc.) and normalized using the following equation: (fluorescence − minimum fluorescence observed)/(maximum fluorescence observed − minimum fluorescence observed). All data shown represent the mean of three runs per mutant variant. PIFE data in Fig. 1 (B, C, and E) were captured using a Tecan M1000 (Tecan Group Ltd.), and either a single representative trace (Fig. 1, B and C) or the mean of three runs (Fig. 1 E) is shown. For GTP kinetics (Fig. 1 D and Fig. 2, C and D), cytoDATL labeled with Cy3 was mixed with GTP in a stopped flow device (Applied Photophysics SX20). Cy3 was excited at 540 nm, and the resulting change in fluorescence emission was observed with a 560-nm long-pass filter at 2.5-ms intervals. Plotted data were the mean of seven runs per mutant variant. Data were normalized and all PIFE data were replicated with similar results from at least two independent protein preps. All PIFE assays were performed at 25°C in SEC buffer with 2 mM β-mercaptoethanol. All data analysis was performed in Microsoft Excel.

Static light scattering measurements as a function of temperature were made in triplicate using a dual emission PTI QM-40 Spectrofluorometer (Horiba Scientific Northampton, UK) equipped with a 4-position cell holder Peltier temperature control device, a high-power continuous 75 W short-arc Xe lamp (Ushio), and a Hamamatsu R1527 photomultiplier tube. Data were collected using FelixGX software (Horiba Scientific) in 10 mm path length quartz cuvettes. RBD samples at 0.2 mg/mL were examined as a function of temperature (10°C-90°C) using an excitation wavelength of 295 nm. Static light scattering signal at 295 nm was collected at 1.25°C interval with a 2 min equilibration at each temperature. The light scattering signal of the buffer was subtracted from all sample measurements and the light scattering intensity at 295 nm was plotted at a function of temperature.