Sfm 300

The setup of stopped-flow spectrophotometry was done as per a previous report [12 (link)]. In brief, liposomes or proteo-liposomes were measured at 436 nm in a stopped-flow spectrophotometer (SFM 300, Bio-Logic SAS, Claix, France). Sample suspensions were quickly mixed with equal volumes of hyperosmotic solution (assay buffer A with 400 mM sucrose). Data obtained from the spectrophotometer was fitted into an exponential rise equation; the initial shrinkage rate (k) was determined by the fitted curve of 6–10 measurements. The water permeability factor (P_f) of the vesicle samples was calculated using the equation described previously [9 (link),12 (link),19 (link)]: $P_f=\frac{k}{(S/V_0)\times V_w\times {∆}_{osm}}$
where S/V₀ is the vesicle’s initial surface-to-volume ratio, V_w is the partial molar volume of water (18 cm³/mol), and ${∆}_{osm}$ is the osmotic driving force. The S/V₀ was calculated by determining the diameters of the proteo-liposomes using dynamic light scattering (ZetaPlus particle sizing software 2.27). The ${∆}_{osm}$ was 200 mM in this case.

Stopped-flow measurements were carried out in a BioLogic SFM 300 instrument. Post-mixing concentrations are stated. Trp fluorescence was detected through a 320-nm cutoff filter at 280-nm excitation with a 4-nm bandwidth. P_i generation from ATP was followed using MDCC-PBP. The setup for MDCC-PBP calibration and single-round translocation experiments were as described earlier24 (link)25 (link)30 (link).

Unless otherwise stated, all measurements were carried out in SF50 buffer at 25°C. Stopped-flow experiments were carried out in KinTek SF-2004 and BioLogic SFM 300 apparatuses. Quenched-flow experiments were performed in a KinTek RQF-3 instrument. Post-mixing concentrations are stated in all experiments. In experiments requiring nucleotide-free RecQ, nucleotide contamination was removed by pre-incubation with 0.02-U/ml apyrase for 15 min at 25°C. P_i liberation from ATP was followed using a fluorescently labeled P_i binding protein (MDCC-PBP) (12 (link)). MDCC-PBP calibration was performed as described earlier (13 (link),14 (link)). mdATP and mdADP were excited at 280 nm and fluorescence emission was detected through a 420-nm long-pass filter utilizing FRET (Förster Resonance Energy Transfer) from aromatic residues of RecQ.
Steady-state ATPase measurements were carried out in SF50 buffer plus 50-μg/ml bovine serum albumin using a pyruvate kinase-lactate dehydrogenase (PK-LDH) linked assay (14-U/ml PK, 20-U/ml LDH, 1-mM ATP, 1-mM phosphoenol pyruvate, 200-μM NADH (nicotinamide adenine dinucleotide, reduced form)). Time courses of NADH absorbance (ϵ_{340 nm} = 6220 M⁻¹cm⁻¹) were followed in a Shimadzu UV-2101PC spectrophotometer.

Rapid mixing experiments were conducted with a thermostated stopped flow apparatus (BioLogic SFM-300). Kinetics of carbon monoxide (CO) binding to determine the
k
_on CO were measured on the deoxy state of mutant and wild type globins at 20ºC. Solutions containing 5 μM protein in a 100 mM sodium phosphate at pH 7.0 were degassed in a nitrogen atmosphere and reduced with an equimolar concentration of sodium dithionite and mixed with increasing CO concentrations. The observed pseudo first-order rate constant (
k
_obs) was determined by fitting the absorbance decay resulting from association of the protein with CO, to a single exponential function. Kinetic rate constants (
k
_on CO) were obtained from the slope of the plots of
k
_obs as a function of CO concentration.

The folding and unfolding kinetics of the CCL11 and CCL24 in guanidine hydrochloride solution were characterized on a stopped-flow system (Bio-Logic SFM300, France). The concentration of CCL11 and CCL24 was set as 100 μM in phosphate buffer (20 mM phosphate, pH 7.0). The intrinsic fluorescence signal of CCL11 and CCL24 was recorded at 320 nm with the excitation wavelength as 280 nm at 25 °C. The dead time of instrument was determined as 3.4 ms. Folding process of CCL11 and CCL24 was initiated by mixing the denatured protein in high concentration of guanidine hydrochloride with corresponding buffer without denaturant. Unfolding process of CCL11 and CCL24 was initiated by mixing the native protein with different concentration of guanidine hydrochloride. The folding and unfolding kinetics traces at different concentration of guanidine hydrochloride were repeated for at least three times and then fitted using an exponential function.