Measurements were conducted using an Applied Photophysics stopped-flow apparatus. Reactions were initiated by mixing equal volumes of 500 nM TbQSOX D-147 and various DTT concentrations at 25 °C. Donor fluorescence was filtered by a Semrock FF01-536/40 filter and monitored as readout of conformational change. Five to ten traces were collected for 0.5 s (using a split-time base of 0.05 and 0.5 s with 1,000 sampling points in each time interval) for each DTT concentration. Rate constants obtained from fitting the traces to a single exponential were averaged. Traces were collected in the absence of DTT as well, to verify that the observed decrease in donor fluorescence was due to TbQSOX activity.
Stopped flow apparatus
Manufactured by Applied Photophysics
Sourced in United Kingdom
The Stopped-flow apparatus is a laboratory instrument used for rapid mixing and analysis of chemical or biological reactions. It allows for the study of fast kinetic processes by rapidly mixing small volumes of reagents and monitoring the resulting changes in the sample over time.
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Lab products found in correlation
Pro k software, by Applied Photophysics (2 mentions)
Mantatp, by Jena Biosciences (1 mentions)
Cary 4000 spectrophotometer, by Agilent Technologies (1 mentions)
V 660, by Jasco (1 mentions)
Tablecurve 2d, by Grafiti LLC (1 mentions)
Prism 5, by GraphPad (1 mentions)
Fluoromax 4 spectrophotometer, by Horiba (1 mentions)
20 protocols using stopped flow apparatus
1
Rapid Kinetic Analysis of TbQSOX
2
Kinetics of gp32 Binding to DNA
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Kinetics of gp32-F and gp32-A-F binding to DNA and dissociation were measured on an Applied Photophysics stopped-flow apparatus equipped with a fluorescence detector. For the experiments involving gp32 proteins binding to DNA loading, the reaction was initiated by mixing the ssM13 DNA (200 nM) with gp32-A-F (1 μM), and the kinetics was monitored by signal change (λem > 515 nm) upon excitation at 480 nm. For the kinetics of gp32-F dissociation from DNA, the reaction was initiated by mixing a preorganized complex of gp32-F●DNA with the appropriate enzyme or enzyme complex, and the kinetics being performed as previously stated.
3
Kinetic Analysis of α1-Antitrypsin Variants
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Kinetic parameters of GSK716 binding to M, Z, S (Glu264Val) and Baghdad (Ala336Pro) (Haq et al, 2016 ) α1‐antitrypsin were measured by detecting intrinsic tryptophan fluorescence of the protein (excitation at 280 nm and detection of emission at 320 nm) on a stopped flow apparatus (Applied Photophysics) (Kim & Yu, 1996 ; Dafforn et al, 1999 ). A competition assay for binding to M α1‐antitrypsin and Z α1‐antitrypsin and Z α1‐antitrypsin polymers (Lomas et al, 1993 ; Irving et al, 2011 ; Haq et al, 2013 ), based on an Alexa488‐labelled analogue of GSK716 (A488‐GSK716), was used to determine the binding affinity of test compounds.
4
Oxygen Dissociation Kinetics of Microcephalus Hemoglobins
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S. microcephalus Hb 1, Hb 2 and Hb 3 were adjusted to 20 μM in a solution containing 0.1 M Tris-HCl, 0.5 mM EDTA, 0.1 M KCl at pH 6.5, in the absence or presence of 2 mM ATP. The solutions were then fluxed with pure nitrogen for 5 minutes to remove unbound O2. The samples were then loaded on a stopped-flow apparatus (Applied Photophysics) and mixed with the same CO-equilibrated solution. The displacement of O2 by CO was monitored at 420 nm and the time courses were analysed as mono-exponential decays. The value of koff,R (O2 dissociation constant of Hb in the R state) is estimated as the reciprocal of the lifetime in the exponential decay. Considering that Hbs at the beginning of the displacement reaction were fully oxygenated, the resulting apparent koffs are referred to the R state. All experiments were carried out at 25°C. For each condition, at least two independent experiments were carried out.
5
Measuring Myosin's Conformational States
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The fluorescence of 3’-O-(N-Methyl-anthraniloyl)-2’-deoxyadenosine-5’-triphosphate (mantATP) (Jena Biosciences) was measured with 290 nm excitation and a 395 nm long-pass emission filter in a stopped-flow apparatus (Applied Photophysics) at 25°C. Each myosin sample was incubated on ice for about 10 minutes in MOPS 20 buffer at varying KCl concentrations (20 – 150mM) prior to each experiment. Single mantATP turnover reactions were performed by incubating 0.25μM myosin constructs (S1, 2HEP, 15HEP, 25HEP) with 1μM mantATP for 30 seconds at room temperature. Subsequently, the complex was mixed with saturating ATP (2mM) in the stopped flow. Fluorescence decays were examined over a 1000 second period and were fitted to a two-exponential function to determine the SRX and DRX rate constants (Suppl. Fig. 1 ). Relative amplitudes of the fast and slow phase rate constants determined the fraction of myosin in the DRX and SRX states, respectively.
6
Actin-Activated ATPase Kinetics Assay
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We utilized the NADH-coupled ATPase assay to examine the actin-activated ATPase activity at a range of actin concentrations (0 – 60 μM). Briefly, each construct (S1, 2HEP, 15HEP, and 25HEP) at 0.1 μM, was assayed in the presence of 1 mM ATP, and the NADH absorbance was monitored for a 200 second period in the stopped-flow apparatus (Applied Photophysics) at 25°C in MOPS 20 buffer (10 mM MOPS, pH 7.0, 20 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 1 mM DTT). For the 0 μM actin condition, 1.0 μM of construct was used to improve signal to noise. The linear fit of the NADH absorbance as function of time was converted to the ADP produced per unit time using a standard curve with known ADP concentrations. The ATPase was expressed as the amount of product produced per second per molar concentration of myosin. The ATPase data plotted as a function of actin concentration was fit to the Michaelis-Menten equation to determine the maximal ATPase activity (kcat) and actin concentration at which ATPase is one-half maximal (KATPase).
7
Measuring COPY^o2-Tubulin Association and Dissociation
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Association and dissociation of COPYo2 and tubulin were measured in a stopped-flow apparatus (Applied Photophysics, Leatherhead, U.K.) with excitation at 535 nm and a 670/30 nm band-pass filter to monitor sensitized emission changes at 25 °C in 80 mM PIPES pH 6.8, 75 mM KCl, 2 mM MgCl2, 20 mM β-mercaptoethanol, 60 µM GTP. COPYo2 or phosphorylated COPYo2 (see “Time-resolved measurements of AuroraB and λ-PPase activity”) was kept constant at 200 nM for the tubulin titration. Dissociation was measured at 200 nM COPYo2 and 4 µM tubulin, displacing COPYo2 with 60 µM unlabeled stathmin. Data were fit in Origin (OriginLab), and error propagation for derived constants was calculated by Gaussian equation taking the errors of the fit as a starting point.
8
Actin-Activated ATPase Assay of M2β-S1
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An Applied Photophysics stopped-flow apparatus was used to perform NADH-coupled ATPase assays to measure steady-state actin-activated ATPase of the human M2β-S1 constructs (De La Cruz and Ostap, 2009 (link); Swenson et al., 2017 (link)). Briefly, 0.1 µM M2β-S1 was assayed in the presence of varying concentrations of F-actin (0, 5, 10, 20, 40, and 60 µM). After the addition of 1 mM ATP, changes in absorption at 340 nm were measured for 200 s, and the transients were fit to a linear function. A standard curve of known ADP concentrations was performed to determine the ATPase rate at each actin concentration, and the data were fit to a Michaelis–Menten relationship to determine the ATPase parameters.
9
Monitoring Ribosome-SRP Recruitment During Translation
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Recruitment of SRP to translating ribosomes was monitored at 37°C in a stopped-flow apparatus (Applied Photophysics) by exciting the donor fluorophore MDCC at 410 nm and recording fluorescence emission after passing through a KV 530 long-pass filter (Schott). Translation elongation was induced by rapid mixing of MDCC-labeled initiation complex (25 μM, final concentration after mixing) with a solution containing EF-Tu (15 μM), EF-G (2 μM), EF-Ts (0.1 μM), total aminoacyl-tRNA, and Bpy-labeled SRP. Leu-tRNALeu in the total aminoacyl-tRNA was 14C-labeled and present at 1.35 μM in translation reactions. This provided a 3-fold excess of Leu-tRNALeu over leucine codons in the longest mRNA, Lep75 mRNA, which encodes nine leucines. All solutions contained GTP (1 mM), PEP (3 mM), and pyruvate kinase (0.1 μg/ml). Between seven and nine replicates of each experiment were carried out for 500 s.
10
Reducing and CO-binding Cytochrome c Oxidase
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The SMPs were dissolved in the sucrose buffer described above to a concentration of ~3.8 mg ml-1. The sample was loaded into a Thunberg cuvette and air was exchanged for nitrogen. The sample was then reduced with 4 mM ascorbate and 1 µM PMS. After the sample was fully reduced, nitrogen was exchanged for CO. The absorbance changes associated with reduction and binding of CO to CytcO were monitored using a Cary 4000 spectrophotometer (Agilent).
The flow-flash measurements were performed essentially as described previously57 (link),58 (link) using a laser flash-photolysis system combined with a stopped-flow apparatus (Applied Photophysics). The mixing ratio of sample and saturated oxygen buffer was 1:1 and the cuvette path length was 1.00 cm. The delay time between mixing and laser flash was 0.8 s. The oxygen-containing buffer consisted of the sucrose buffer used to dissolve SMPs and it also contained either only ATP or ATP, 200 nM FCCP and 10 µM valinomycin. The ATP concentration after mixing was 25 mM.
The flow-flash measurements were performed essentially as described previously57 (link),58 (link) using a laser flash-photolysis system combined with a stopped-flow apparatus (Applied Photophysics). The mixing ratio of sample and saturated oxygen buffer was 1:1 and the cuvette path length was 1.00 cm. The delay time between mixing and laser flash was 0.8 s. The oxygen-containing buffer consisted of the sucrose buffer used to dissolve SMPs and it also contained either only ATP or ATP, 200 nM FCCP and 10 µM valinomycin. The ATP concentration after mixing was 25 mM.
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