Stopped Flow is your key to understanding the rapid kinetics of many biological processes, crucial in drug discovery for complex targets like GPCRs. By extreme fast mixing of the target protein with its ligand molecules and monitoring the ensuing reactions in milliseconds, Stopped Flow illuminates the critical early stages of interactions, providing invaluable mechanistic insights for drug development.
We run Stopped Flow on the preferred instrument of researches across the world: The SX20 from Applied Photophysics. Its outstanding sensitivity, opto-mechanical performance, and robustness together with 2bind’s customized solutions and expert data interpretation, ensures high-quality data for delivering actionable results.
Stopped Flow operates by swiftly mixing two or more reactants, initiating a reaction whose progress is immediately monitored by means of a change in an optical signal (e.g. absorbance, fluorescence). For fast, millisecond to second timescale reactions, the course of reactions is continuously observed, making it possible to elucidate the details of the reaction mechanism that are otherwise invisible with slower techniques.
The SX20 from Applied Photophysics elevates Stopped Flow with advanced features like:
This combination of speed, sensitivity, and versatility makes Stopped Flow an indispensable tool in drug discovery, where understanding drug-target binding interactions is paramount. Stopped-flow based platforms have been used to develop therapeutic agents as inhibitors of receptors, such as GPCRs or receptor tyrosine kinases, and various enzymes, such as protein kinases and methyl transferases.
Separate and analyze individual reaction steps
Determine rate constants for substrate or product association and dissociation (kon, koff)
Infer dynamics of conformational state changes
Understand reaction kinetics of individual chemical intermediates (e.g. state of cofactors during multistep reaction mechanisms)
Derive binding affinities (KD values) and catalytic efficiencies (kcat/KM) by fitting stopped-flow data to kinetic models
Investigate interactions with many types of ligands (small molecules, oligonucleotides, peptides, proteins) on a millisecond time scale
Measure rates of ligand association and dissociation (kon, koff)
Determine the potency of inhibitors (IC50) and distinguish the type of inhibition
Evaluate the structural integrity and oligomeric state of proteins and ligands (via Chevron plots)
Drug binding mechanisms: How drugs interact with their targets.
Lead optimization: Identifying and improving potential drug candidates.
Signal Proteins: Elucidate how drugs affect fast changes in signaling pathway proteins, e.g. GPCRs.
Fast Enzyme kinetics: Understanding how enzymes catalyze reactions.
A stopped flow apparatus consists of two or more loading syringes containing separate reactants (e. g. protein and ligand) and a stop syringe that rapidly stops the flow of the reactants in an observation cell. Solutions are driven from the syringes to a high efficiency mixer just before passing into a measurement flow cell. As the solutions flow through, a steady state equilibrium is established and the resultant solution is only a few milliseconds old as it passes through the observation cell. The mixed solution then passes into a stopping syringe where the flow of mixed solution can be instantaneously stopped. A fraction of the solution rests in the observation cell and as the reaction of the two solutions proceeds, the kinetics can be continuously monitored using an optical signal change, e.g. in absorbance, fluorescence, static light scattering. Our instrumentation has a dead time in the 1 ms range allowing for determination of rate constants kon ≤ 107 M-1 s-1 and koff ≥ 0.01 s-1.
The setup of the experiment depends on the specific question. For simple cases of protein-ligand interactions, the kinetics of the binding interaction between two reactants is measured directly by the difference in optical signature between the free and bound forms of the reactants. In a one-step binding interaction where one reactant (here the nonfluorescent ligand) is in large excess, this interaction follows a pseudo-first order exponential change with the apparent rate constant kobs. Kinetic constants can be derived by observing the change in kobs that occurs as the concentration of one of the reactants is varied. Plotting the kobs versus reactant concentration will yield a straight line with the slope indicating the kon and the y-intercept representing the koff. A hyperbolic line in the secondary plot would indicate a 2-step kinetic mechanism such as association before or after an isomerization step. More complex binding mechanisms result in multiphase time traces and complex concentration dependencies and require analysis by global fitting methods.
Data acquisition in the stopped flow experiment is based on the change in an optical signal (absorption, fluorescence, static light scattering). Ideally, the ligands of the reaction contain chromophores or the intrinsic tryptophan fluorescence of the protein binding partner changes upon ligand binding. It is also possible to generate an optical signal by site-specific labeling of a fluorescent dye onto a protein, oligonucleotide or antibody. A special application is the use of fluorescent competitive ligands as a method to analyze the interaction with non-fluorescent ligands indirectly. If the change in the optical signal is large enough, Stopped flow can be used to study reactions in biological systems ranging in complexity from whole organisms to purified proteins.
Reactions are carried out using the SX20 fitted with a 20 µl optical cell and drive volumes of ~ 100 µl. Normally, 4 to 8 time traces are averaged to achieve an adequate signal/noise ratio. Consequently, to fill the flow circuit and obtain the averaged trace a minimum volume of ~ 800 µl is required for each time trace measured. For the investigation of ligand binding kinetics, pseudo-first order conditions are often applied, whereby the reactant that is not the optical signal transmitter is present in large excess (~ µM concentration). The required concentration of the signal-transmitting reactant is usually in the nM-µM range, depending on its optical signature.
The sequential (or double) mixing mode of the Stop Flow is particularly suitable to study the reactivity of intermediate and transient species. The sequential mixing configuration equips the instrument with four syringes and two drive rams. The first drive mixes two reagents (A and B) into an aging loop and, after a user defined aging period (within the range of 14 ms to 1000 s), a second drive mixes the aged solution with a third reagent (C) in the stopped-flow cell.