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Investigate the interactions with many types ligands on a milli-second time scale

Protein-Ligand Interaction Kinetics

The kinetics of protein ligand interactions often play an important role in biological processes. We can use Stopped-Flow spectroscopy to measure association and dissociation rate constants which help us understand the mechanisms of action of many different types of protein-ligand interactions.

In order to measure these interactions, we need to observe a spectroscopic signal change. Sometimes ligands will contain a chromophore whose signal will change over the course of the interaction or we can see a change in the intrinsic fluorescence of the protein from tryptophan residues.

In cases where we do not have a naturally occurring signal, it is common to study protein-ligand interactions by labelling the ligand or the protein with an external fluorescent probe. For example, oligonucleotides can be labelled with various dies which change signal on interacting with a protein due to the change in environment. Another approach is to use a fluorescent competitive ligand as a way to indirectly measure the interaction with our non fluorescent ligand.

We can also design and perform sophisticated FRET experiments that give information about the distance relationships between protein and ligand. Here we need to choose a donor-acceptor pair and choose corresponding optical filters to observe the two signals simultaneously.

Typically we wish to measure the binding kinetics at various concentrations of ligand in pseudo-first order conditions whereby the concentration of one of the components is significantly greater than the other. We can then analyse the data to obtain the rate of association and or dissociation.



Showing simultaneously acquired kinetic fluorescence signals at 2 wavelengths of a ligand 8-anilino-1-napthalene sulphonate (ANS) to the protein, bovine serum albumin (BSA)
SX20 FP Teaser

Stopped-Flow Fluorescence Polarisation/Anisotropy

Stopped-flow fluorescence polarisation/anisotropy is a highly useful technique for obtaining valuable kinetic information about biomolecular interactions and structural changes of biomolecules. An example from the literature illustrates how the SX20 Fluorescence Polarisation accessory is used to gain insights into the kinetics of the interaction between a helicase and its RNA binding partner.

SX20 FRET Teaser

Stopped-Flow Fluorescence (Förster) Resonance Energy Transfer (FRET)

Stopped-flow FRET is a useful technique for studying many types of biomolecular interactions. The method provides kinetic information regarding distance changes occurring on the millisecond timescale. This application note gives an introduction to the technique along with a review of some application experiments from the scientific literature.

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