Small molecule binding is measured by following a property of the target that changes when the compound binds. Flow Induced Dispersion Analysis (FIDA), an efficient, in-solution method for protein characterisation, measures it in free solution without needing the mass to change, which is the usual obstacle for small molecules. In one run FIDA reads the molecular size of the target, the intensity of its fluorescence, and, on a Fida Neo, the shift in the fluorophore's emission wavelength through Lambda Dynamics. Where none of those responds, a separate chemical unfolding measurement confirms or rules out the interaction.

Why is small molecule binding hard to measure?

The difficulty is the small mass change between the free and the bound state of the target. A compound that binds adds very little, which is an obstacle for every method that detects complex formation through a change in mass, including surface plasmon resonance (SPR), biolayer interferometry (BLI), mass photometry, analytical ultracentrifugation and microscale thermophoresis. Isothermal titration calorimetry (ITC) sidesteps the mass problem by measuring the heat of binding, but it consumes a large amount of sample. Surface methods add a further complication, because immobilising one partner can perturb the interaction. FIDA avoids both problems by measuring in free solution and by reading signals other than mass.

How does FIDA measure small molecule binding?

FIDA takes a titration in free solution and reads three responses from the same measurement, so one experiment gives three ways to see the binding.

The first is molecular size. Where the compound changes the shape of the target, that shows directly as a change in hydrodynamic radius, and the binding curve is built from size as in any FIDA binding affinity assay. The change is not always a simple increase, and its pattern can reveal how the compound reshapes the protein and how many sites it binds. Source: Fidabio, small molecule interactions with membrane proteins.

The second is fluorescence intensity, the Binding Related Intensity Change (BRIC), which follows the quenching or enhancement of the signal on binding and is fitted with a standard one to one model. This is the common case, where the compound causes little conformational change but the target's fluorescence still shifts. Source: Fidabio, small molecule interactions with membrane proteins.

The third, on a Fida Neo, is Lambda Dynamics, which detects a shift in the fluorophore's emission wavelength when binding changes its local environment, so it often sees binding even when the size does not move. In a worked example, carbonic anhydrase titrated with its inhibitor furosemide shows no change in hydrodynamic radius, yet the Lambda Dynamics ratio changes and the binding curve is read from it. Source: Fidabio, in-solution quantification of small molecule interactions with Lambda Dynamics.

What is Lambda Dynamics?

Lambda Dynamics is Fidabio's term for ratiometric fluorescence: the Fida Neo detector records fluorescence in two channels and plots their ratio, which moves when the fluorophore's emission wavelength shifts on binding. Because it is a ratio of two channels on the same sample, it does not depend on how much protein is in the well, whereas BRIC depends on total protein concentration and so on pipetting. The two are read side by side, since a change in one does not always come with a change in the other, and Lambda Dynamics is well suited to protein and small molecule interactions. Source: Fidabio, Enhanced FIDA analysis of small molecule protein interactions.

What if there is no change in size or fluorescence?

No change in size and no change in BRIC does not prove there is no interaction. A compound can bind at a site that neither reshapes the protein nor sits near a reporting fluorophore. Such an interaction still changes the energy of folding, which FIDA measures by unfolding the protein with urea or guanidinium hydrochloride, with and without the compound, and comparing the two. Fidabio's application note small molecule interactions with membrane proteins shows a worked example, a chemical unfolding assay in which a compound invisible to both size and fluorescence still shifted the protein's stability and confirmed binding. Read the full assay and its results in the application note. Source: Fidabio, small molecule interactions with membrane proteins.

How does FIDA compare with SPR and ITC for small molecules?

FIDA measures in free solution with nothing immobilised, so the target stays in its native state, and it does not depend on a mass change, because the reporters are size, fluorescence intensity and emission wavelength rather than the weight of the complex. A full titration curve takes microlitres of sample rather than the much larger volumes calorimetry needs, and the affinities agree with the established methods, tracking closely with SPR. See the affinity readout. Source: Fidabio, Enhanced FIDA analysis of small molecule protein interactions.

Common questions

Can FIDA measure small molecule binding?

Yes. Where the compound changes the target's size, the hydrodynamic radius reports it. Where it does not, which is the usual case, BRIC reads the change in fluorescence intensity and Lambda Dynamics reads the shift in emission wavelength, both from the same run. Where none of those moves, a chemical unfolding assay confirms or rules out binding.

What if the small molecule causes no change in hydrodynamic radius?

That is expected, and it does not stop the measurement. BRIC and Lambda Dynamics are the readouts for that case, and both fit a standard one to one binding model.

What is the difference between Lambda Dynamics and BRIC?

Lambda Dynamics follows a shift in emission wavelength; BRIC follows a change in fluorescence intensity. They are not exclusive and do not always move together. BRIC depends on total protein concentration, and so on pipetting, while Lambda Dynamics, being a ratio of two channels, does not.

Related resources

The blog Enhanced FIDA analysis of small molecule protein interactions introduces Lambda Dynamics and the three assays in one run, and the webinar In-solution quantification of small molecule protein interactions with FIDA Lambda Dynamics covers the method in depth. Two further Fidabio pieces show the membrane protein work in full, Small molecule interactions with membrane proteins and FIDA-based quantification of small molecule binding to membrane proteins in solution.

On this site: What is K_D?, How to perform a binding affinity assay, What is hydrodynamic radius? and the affinity readout.