I'm afraid, that for using intrinsic fluorescence of a protein to judge about any changes in its structure or oligomeric state, first, you have to find any spectral change during titration (shift of lambda max, amplitudes at different nm, change in the width of the peak, its shape, etc., provided that these changes are saturable upon titration), otherwise this method is insensitive to changes in the association/dissociation. Also, what do you mean by 'high affinity protein'? Affinity to what? Is it an oligomer and then you mean Kd for dissociation of its oligomeric species or what? Check the location of fluorophores in your protein structure if it is available, because it can give you some clues what to expect from your titration.

The general rule for affinity measurement is "Kd, protein concentration, and ligand concentration are in the same order (dimension is M)."

1. Therefore, "fluorescence photon counting" will be suitable for your experiment.

2. Another method is the competitive experiment such as Tanaka, T. et al. (2012)

FEBS J. 279 (No.6), 1014-1029.

You have to start with both protein and ligand concentrations below Kd. Otherwise you basically titrate one of the two and you'll get a number, which says "The Kd is somewhere below that." but not more.

Competition assays are helpful (see also http://www.ncbi.nlm.nih.gov/pubmed/12069420, http://www.ncbi.nlm.nih.gov/pubmed/17672523 or similar)

Hi Archishman. I am afraid that using intrinsic tryptophan fluoresence for Kd determination will be problematic, especially if you are trying to publish the results or to get a grant.

If your protein has only one Trp and nothing in your polymer solution fluoresces, you might be able to make a case. If your protein has more than one Trp's, then all bets are off. Critics can point to differential quenching, blueshift, solvent exposure, etc. to shoot down your argument.

If your protein is an enzyme, is there a way to covalenly label the active site with a fluorophore with a longer wavelength than Trp? In this way you are only looking a 1 fluorophore in 1 protein.

Hi Archishman, have you though about fluorescence life time. Life time technique is very sensitive to local environmental changes tryptophan have been shown to be a sensitive probe for it.

Thank you everyone for you comments. I cannot label my protein because I feel labeling (like FRET) is too invasive for the kind of project that I am doing. When I mentioned high affinity protein I meant that my protein has a high affinity for its ligand.

Eduardo, could you elaborate ? I understand tau will be different for different concentrations of ligand. But will it be sensitive enough to give me proper Kd upon analysis, at concentrations of protein higher than it's Kd ? For example if I do my experiments at 100 nM concentrations while the Kd is 5 nM.

Could you cite a reference ?

You can try using MicroScale Thermophoresis. It can be used entirely label-free and is very sensitive. Also, buffer viscosity has little to no effect on the experiment. I already measured nM Kds at all kinds of viscosities and crowded solutions. SInce the sample consumption is tiny as well and not much preparation is needed, it might be worth a try.

I think your main limitation is the large binding constant of your protein-ligand complex. If you could diminish this affinity by changing enviromental variables such as pH or ionic strength, you would have more techniques available to follow the binding process and to estimate the binding constant by extrapolation.

Firstly I will perform some CD experiments with a longer path length cell to understand the structural features of my protein. The longer path length cell will compensate the low concentration of protein. Then perform titration with my ligands and collect CD spectra. We have done a similar study last year where we used CD to measure the binding constant. Fluorescence lifetime study can be another option, but if the microenvironment of the ligand in the protein does not change, there will be no change in lifetime.

Permyakov, E.A. Luminescent Spectroscopy of Proteins. CRC Press, Boca Raton, Ann Arbor, London, Tokyo, 1993

One way to do that is by measuring the folding free energy in the absence and presence of ligand , by performing denaturations of the protein in the absence and presence of varying concentration of ligands or other proteins. This article may help...

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728349/

I do not think that you can use the quenching of intrinsic trp fluorescence at these concentrations.....

Tryptophan Fluorescence Quenching is a possibility, I don't think that you have to use such a low concentration for titration, please see the following article:http://www.ncbi.nlm.nih.gov/pubmed/14530451

I am wondering how do you estimate that your protein-ligand dissociation constant is 4-5 nM. How did you determine that?

Another possibility is to radioactive ligand (if available) and do a filter-binding assay.

Hi Archishman. I think that the fluorescence lifetime is a way to go if the interactions with the ligand change the lifetime. A way to overcome a need of working at very low protein concentrations is "titration by dilution" - an experiment where you change the concentrations of both reagents simultaneously while keeping their molar ratio constant (at 1:1 for stoichiometric binding) - i.e., dilute an equimolar mixture. In this kind of titration you will reach 90% saturation at ~100*Kd, so that a titration curve in the range of 10 - 500 nM will give enough information for determining Kd in your case. Example of using this strategy with life time fluorescence you can find in: http://www.ncbi.nlm.nih.gov/pubmed/22194603. Another possible strategy is Job's (continuous variation) titration - an experiment where you change the concentrations of both the protein and the ligand, while keeping their total concentration constant(i.e., increase the ligand while decreasing the protein). Here the total concentration of 100-200nM will work well for Kd=5nM. Example of using this technique (with absorbance spectroscopy, though) you can find in: http://www.ncbi.nlm.nih.gov/pubmed/16701937.

Scattering from 300 g/L of a polymer will make any optical spectroscopy very difficult. Radiolabeling or microscale thermophoresis may be your only options.

Dear Colleague,

Estimating the dissociation constant (Kd) of a high-affinity protein-ligand interaction using intrinsic tryptophan fluorescence spectroscopy is a sophisticated approach that leverages the sensitivity of tryptophan residues to their microenvironment. This method is particularly useful when tryptophan residues are located near the binding site, as their fluorescence properties will change upon ligand binding, providing a quantitative measure of the interaction. Below is a structured methodology to accurately determine the Kd of a high-affinity protein using this technique:

Intrinsic tryptophan fluorescence spectroscopy provides a powerful and sensitive method for investigating protein-ligand interactions, especially for high-affinity complexes. The precision in preparation, measurement, and analysis is paramount to accurately determining the Kd value, offering insights into the binding dynamics and molecular mechanisms governing the interaction.

Yours sincerely,

With this protocol list, we might find more ways to solve this problem.