In my case, I dock different substrates (ligand) to an enzyme (receptor). Each substrate gives different binding energy. How can I relate the binding energy with the substrate affinity? Is the lower the binding energy means the higher binding affinity?
yep you are right....the more negative of the free binding energy results in the formation of stronger complexes....therefore, when a ligand have a low free binding energy (dG) its affinity towards the target enzyme is bigger in comparison with a ligand with higher dG
As I understand it, the accuracy of prediction of affinity by docking is not very high, so it is a good practice to follow up the computational effort with empirical measurements in order to test the quality of the docking results. When doing so, however, bear in mind that for a 1-substrate enzyme, you can measure the Km for the substrate, but the Km is not always equivalent to the affinity (Kd).
I must agree with Adam, the results from docking dG values do not correlates good with experimental results.
Nor, it is important to be quantitative. This page has the formula to convert energy to affinity:
https://en.wikipedia.org/wiki/Binding_constant
Just make sure you use the right units for the ideal gas constant (consistent with your temperature and energy units).
Which practical experimentation can be carried out to determine the binding energy of ligands with its receptors?
If you have the receptor in a purified, native form, there are many biophysical experiments that are possible to measure the affinity of the receptor-ligand complex. If you have a membrane preparation containing the receptor, some binding measurement are possible, especially if you have a radiolabeled version of the ligand. If you are working with cells expressing the receptor on the surface, you can use a biological response to measure the concentration-effect relationship of the ligand.
Thanks for the answer sir. I searched and got Isothermal calorimetry (ITC), Differential scanning calorimetry (DSC), and Thermal shift assay (TSA).
I have the following two doubts sir:
1. I understood the difference between Ki and kd. In one paper I saw Km values for Hsp90. They used Eadie-Hofstee linear transformation for calculating Km vales of Hsp90 inhibition. What is the difference between Km and Ki/Kd.
2. In one paper they wrote that IC50 value of Hsp90 inhibition (determined by scintillation proximity assay, Kung et al., BMCL, 2008, Article Dihydroxylphenyl amides as inhibitors of the Hsp90 molecular chaperone
) was converted to competitive Ki using Cheng-Prusoff equation. Can we apply the same to convert the IC50 determined by malachite green assay (Article High-throughput screening assay for inhibitors of heat-shock...
)
1. Km is the substrate concentration at which the rate of the enzyme reaction is 50% of the maximal rate when the enzyme is saturated with substrate. Km can also be defined in terms of rate constants for individual steps in the reaction. For a single-substrate enzyme where the reaction is E + S ESE + P,
Km = (k-1 + k+2)/k+1. Only in the limiting case where k-1>>k+2 (rapid equilibrium), Km = k-1/k+1, which means that Km is the dissociation constant Kd of the substrate. Otherwise, Km is not the dissociation constant of the substrate.
Ki is the dissociation constant of the inhibitor.
2. You can use the appropriate Cheng-Prusoff relationship to convert IC50 to Ki. It doesn't matter how you measured the IC50. You must understand the kinetic mechanism of the inhibitor on order to apply the right equation. In the case of a competitive inhibitor, IC50 = Ki(1+ [S]/Km), so you also need to know the Km of the substrate. Other equations apply for other types of inhibitors (e.g. competitive, noncompetitive, mixed).
Make sure that the inhibition you measure is real and specific. Compounds can interfere with the assay readout sometimes, causing false positives (see the attached file). Also, compounds can inhibit enzymes non-specifically, by which I mean in a manner that does not involve 1:1 binding to a binding site on the protein. Examples include inhibitor aggregation, protein denaturation, and covalent reactions by contaminating substances.
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