One interpretation is that ATP and analogs don't bind to the protein under the tested conditions. Was Mg2+ present? ATP binding could be dependent on something else binding first if the protein has multiple substrates and uses a strictly ordered kinetic mechanism. Do you have any independent evidence that ATP and the analogs bind to this protein?

Another interpretation is that ATP binds but has no effect on thermal stability of the protein.

One interpretation is that ATP and analogs don't bind to the protein under the tested conditions. Was Mg2+ present? ATP binding could be dependent on something else binding first if the protein has multiple substrates and uses a strictly ordered kinetic mechanism. Do you have any independent evidence that ATP and the analogs bind to this protein?

Another interpretation is that ATP binds but has no effect on thermal stability of the protein.

Hi Ruigang

Please verify by other methods if the ATP as well as analogs are binding to your protein. You can use Isothermel Titration Calorimetry for instance. Also test with varing concentrations of divalent metals.

Thanks for answering my questions. I was using 1mM Mg2+ in the thermo shift assay. The ATP/ADP bind crystal structures of proteins from other species were solved and published already. The protein I am working on has the NTPase Walker A/B motifs etc, according to the sequence and their secondary structures are similar from prediction. Isothermel Titration Calorimetry is worth to try to see if I could see any binding. 

There are some points to check before further speculating:

1. Is your protein properly folded at the experimental conditions you did the assays? Did you check the fluorescence and/or the CD spectra? Did you check if your protein is OK in solution (e.g. DLS or SEC)?

2. Do you have any evidence for interaction with ATP analogs at those experimental conditions? It is not enough to know your protein is an ATPase; binding interactions are modulated by pH, ionic strength... Therefore, the selected experimental conditions will be very important.

3. Are any cofactors or effectors required (e.g. Mg++ as already mentioned) for the interaction?

4. Did you perform control assays? That is, did you check if the Tm you are obtaining with the free protein in the thermal denaturation assay is similar to that obtained in a conventional fluorometer following the intrinsic tryptophan fluorescence or a in circular dichroism instrument following the secondary structure?

In general, any ligand (cofactor, metal ion, protein, denaturant...) binding to a protein will stabilize the protein. The point is what protein conformations are being stabilized, i.e, what conformational states are increasing their relative populations. A cofactor may stabilize the native state because is binding preferentially to the native folded state, but a denaturant is stabilizing the unfolded state becuase is binding preferentially to the unfolded state. Thus, simplifying the system to a two-state conformational equilibrium, if a ligand binds to the native and the unfolded states with similar affinity, there will be no net stabilization. Only if the ligand binds to one of them preferentially you will see a net stabilization (higher Tm if native state is stabilized preferentially, or lower Tm if unfolded state is stabilized preferentially).

In addition, even if the ligand binds preferentially to the native state, and, therefore, it should stabilize the native state and increase the Tm, there may be some subtleties precluding the observation of a clear Tm increase. If the binding enthalpy and the binding heat capacity of the ligand are very large and negative at 25 degrees, the strong temperature dependence of the ligand binding affinity will cause a marked binding affinity reduction as son as the temperature increases in the unfolding assay, and a very small Tm change (maybe within the experimental error) could be elicited. The large and negative binding enthalpy is typical of binding interactions with good complementarity in polar interactions (e.g. h-bonds, electrostatics), and a large and negative heat capacity is typical of binding interactions where a large conformational change is coupled to ligand binding.

Summarizing, I would check if your protein is in good shape:

- Fluorescence and CD spectra, for proper folding

- DLS and SEC for potential aggregation

- Fluorescence and/or CD temperature unfolding, for proper folding

Then, I would check if your protein interacts with ATP and its analogs:

- ITC is the gold-standard, but you may think of spectroscopy, SPR, MST... that is, any appropriate binding technique

Then, I would check if the results of the thermal unfolding of the ligand-free protein in your thermal-shift machine are in agreement with those of the ligand-free protein in conventional equipments. This will inform you if the concentrations of protein and fluorescent dye are appropriate.

By the way, the Tm increase in a protein induced by ligand binding to the native state may exhibit a saturation behavior with ligand concentration (for example, if the ligand binds to the native and the unfolded states with 1:1 stoichiometry, but higher affinity for the native state) or not (if the ligand binds uniquely to the native state). In the latter case the increase in Tm will depend only on the liagand binding affinity and the (free) ligand concentration, and, in principle, does not have maximal value. Increases in Tm larger than 10 degrees are not exaggerated (e.g. if ligand binds with picomolar affinity and it is added at sufficiently high concentration).