A micellar solution of protein (in PBS, pH 7.4) was loaded into the sample cell of the calorimeter. The ligand was prepared in 10% DMSO and was loaded in the injector-stirrer syringe.
I have also performed a control experiment to consider the heat of dilution of the ligand solution. A similar addition of the ligand solution was performed under the same experimental conditions keeping PBS in the sample cell. Before evaluating the data, the control data were subtracted from the actual experimental data.
N = 0.4
Model: One set of sites
The presence of the micellar solution could influence the interaction between the ligand and the protein. Micelles can affect the solubility, stability, and interactions of molecules. A change in micelle properties or interactions with the ligand could lead to changes in the binding behavior.@ Priti Sengupta
Dear Priti Sengupta
You may have achieved a critical detergent concentration after the 29th injection of 10% DMSO solution. That would cause the disintegration of micelles, leading to changes in the signal.
Regards
Did you add DMSO 10% to the protein solution in the calorimetric cell?
If not, DMSO dilution heat signal will dominate and mask any other processes.
Dear Prof. Tomasz Fraczyk
Thank you for the suggestion. I took the micellar solution with almost a ten-fold higher concentration of its critical detergent concentration (CDC) into the sample cell of the calorimeter. I also considered the dilution due to adding solution from the syringe. Even after the addition of 39 µL of the solution, the concentration of micellar solution remained much above its CDC and thus, there should not be any possibility of disintegration of micelles. If you have any further suggestions I would be happy to consider them.
Regards.
Prof. Adrian Velazquez-Campoy, thank you for the answer. No, I did not add DMSO to the protein solution. But, as I mentioned in the question description, I did consider the DMSO dilution event and thus, performed a control experiment and corrected my experimental data with the results of the controlled experiment.
No, that is not enough. The dilution heat effect with and without ligand will be very high, but somewhat different. At the end, you will not get the heat of interaction, but the difference between the DMSO dilution heat under two conditions.
You must add DMSO to both solutions at the same percentage.
In fact, the solutions for ligand and protein must have the same composition (buffer, ionic strength, pH, solutes, additives…), except, obviously, ligand and protein.
Dear Priti Sengupta
Did you consider DMSO influence on critical detergent concentration (CDC)? I am not sure, but I would expect lowering of CDC in the presence of this solvent.
Regards
Thank you for the suggestion Prof. Adrian Velazquez-Campoy
I will try this experiment. I hope that 10% DMSO will not affect the structure of the protein micelles.
Dear Prof. Tomasz Fraczyk
No, I did not particularly consider the influence of adding DMSO on the CDC of the micelle. I will definitely consider your suggestion and perform the experiments further. I hope that will help. Thank you.
Regards.
I have another observation on the ITC raw data - in the top view in the figure, I notice there are exothermic (deflection below the baseline) and endothermic (deflection above the baseline) heat changes with each injection. You have a very exothermic reaction - what does the raw ITC titration data look like before the integration baseline is set to zero? The baseline should be well above 0, so all of the DP values for the heat changes are above 0 through the experiment. I suspect the DP values are going below zero - this will cause the ITC cell heaters to over-compensate and result in that endothermic "spike" afterwards. This will impact the net heat change/injection and the fit data This effect has been observed in MicroCal ITCs.
Matching the DMSO in the cell and syringe should help eliminate this large exothermic heat change. Make sure your reference power setting is as high as possible in the ITC experimental settings. If the DP value is still going below zero, even after matching the DMSO concentrations, you should decrease sample concentrations and/or injection volumes to decrease the overall heat change.
Note it is not uncommon to have both exothermic and endothermic heat changes in a single injection. You need to make sure that this is not caused by the DP going below 0.
Thank you for such a detailed answer Verna Frasca .
I have attached the raw data before setting the baseline and as you can see, the DP values varied between the range of nearly +20 to -20.
And yes, I did perform the experiment in MicroCal ITC.
Priti Sengupta How are your protein micelles formed? You only mention PBS, do you also have your protein in a surfactant to have the micelle formation? As others have mentioned, for ITC it is important to have your ligand and protein micelles be in identical buffer, pH and concentrations, including PBS, DMSO, and any surfactant and additives.
Your ITC experiment has several possible sources of detectable heat change, that make data interpretation and analysis difficult. These can include:
-Binding of ligand to protein (this is what you want to measure)
-Heat change due to mismatch of DMSO, buffer, pH or anything else in the samples that is different between the cell and syringe. Control experiments can help identify mismatches (ligand in syringe and buffer in the cell, and buffer in the syringe and protein in the cell) -however it is often not possible to correct for this by simply subtract the control titration from the "binding" titration
-Heat change due to demicellization - this is seen of the micelles are in the syringe and are injected into only buffer, one can determine CMC from this type of experiment
-Is there an "outside" and "inside" of the micelles - as well as ligand binding to the "outside" could ligand be transported to the "inside"?
You do need to match the DMSO in both the protein and the ligand, and if you can use less than 10% (v/v) DMSO that would be better. There are specific ways to prepare the samples to have the matching be the closest.
You can also raise the reference power to about 10-12 ucal/sec (that is the highest you can go with a MicroCal PEAQ-ITC). You also need to reduce the heat change per injection to not have the DP go below 0 ucal/sec fir the injections. The DMSO matching may do that. If not, you will need to decrease concentrations of your samples. It looks like you are already doing 1 uL injections, with extremely large heat changes you could reduce that to 0.5-0.8 uL injection volume.
Thank you for the suggestions Verna Frasca
- The protein system being studied has the ability to form a micelle without the need for any additional surfactant or substances.
- The syringe contains ligand (prepared in 1:9 DMSO:PBS) and the cell contains protein micelle (prepared in only buffer).
- The micellar concentration was kept 10 times higher than its CMC value, as reported in the literature for PBS buffer, to prevent demicellization.
- Although I am unsure of how the 10% DMSO affects the protein micelle's CMC, I have confirmed that the micellar system remains intact with ~5% DMSO based on TEM images.
- When continuously stirred, the ligand becomes encapsulated within the micellar cavity.
Based on your suggestions, I could plan an ITC experiment with 5%DMSO, keeping the reference power to about 10-12 µcal/sec and also reducing the injection volume to 0.5-0.8 uL per addition.
I have a small query: Should the concentration of the sample in the syringe be 5 times greater than in the cell sample?
Priti Sengupta you will have a bit of dilution of the micelles during the ITC experiment, but should not be demicellization (unless there is another factor).
-With protein micelles, you may need to try different ratios of syringe:cell concentrations. For a typical protein-ligand binding experiment, with 1 binding site on the protein, the recommended molar concentration of ligand is 10 times the protein molar concentration. For your experiment are you using the monomer protein concentration? Or are you estimating the micelle concentration? You may have something other than one binding site per protein monomer, or more than one ligand binding site per micelle.
-for 5% DMSO final concentration: Dilute stock solution of ligand (in 100% DMSO) with PBS to be 5% DMSO and desired ligand concentration for ITC. Make micelles as usual with PBS to be slightly above desired final concentration, then right before ITC experiment add 100% DMSO at appropriate volume to have final DMSO of protein be 5%.
The thermogram shows a huge heat effect, very likely stemming from DMSO dilution. It is not enough performing the control experiment and then subtracting it. You will be subtracting large quantities and the residual amounts may be meaningless.
Imagine you have a scale which is not well adjusted having two unbalanced arms and, to get it even, you have to put a 1 kg weight in one side. Now, you want to detect a 1 g weight placed on any arm. Will you be able to detect that small weight?
I recommend you to follow the suggestions provided by Verna Frasca
The amount of the protein was taken based on its molecular weight (in the order of kDa) and then the micelle formation procedure was carried out in PBS.
A big thanks to Verna Frasca for your help, as it provided me with a clear understanding of the experimental setup. Also, thanks to Adrian Velazquez-Campoy for your suggestion. I will be implementing these tips and remain hopeful for successful outcomes.
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