This method will allow the researcher to probe hard to study systems, like peptides and first row transition metal ions, both of which are generally challenging using more standard applications. The main advantage here is using orthogonal techniques because they complement each other well, and that can give more insight into the system and help us to not miss any phenomenon. The method detailed in this video is very well suited for studying other systems.
Although we show it with Cu(II)and a peptide, it can be used for many different metal peptide and metal protein interactions. I think the hardest part will be finding a good buffer choice for your metal ion and peptide. I would consult literature to see what buffers have been used by others before.
demonstrating the procedure will be Sohee Choi, a graduate student in my research lab. To begin, turn on the electronic absorption spectrophotometer and let it warm up for approximately 15 to 20 minutes before use. Then launch the spectrophotometer software and configure the parameters, such as scanning range of 200 to 900 nanometers, the scan range of 200 nanometers per second, and double beam baseline corrected.
Next, collect a baseline with no cuvettes or samples in the beam paths. Using two matched cuvettes in the double beam spectrophotometer, load one cuvette with 115 microliters of ultrapure water and the other cuvette with 115 microliters of the peptide sample while ensuring there are no air bubbles in the cuvettes as these will interfere with the signal. Afterward, place the cuvette with ultrapure water in the reference beam and the cuvette with peptide in the sample beam.
Then collect the absorption spectrum of the metal-free peptide. Add a substoichiometric amount of the Cu(II)solution into the cuvette with the peptide sample and record the volume for analysis later. Gently pipe that up and down to mix the solution while avoiding the generation of air bubbles.
After equilibrating for five minutes put the cuvette back into the sample cell holder and record the absorption spectrum. Then repeat the addition of Cu(II)aliquots into the peptide solution for various equivalents and record the total volume of Cu(II)added to the cuvette. After initializing the software as described earlier in two matched cuvettes load one cuvette with 115 microliters of ultra pure water and other cuvette with 115 microliters of the copper-phen complex solution.
Place the cuvette with water in the reference beam and the cuvette with copper-phen complex solution in the sample beam. Then collect the absorption spectrum of the metal ligand complex. Afterward, add a stoichiometric amount of peptide to the copper-phen complex solution and gently pipe that up and down to mix thoroughly while being careful not to introduce any air bubbles.
Next, incubate the solution for five minutes to reach equilibrium. Put the cuvette back into the sample cell holder and record the absorption spectrum. Later, repeat the addition of peptide aliquots into the copper-phen complex solution for various equivalents, and record the volume of peptide added so that the diluted concentration can be determined.
First turn on the ITC and launch the ITC software to run the instrument. After the first initialization, rehome the burette and follow the instructions on the screen. Then remove the burette and the cover from the reference cell.
Next, remove any water from the reference cell and rinse three times with 450 microliters of degassed ultra pure water. Slowly draw up ultra pure water to the 450 microliters mark of a loading syringe taking care not to introduce air bubbles into the syringe. Then insert the loading syringe into the reference cell until it is approximately one millimeter from the bottom and slowly inject part of the solution until 150 microliters remain in the loading syringe.
Afterward, move the loading syringe plunger quickly up and down by approximately 25 microliters several times to dislodge any bubbles on the cell surface. Then slowly inject until the plunger reaches the 100 microliters mark on the loading syringe, thereby dispensing a total of 350 microliters of ultra pure water into the reference cell and replace the reference cell cover. After removing any residual solution from the sample cell, load 450 microliters of 10 millimolar EDTA using a loading syringe and soak for 10 minutes to ensure trace metal ions are removed because the EDTA will bind trace metals.
After removing the EDTA solution thoroughly rinse the loading syringe with copious amounts of ultra pure water. Clean the ITC in accordance with the manufacturer's directions by rinsing the sample cell with ultra pure water. Remove any residual water from the sample cell and then condition the sample cell by rinsing with 450 microliters of buffer at least three times.
Next, remove the residual buffer that is conditioning the sample cell and load the peptide solution into the sample cell using the loading syringe. Then rinse the titration syringe with 200 microliters of the buffered solution by first removing the plunger. Using a micro pipette, pipette the buffer solution through the hole at the top of the glass titration syringe through the syringe and out the needle below.
Later, fully insert the plunger into the titration syringe. Then dip the tip of the titration syringe needle into the metal solution and slowly pull the plunger up causing the metal solution to fill the syringe and resulting in a void volume at the top of the glass part of the titration syringe. To remove the void volume rotate the titration syringe parallel to the floor.
Then remove the plunger and slightly tilt the glass part toward the floor. Gently shake the titration syringe so that the solution moves to the glass part of the titration syringe and fills most of the void volume. But ensure that two to three microliters of void volume remain.
While keeping the syringe parallel to the floor reinsert the plunger. Then hold the titration syringe upright and dip the tip of the needle back into the metal solution. Next, push the plunger down until air ceases to come out of the needle and load the titration syringe by slowly pulling up the plunger to just above the 50 microliters mark while keeping the tip of the needle in the solution.
Carefully insert the glass part of the titration syringe into the burette and screw until finger tight. Afterward, using a light duty delicate wiper absorb the solution that comes out of the titration syringe due to compression of the plunger without touching the tip of the needle. Next, insert the burette with a titration syringe into the sample cell and fasten it securely.
To set up the parameters of the ITC software, select the Instrument Control. Set the stirring rate and the temperature. Then under Experiment Details, enter the syringe and cell concentrations in millimolar units.
Next, in the Experiment Method section select Incremental Titration. Click on Setup and specify 20 injections of 2.5 microliters and if more resolution is required to observe the binding event increase the number of injections and decrease the volume per injection. Afterward, input the time spacing between each injection so that it is long enough for the signal to equilibrate and return to baseline, typically 300 seconds.
Then click the Run button to start the experiment and specify where the data are saved. Titration of Cu(II)was performed into C-Peptide demonstrating that the addition of 150 micromolar of Cu(II)caused an immediate increase at the band at 600 nanometers attributed to the d-d band of Cu(II)and continued to increase until 300 millimolar Cu(II)was added. Further addition above 300 micromolar Cu(II)did not increase the absorption of the d-d ban indicating saturation and that Cu(II)binds to C-Peptide in a 1:1 complex with a log K value of more than six.
C-Peptide was titrated into approximately 10 micromolar copper-phen complex and the absorption from the charge transfer band at 265 nanometers decreased indicating that the C-Peptide was able to chelate Cu(II)from the phenanthroline ligand with a log K value of 7.4 to 7.8. The ITC thermogram shows the titration of 1.4 millimolar Cu(II)into 154 micromolar C-Peptide in 15 millimolar MOPS buffer. The binding affinity was found to have a log K value of eight with change in enthalpy as 8 kilojoules per mole.
Gibbs free energy as minus 46 kilojoules per mole, and change in entropy as 120 joules per mole per kelvin. A controlled titration of 1.4 millimolar Cu(II)into 15 millimolar MOPS buffer in the absence of C-Peptide shows no binding in the thermogram. I would say making sure everything is clean.
If there are metal ions or peptides left over from previous experiments it can drastically affect the stoichiometry and provide unknown competition in the binding experiments. Circular dichroism is another method to study metal peptide or metal protein interactions because it looks at chirality. Mass spectrometry can also interrogate these complexes by looking at changes in mass.
This set of techniques has been used by many labs who study metal ions interacting with peptides or proteins.
