Begin by preparing a sample with a protein-ligand complex. Then, using a pipette, carefully transfer 0.6 milliliters of the prepared solution into a five milliliter NMR tube. To set up the NMR instrument to the required temperature, use the EDT command to open the temperature control monitor and adjust the desired temperature.
Then, place the sample at the sample changer. Use an autosampler to insert the sample into the magnet and run the command sx, followed by the position number, N, corresponding to the position of the NMR tube in the autosampler tray. After this, the sample enters the NMR magnet.
To lock on the solvent signal, type the lock command and select the appropriate solvent from the menu. Use either the automatic module, atma, or the manual module, atmm, to complete the tuning and matching process. Now, using the topshim command, start the automatic shimming, then execute the pulsecal command to determine the proton 90 degree pulse.
After that, create a new data set and upload the STD NMR pulse sequence. Define the off and on resonance frequencies for the STD NMR experiment under the FQLIST entry in the ACQUPARS window. Set the off resonance frequency in a region without any ligand or protein proton signals.
Then, choose an on resonance frequency in a spectral region devoid of glycan signals. Define the shaped pulse to be used during the saturation time in the ACQUPARS parameters of the ASED window. Afterwards, set the proton 90 degree pulse length, adjust the total saturation time and the relaxation delay to three seconds.
Set the number of scans to a multiple of eight and dummy scans to eight. Then, set the number of points in F2 to 16K, 32K, or 64K, and an F1 to two. Now, using the automatic command rga, set the receiver gain to avoid overflow.
Calculate the total experiment time using the experiment command. Finally, use the zg command to send the experiment for acquisition. After the experiment, perform the Fourier transform of the first FID, and select the destination of the processed spectra.
Then, using the lb command, adjust the line broadening factor. To manually phase the spectrum, access the process tab, and then the adjust phase sub-menu. Click and drag on the corresponding button to perform zero and first order corrections and save the phasing results.
After performing the Fourier transform for the second experiment, save the processed spectrum with a different code. Load the two processed spectra with the multiple functions, and, using the subtraction button available in the multiple visualization, subtract them. Then, open the off resonance spectrum and execute the MD command to initiate the multiple display window.
Subsequently, upload the STD spectrum. Next, conduct a comparative analysis of the frequencies and intensities of the signals present in the STD NMR spectrum. In the off resonance experiment, measure the signal intensities.
Navigate through the menu to select process, and then integrate. Carefully define the regions of interest and record the integrals in a file. Similarly, measure the intensities in the STD NMR experiment, ensuring that identical parameters are used, and document these integrals in a separate file.
Alternatively, STD values can be determined by comparing signal intensities between the STD and off resonance spectra. To calculate the relative STD as a percentage, assign a 100%value to the proton exhibiting the maximum STD intensity. The proton STD NMR spectrum for the interaction of N-acetyl lactose amine, with human galectin 7, showed STD NMR signals indicating binding.
Moreover, signals belonging to protons in close contact with the protein showed up, allowing delineation of the binding epitope.
