Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue

Our research delves into the study of diverse glycan lectin systems, exploring their connections to health and disease. We merged methodologies from chemistry, biology, and biomedicine to deeply investigate the roles in immune responses to pathologists from cancer to bacterial and viral infections. Recently, glycosciences have marked groundbreaking milestones by transforming glycan design synthesis and analysis of a structural molecular recognition aspects from the atomic to the cell level.

These advances collectively set the stage four flourishing of advanced glycan-based therapies. This protocol outlines the procedures for acquiring, processing, and analyzing two of the most powerful NMR methodologies employed in the glycan field. These methodologies, saturation transfer difference, and chemical safe perturbation analysis complimentarily offer precise information of the structural and dynamic aspects of molecular recognition of glycans.

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 edte command to open the temperature control monitor and adjust the desired temperature.

Then place the sample at the sample changer. Use an auto sampler to insert the sample into the magnet and run the command sx followed by the position number and corresponding to the position of the NMR tube in the auto sampler 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 FQ list 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-acetyllactosamine with human galectin-7 showed STD NMR signals indicating binding. Moreover, signals belonging to protons and close contact with the protein showed up allowing delineation of the binding epitope. Begin by preparing the sample using the lectin of interest.

To create the buffered solution, use a mixture comprising 90%water and 10%deuterium oxide. After acquiring the proton NMR spectrum, create a new dataset and select the pulse program HSQCETFPF3GP, then define the required experimental parameters. Send the experiment for acquisition and process the fid using the command xfb.

Next, the same experiment is acquired for different 15 N-galectin-7 bound to N-acetyllactosamine ratios in the spectra are compared. Then using supplementary software, generate a comprehensive list of protons and nitrogen 15 frequencies for all cross peaks observed. After that, calculate the maximum chemical shift perturbations.

Construct a 2D plot with the maximal chemical shift perturbations plotted on the vertical y-axis against the corresponding amino acid residue on the horizontal x-axis. If the protein's 3D structure's available, open the corresponding PDB file using the appropriate software. Highlight the residues showing the highest maximum chemical shift perturbations in a distinct color to identify the probable binding site.

Super imposition of the proton nitrogen 15 HSQC spectra recorded for the titration of N-acetyllactosamine into human galectin-7 solution depicted several cross peaks experiencing chemical shift changes indicating interaction. The most perturbed amino acids of human galectin-7 are mapped into the five gal PDB structure. The colored region likely represents the binding site.