Name: Vidéo: 15N-Heteronuclear Single Quantum Coherence (HSQC) Titration Experiments to Monitor the Chemical Shifts of the 1H and 15N NMR Resonances
Uploaded: 2024-05-17T14:25:00.000+00:00
Duration: 2 min
Description: 294 vues. 15N-Heteronuclear Single Quantum Coherence (HSQC) Titration Experiments to Monitor the Chemical Shifts of the 1H and 15N NMR Resonances

15n-heteronuclear single quantum coherence (hsqc) titration experiments to monitor the chemical shifts of the 1h and 15n nmr resonances

Advanced NMR Techniques for Glycan-Protein Interaction Analysis

Molecular recognition of glycans is essential for many processes related to health and disease. The specificity and selectivity of biological receptors (lectins, antibodies, enzymes) for glycans heavily depend on adjusting the precarious balance between the diverse components of enthalpy (CH-&#960; and van der Waals, hydrogen bonds, electrostatics) and entropy (hydrophobicity, dynamics, solvation-desolvation)1.
Given the large chemical diversity and dynamic nature of glycans, NMR methods have been widely employed to dissect glycan interactions for more than 25 years2, since these methodologies afford superb information on molecular recognition events with precise details, at atomic resolution3,4, even when the required interaction evidence cannot be retrieved by employing other methodologies. As key point, NMR is versatile and allows studying dynamic events, at the atomic level, at different time scales, constituting the best technique by far for studying the structure, conformation, and dynamics of glycans in solution. Nevertheless, disentangling this information may be a rather complex process that requires the employment of well-defined strategies together with careful data analysis5.
NMR techniques are diverse and, indeed, there are many methodologies that can be employed to unravel glycan-protein interactions6. We herein describe two basic NMR approaches that are currently employed to decipher glycan-receptor interactions7,8, making emphasis on how to untangle the presentation of the key glycan epitope as well as the protein binding site9.
In any molecular recognition event, when a receptor binds to a given ligand, there is a chemical exchange process that affects many NMR parameters of the participants in the binding10. Therefore, from the NMR perspective, the interaction can be monitored either from the point of view of the glycan ligand or from that of the protein receptor11. Generally speaking, the protein receptor is a large biomolecule (slow rotational motion, with rates in the ns timescale, and therefore, fast transverse relaxation), while the interacting glycan can be considered as a small-medium size molecule (fast rotational motion, with rates in the ps timescale, and slow transverse relaxation)12. From a standard perspective, the NMR signals of the glycan are narrow, while those of the receptor are broad13.
Ligand-based NMR methods rely on the dramatic change that many glycan NMR parameters experience when passing from the free to the bound state14. STD-NMR is the most employed experimental NMR technique to assess diverse glycan binding features15, from deducing the existence of binding in the solution state to the determination of the glycan binding epitope; that is, the atoms of the ligand that are in contact with the protein receptor16.
Alternatively, receptor-based NMR methods monitor the changes that take place in the signals of the protein receptor in the presence of the glycan with respect to those recorded for the apo state17. These are mainly focused on screening the chemical shift perturbations of the protein signals between both states. The most commonly employed experiment is 1H-15N HSQC, or its TROSY alternatives18.
The combination of both approaches allows applying NMR to many diverse systems that display a wide range of affinities. However, for the receptor-based NMR methods, in contrast to those based on the ligand, a relatively large amount of soluble, non-aggregated, stable isotope-labelled (15N) protein must be available.
We herein describe both methods, highlighting their strengths, and weaknesses. Note that the basic steps described in the protocol serve as examples for the use of Bruker spectrometers. Consequently, commands and parameters names align with those utilized in TopSpin (Bruker&#39;s spectrometers control software).

Author Spotlight: Unveiling the Structural and Dynamic Aspects of Glycan Molecular Recognition

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, glyco sciences 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 for fluorescent or 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 lichens.

15N-Heteronuclear Single Quantum Coherence (HSQC) Titration Experiments to Monitor the Chemical Shifts of the 1H and 15N NMR Resonances

15n-heteronuclear-single-quantum-coherence-hsqc-titration-experiments

Research

JoVE Journal

Chemistry

294 vues. 15N-Heteronuclear Single Quantum Coherence (HSQC) Titration Experiments to Monitor the Chemical Shifts of the 1H and 15N NMR Resonances

Vidéo: 15N-Heteronuclear Single Quantum Coherence (HSQC) Titration Experiments to Monitor the Chemical Shifts of the  1H and  15N NMR Resonances

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.

Here, we present a protocol detailing the acquisition, processing, and analysis of a series of NMR experiments aimed at characterizing protein-glycan interactions in solution. Most common ligand-based and protein-based methodologies are outlined, which undoubtedly contribute to the fields of structural glycobiology and molecular recognition studies.

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and ...

Acquisition, Processing, and Analysis of 1H-Saturation Transfer Difference (STD) NMR to Characterize Protein-Glycan Interactions

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.