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

Research

JoVE Journal

Chemistry

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