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NMR, MS and SPR for Protein─Ligand Analysis

Surface plasmon resonance (SPR) analysis is an optical detection method that is used to study ligand binding interactions with membrane proteins, the main targets in drug research and development. The technique offers significant benefits when measuring affinity and kinetics in the analysis of biomolecular interactions. Since SPR only enables analysis of binding processes, technologies have been developed that combine SPR with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy so that bound molecules can also be examined.

At Saitama University in Sakura, Japan, Deepti Diwan and colleagues from the Division of Material Science have used nuclear magnetic resonance (NMR), MS and SPR to investigate the effect of multivalent binding on the specificity of protein─saccharide interactions. Several instruments supplied by Bruker were used for the study, including the AV400 + Cryo spectrometer, the AVANCE 500 spectrometer and the Autoflex III spectrometer. The latter enables matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) analysis, which is used when biomolecules of interest are fragile and prone to fragmentation with alternative ionization techniques.

MALDI autoflex speed
MALDI autoflex speed

Protein─saccharide interactions enable essential intercellular recognition events in important physiological processes such as fertilization, development and the immune response. Such reactions are often weak, but are amplified by the so called “glyco-cluster effect,” the multivalent effect that a clustered saccharide has on account of densely packed saccharides along the polymer backbone.

As well as enhancing the affinity of protein─saccharide interaction, multivalancy gives rise to other phenomena such as agglutination. Synthetic glycopolymers that exhibit significant multivalency have therefore become attractive as potential tools for use in biomedical applications.

As reported in the journal Molecules, Diwan and team synthesized mannose glycosides as a novel type of glycosylated monomer and, using radical polymerization, created its polymers, compounds 8a and 8b. For the polymerization reaction, H-NMR spectra were used to estimate the degree of polymerization of products to ascertain the copolymer composition. NMR spectra analysis was also used to determine the chemical structure of the glycomonomer and glycopolymers synthesized.

AVANCE NMR
NMR system with CryoProbe

 

Using SPR detection to obtain real-time binding profiles, the team then investigated the interaction of the synthesized glycopolymers with the mannose/glucose binding protein, concanavalin A (Con A). To test the Con A–glycopolymer interaction, the lectin was immobilized on the sensor surface of a carboxymethylated dextran-coated (CM5) sensor chip and the polymers 8a and 8b were used as analytes.

The results showed that Con A sequestration was enhanced as the concentration of the glycopolymer increased. The kinetic affinity for the synthesized polymers displayed strong, specific molecular recognition abilities with lectin.

Further analysis suggested that mannose residues are the main contributors in enhancing multiple interactions for the binding stoichiometry, the binding rate, the potency, and the stability of Con A clustering.

“We anticipate that this synthetic scaffold will offer new means to define the structures of multivalent ligands and densities of binding epitope for specific functions in lectin–glycan interactions,” write the researchers.

References

Dian D, et al. Synthetic Assembly of Mannose Moieties Using Polymer Chemistry and the Biological Evaluation of Its Interaction towards Concanavalin A. Molecules 2017;22(1), 157; doi:10.3390/molecules22010157

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