Name: Exploring Protein-Glycan Interactions: Advances in Nuclear Magnetic Resonance (Video) | JoVE Journal
Uploaded: 2025-08-26T14:00:00.000+00:00
Duration: 10 min 7 s
Description: Explore protein-glycan interactions using NMR techniques to study structural dynamics and surface forces in molecular recognition. This method reveals binding mechanisms for integrin-disintegrin complexes.

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5 minutes

We thank the Centro Nacional de Resson&#226;ncia Magn&#233;tica Nuclear Jiri Jonas (CNRMN) for the use of the NMR spectrometers. We are grateful to Dr. Carole Bewley for sharing with us the chemical shift assignments of CVN in complex with Man&#945;1-2Man&#945;. We also thank the funding agencies FAPERJ and CNPq.

The authors declare that they have no competing interests.

NMR spectroscopy represents a versatile approach for investigating protein-glycan interactions, particularly because of its ability to detect weak and transient interactions, such as those observed between CVN and D-mannose. The protocol described here combines ligand- and protein-based NMR methods, allowing for a more comprehensive characterization of the interaction interface.
The critical steps of the protocol include careful sample preparation with precise control of protein and ligand con...

Protein-glycan interactions are fundamental to a wide range of biological processes, including cell-cell recognition, immune response modulation, and pathogen-host interactions1,2. These molecular interactions are highly specific and dynamic and play crucial roles in both physiological and pathological mechanisms3,4,5. A classic example of such interactions occurs with lectins, proteins that specifically recognize and bind to glycans. Cyanovirin-N (CVN), for example, has been widely studied as an experimental model because of its ability to interact in high-affinity with high mannose oligosaccharides5,6,7.
In addition to lectins, many glycosylated proteins, such as integrins, also play essential roles in recognition and cellular signaling. Integrins are transmembrane receptors that frequently undergo glycosylation of their subunits, influencing their stability and affinity for ligands, including glycans5,8,9,10. However, the structural characterization of these interactions remains challenging owing to the intrinsic heterogeneity and flexibility of glycans, which complicates traditional structural biology approaches. In this context, the development of advanced methodologies capable of capturing these interactions in solution is of great value to this field, particularly for glycobiology11,12.
Nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful tool for investigating protein-ligand interactions at the atomic level. Unlike other techniques, such as X-ray crystallography and cryo-electron microscopy, NMR allows the study of biomolecular interactions under near-physiological conditions, providing insights into both structure and dynamics. This flexibility enables researchers to modulate environmental parameters such as pH (typically between 4.0-8.0), ionic strength (e.g., 0-500 mM NaCl), and temperature (generally 273-330 K), thereby tailoring conditions to the specificities of protein-glycan complexes. Additionally, NMR is particularly advantageous for analyzing transient and weak interactions, which are often characteristic of protein-glycan recognition events13,14.
Several NMR techniques have been employed to investigate protein-glycan interactions, utilizing both protein- and ligand-based approaches. From the protein perspective, heteronuclear single quantum coherence (HSQC) titration is widely used to map binding sites by monitoring chemical shift perturbations upon ligand addition. Relaxation dispersion experiments enable the detection of conformational and chemical exchange processes occurring on the micro- to millisecond timescale, providing insights into the dynamic aspects of glycan recognition15,16. These protein-based techniques typically require sample concentrations in the range of 50 &#181;M to 2 mM, depending on protein stability and labeling efficiency17,18.
Ligand-based NMR techniques offer complementary information by focusing on glycan changes during binding. Saturation transfer difference (STD-NMR) is particularly useful for identifying glycan epitopes involved in recognition, as it selectively saturates the NMR signals of ligand regions in close contact with the protein19,20. Water-Ligands Observed via Gradient Spectroscopy (WaterLOGSY)21,22 and Transferred Nuclear Overhauser Effect Spectroscopy (Tr-NOESY) provide additional means to assess ligand binding and conformational changes during interactions23. Furthermore, Carr-Purcell-Meiboom-Gill (CPMG) relaxation experiments aid in identifying weak and transient interactions that are difficult to detect with conventional methods24. Ligand-based experiments are often performed at protein concentrations of 10-50 &#181;M and may require optimization of mixing times and saturation parameters25. Notably, these methods can be limited by the low solubility of glycans or the poor signal-to-noise ratio when working with small ligands.
Together, these NMR methods provide a comprehensive framework for elucidating the structural and dynamic properties of protein-glycan interactions. With ongoing advancements in sensitivity and isotopic labeling strategies, this technique is becoming increasingly essential in glycobiology, offering new perspectives on molecular recognition mechanisms with potential applications in drug development and biomarker discovery. Nonetheless, the success of these approaches depends on factors such as sample homogeneity, glycan complexity, and the presence of flexible or disordered regions that may broaden NMR signals or hinder interpretation26. NMR is a powerful method for studying transient events, which is a major challenge in structural biology.
CVN recognizes &#945;(1,2)-linked mannosyl residues present in high-mannose oligosaccharides with nanomolar affinities27,28,29. However, it poorly recognizes the monosaccharide D-mannose. In the present work, we studied the interaction of CVN with D-mannose as a way to illustrate how NMR is powerful in understanding transient interactions.

<table><tbody><tr><td>13C Glucose</td><td>Cambrige Isotopic Lab.</td><td>CLM-481</td><td></td></tr><tr><td>15N Ammonium Chloride</td><td>Cambrige Isotopic Lab.</td><td>NLM-467</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Coquetel inhibit protease</td><td>Sigma-Aldrich</td><td>4693159001</td><td></td></tr><tr><td>D&lt;sub&gt;2&lt;/sub&gt;O</td><td>Cambrige Isotopic Lab.</td><td>DLM-4</td><td></td></tr><tr><td>D-Mannose</td><td>Sigma-Aldrich</td><td>M2069-5G</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>NMRPipe</td><td></td><td></td><td>Link to the software tutorial: https://www.ibbr.umd.edu/nmrpipe/demo.html</td></tr><tr><td>Potassium Chloride (K2HPO4)</td><td>Sigma-Aldrich</td><td>P3786</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium Chloride (KH2PO4)</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Shigemi tube</td><td>Bruker</td><td>Z1Z10684A</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>Sodium phosphate (Na2HPO4)</td><td>Sigma-Aldrich</td><td>567545</td><td></td></tr><tr><td>Sodium phosphate (NaH2PO4)</td><td>Sigma-Aldrich</td><td>S97263</td><td></td></tr><tr><td>Spectrometer Bruker 600 MHz</td><td>Bruker</td><td></td><td></td></tr><tr><td>Spectrometer Bruker 800 MHz</td><td>Bruker</td><td></td><td></td></tr><tr><td>Tiamine</td><td>Sigma-Aldrich</td><td>T1270</td><td></td></tr><tr><td>Triple resonance TXI probe</td><td>Bruker</td><td></td><td></td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>93657</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>Y1625</td><td></td></tr></tbody></table>

exploring protein-glycan interactions: advances in nuclear magnetic resonance

Exploring Protein-Glycan Interactions: Advances in Nuclear Magnetic Resonance

Protein-glycan interactions are central to many biological processes and are increasingly recognized as promising targets for diagnostic strategies in infectious, inflammatory, and neoplastic diseases. However, characterizing these interactions, especially when they are weak and transient, remains technically challenging. Nuclear magnetic resonance (NMR) spectroscopy offers unique advantages for studying such interactions under near-physiological conditions, providing atomic-resolution insights into both structural and dynamic aspects. In this study, we present an integrated NMR protocol designed to investigate low-affinity protein-glycan interactions using the mannose-specific lectin cyanovirin-N and the monosaccharide D-mannose as a model system. The protocol combines ligand- and protein-based NMR approaches to map binding sites comprehensively and detect subtle binding events, including potential allosteric effects or encounter complexes. The critical steps of the protocol include careful sample preparation, precise control of concentrations, and spectral standardization to ensure data reproducibility and reliability. NMR in solution enables the detection of transient interactions that are often inaccessible by other techniques. Although weak interactions are essential for life, there is a bias in the literature toward higher-affinity complexes, even though low-affinity complexes are relevant in many important biological functions, such as signal transduction and cell-cell communication. This technique provides a powerful platform for investigating protein-glycan recognition and may serve as a valuable tool for identifying novel diagnostic markers on the basis of glycan-specific interactions.

In the present work, we presented NMR experiments to investigate protein-glycan interactions. Protein-glycan molecular recognition underlies numerous biological processes involving the extracellular matrix (ECM), including the regulation of cell adhesion, migration, proliferation, and various other forms of cellular communication.
Here, we chose to work with the monosaccharide D-mannose, which is described as a weak binder. The goal was to describe methods for measuring transient interactions ...

This NMR-based protocol investigates weak protein-glycan interactions using cyanovirin-N and D-mannose. Combining ligand- and protein-detected methods, it maps binding sites, detects allosteric effects, and identifies encounter complexes. The approach outlines sample preparation and data analysis, offering structural and dynamic insights valuable for glycan-specific diagnostics and recognition mechanisms.

Protein-glycan interactions are central to many biological processes and are increasingly recognized as promising targets for diagnostic strategies in ...

We study protein-protein and protein-glycan interactions, focusing on integrin-disintegrin complex. Our model explores structural dynamics and highlight the role of surface force in mediating complex formation and interaction stability. Our group is dedicated to the study of the role of surface forces in molecular recognition and evolution of binding sites by studying surface hydrophobic clusters present in proteins.NMR solution enables the detection of protein interactions. Although weak interactions are essential for life, there is a bias towards high-affinity complexes, even though they are present in many important biological functions. Our group is focusing on using integrative structural biology approaches to study large protein complex.We are using NMR as the main tool to study dynamics and functional aspects. To begin, prepare a four millimolar solution of D-mannose in sodium phosphate buffer at pH 7.4 with 50 millimolar sodium chloride and 5%deuterium oxide. Divide the solution into two samples, one containing 80 micromolar CVN, and the second without.For mapping the ligand binding through chemical shift perturbation on the NMR spectrometer, set up the HSQCTOPSI pulse sequence for H1C13 HSQC acquisition. Configure the time domain, the spectral width, carrier frequency, and number of scans. Calculate chemical shift perturbation using the equation provided.To map ligand binding through saturation transfer difference, first, create a new dataset. Configure the parameters for STD NMR experiments by either screening different saturation frequencies or fixing the frequency while varying saturation times to evaluate buildup. Set up 1D H1 NMR experiments using the ZGPR pulse sequence.Tune the spectrometer for H1, perform shimming, and measure a hard 90 degree pulse. Center the carrier frequency approximately at 4.7 parts per million, corresponding to the water signal, then select the STDDIFFESGP. 3 pulse sequence and set the acquisition parameters.Set the spectral width as required, interscan delay D1 to four seconds, and define saturation time D20, as per experiment goals. Load the FQ2 list with off-resonance frequency at minus 40 parts per million and on-resonance frequencies to saturate protein only. Test the on-resonance frequencies of minus 0.59, 0.73, and 8.1 parts per million to determine optimal conditions.Use the optimal frequency to acquire STD spectra at saturation times of 0.5, one, 1.5, two, 2.5, three, and four seconds. Plot the corresponding ASTD values as a function of saturation time. Now, set number of scans to 64 and average the experiment loop four times.Then, calculate the total number of scans. Set interscan delay to four seconds and acquisition time to 2.7262976 seconds. Configure saturation pulse by setting the duration to 50 milliseconds and control of the Gaussian shape via the shaped program 9SP9.Apply a T1 row filter in STD NMR variant, then, set the spin lock time D29 based on protein size. To map the ligand binding H1 R2, first, select the CPMG_ESGP2D pulse program from the Bruker standard library. Set the experiment as a 2D acquisition.Configure the acquisition parameters as shown. Then, run the experiment for 200 cycles of eight scans each. Adjust the variable counter list according to the desired total time of the CPMG cycle.Run two experiments, one for the sample with the protein and one for the ligand-only sample. Plot the H1 spectra for each TCPMG using the command EFP or sine M followed by FP, then adjust the window function according to the best processing strategy. Calculate the CPMG quotient Q using the equation.Acquire H1N15HSQC spectra of N15-labeled CVN at 298 Kelvin. Titrate with D-mannose to reach the final concentrations of zero, one, two, five, 10, 20, 40, and 60 millimoles. Use phase-sensitive FHSQCF3GPPH fast HSQC pulse sequence from the Bruker standard library.Then, calculate the chemical shift perturbation using the formula. Plot CSP values for each residue as a function of D-mannose concentration to determine dissociation constant KD using equation five. Then, fit the resulting data to a single binding isotherm using the formula.Use phase-sensitive HSQCT2ETF3GPSI3D pulse sequence and configure parameters as shown to measure the N15 R2 values of the individual residues of CVN residues in absence and presence of 60 millimolar D-mannose at 298 Kelvin. Process pseudo-three-dimensional spectra using NMRPipe to generate HSQC-like spectra for each relaxation delay. Import the processed spectra into an analysis platform.Then, select all spectra and apply the follow intensity changes tool. Plot the intensity of each cross peak as a function of T relax and fit the decay to a monoexponential function to extract N15 R2 values for each residue. Calculate the experimental error from the signal-to-noise ratio of the HSQC-like spectra at 67.84 milliseconds.Process a region of the spectrum containing only noise and convert it to a text file using the given command. Then, determine the standard deviation of the noise region or noise intensity using statistical software. Compute the experimental error using the given equation.Chemical shift perturbations were observed for both anomeric forms of D-mannose in the presence of CVN, with greater shifts seen in beta-D-mannose, indicating preferential binding. Two distinct peaks were observed for most D-mannose hydrogens in the presence of CVN, corresponding to free and bound states. The hydrogen nuclei exhibiting the highest ASTD values were associated with beta-D-mannose, consistent with strong bipolar interactions with CVN.In the transverse relaxation rate analysis, H beta four was the only proton that exhibited a notable increase in relaxation upon binding to CVN. The H1 and N15 chemical shift perturbations induced by the addition of a ligand was highest at concentration up to 10 millimolar. Residues I40, E41, N42, V43, D44, and G45 within the beta strand showed significant chemical shift perturbations, identifying them as the high-affinity binding site for D-mannose.Binding isotherms showed residue D44 had the highest affinity among the beta strand residues, with a dissociation constant of approximately one millimolar. Measurement of N15 R2 relaxation rates showed both increases and decreases in delta R2 values upon D-mannose binding, suggesting a mix of conformational stabilization and exchange processes. Residue C58, R59, K74, and R76 demonstrated increased delta R2 values, confirming their involvement in the high-affinity binding site, while residues F4, C8, R24, and G27 represented the low-affinity region.

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