We thank Agencia Estatal de Investigaci&#243;n of Spain for the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, funded by MCIN/AEI/10.13039/ 501100011033, and CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII, Madrid, Spain).&#160;We also thank the European commission for the GLYCOTWINNING project.

Advanced NMR Techniques for Glycan-Protein Interaction Analysis

<ol>
	<li>Sol&#237;s, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+into+glycosciences:+How+chemistry,+biochemistry+and+biology+cooperate+to+crack+the+sugar+code.">A guide into glycosciences: How chemistry, biochemistry and biology cooperate to crack the sugar code.</a> Biochim Biophys Acta. 1850 (1), 186-235 (2015).</li><li>Poveda, A., Jimenez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+studies+of+carbohydrate-protein+interactions+in+solution.">NMR studies of carbohydrate-protein interactions in solution.</a> Chem Soc Rev. 27, 133-144 (1998).</li><li>Kogelberg, H., Sol&#237;s, D., Jim&#233;nez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+structural+insights+into+carbohydrate-protein+interactions+from+NMR+spectroscopy.">New structural insights into carbohydrate-protein interactions from NMR spectroscopy.</a> Curr Opin Struct Biol. 13 (5), 646-653 (2003).</li><li>Widmalm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+shape,+motions,+and+interactions+explored+by+NMR+spectroscopy.">Glycan shape, motions, and interactions explored by NMR spectroscopy.</a> JACS Au. 4 (1), 20-39 (2024).</li><li>Marchetti, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Rules+of+Engagement&amp;#34;+of+protein-glycoconjugate+interactions:+A+molecular+view+achievable+by+using+NMR+spectroscopy+and+molecular+modeling.">34;Rules of Engagement&amp;#34; of protein-glycoconjugate interactions: A molecular view achievable by using NMR spectroscopy and molecular modeling.</a> ChemistryOpen. 5 (4), 274-296 (2016).</li><li>Ard&#225;, A., Jim&#233;nez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recognition+of+glycans+by+protein+receptors.+Insights+from+NMR+spectroscopy.">The recognition of glycans by protein receptors. Insights from NMR spectroscopy.</a> Chem Commun. 54 (38), 4761-4769 (2018).</li><li>Gimeno, A., Valverde, P., Ard&#225;, A., Jim&#233;nez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+structures+and+their+interactions+with+proteins.">Glycan structures and their interactions with proteins.</a> A NMR view. Curr Opin Struct Biol. 62, 22-30 (2020).</li><li>Ca&#241;ada, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+and+structural+characterization+of+carbohydrates+and+their+interactions+studied+by+NMR.">Conformational and structural characterization of carbohydrates and their interactions studied by NMR.</a> Curr Med Chem. 29 (7), 1147-1172 (2022).</li><li>Valverde, P., Quintana, J. I., Santos, J. I., Ard&#225;, A., Jim&#233;nez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+NMR+avenues+to+explore+the+conformation+and+interactions+of+glycans.">Novel NMR avenues to explore the conformation and interactions of glycans.</a> ACS Omega. 4, 13618-13630 (2019).</li><li>Jim&#233;nez-Barbero, J., Asensio, J. L., Ca&#241;ada, F. J., Poveda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+and+protein-bound+carbohydrate+structures.">Free and protein-bound carbohydrate structures.</a> Curr Opin Struct Biol. 9 (5), 549-555 (1999).</li><li>Quintana, J. I., Atxabal, U., Unione, L., Ard&#225;, A., Jim&#233;nez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+multivalent+carbohydrate-protein+interactions+by+NMR.">Exploring multivalent carbohydrate-protein interactions by NMR.</a> Chem Soc Rev. 52 (5), 1591-1613 (2023).</li><li>Rold&#243;s, V., Ca&#241;ada, F. J., Jim&#233;nez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate-protein+interactions:+a+3D+view+by+NMR.">Carbohydrate-protein interactions: a 3D view by NMR.</a> Chembiochem. 12 (7), 990-1005 (2011).</li><li>Unione, L., Ard&#225;, A., Jim&#233;nez-Barbero, J., Millet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+of+glycoproteins:+profiling,+structure,+conformation,+and+interactions.">NMR of glycoproteins: profiling, structure, conformation, and interactions.</a> Curr Opin Struct Biol. 68, 9-17 (2021).</li><li>del Carmen Fern&#225;ndez-Alonso, M., D&#237;az, D., Berbis, M. A., Marcelo, F., Ca&#241;ada, J., Jim&#233;nez-Barbero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-carbohydrate+interactions+studied+by+NMR:+from+molecular+recognition+to+drug+design.">Protein-carbohydrate interactions studied by NMR: from molecular recognition to drug design.</a> Curr Protein Pept Sci. 13 (8), 816-830 (2012).</li><li>Angulo, J., Nieto, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STD-NMR:+application+to+transient+interactions+between+biomolecules-a+quantitative+approach.">STD-NMR: application to transient interactions between biomolecules-a quantitative approach.</a> Eur Biophys J. 40 (12), 1357-1369 (2012).</li><li>Mayer, M., Meyer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+epitope+mapping+by+saturation+transfer+difference+NMR+to+identify+segments+of+a+ligand+in+direct+contact+with+a+protein+receptor.">Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor.</a> J Am Chem Soc. 123 (25), 6108-6117 (2001).</li><li>Williamson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+chemical+shift+perturbation+to+characterise+ligand+binding.">Using chemical shift perturbation to characterise ligand binding.</a> Prog Nucl Magn Reson Spectrosc. 73, 1-16 (2013).</li><li>Fern&#225;ndez, C., Adeishvili, K., W&#252;thrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+relaxation-optimized+NMR+spectroscopy+with+the+outer+membrane+protein+OmpX+in+dihexanoyl+phosphatidylcholine+micelles.">Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles.</a> Proc Natl Acad Sci U S A. 98 (5), 2358-2363 (2001).</li><li>Yee, A., Gutmanas, A., Arrowsmith, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+NMR+in+structural+genomics.">Solution NMR in structural genomics.</a> Curr Opin Struct Biol. 16 (5), 611-617 (2006).</li><li>Skinner, S. P., Fogh, R. H., Boucher, W., Ragan, T. J., Mureddu, L. G., Vuister, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCPNMR+AnalysisAssign:+a+flexible+platform+for+integrated+NMR+analysis.">CCPNMR AnalysisAssign: a flexible platform for integrated NMR analysis.</a> J Biomol NMR. 66 (2), 111-124 (2016).</li><li>Gimeno, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+the+entropy+penalty+for+ligand+binding:+Lessons+from+the+molecular+recognition+of+the+histo+blood-group+antigens+by+human+Galectin-3.">Minimizing the entropy penalty for ligand binding: Lessons from the molecular recognition of the histo blood-group antigens by human Galectin-3.</a> Angew Chem Int Ed Engl. 58 (22), 7268-7272 (2019).</li></ol>

The authors have nothing to disclose.

Saturation transfer difference NMR (STD-NMR) has become the most used and versatile NMR method for studying ligand-protein interactions. As shown above, it relies on the saturation transfer phenomenon, and the experimental setup involves the acquisition of two one-dimensional (1D) 1H spectra: the on-resonance&#172; and the &#172;off-resonance spectra. During the on-resonance experiment, saturation of specific protons of the protein is achieved by applying a train of low-power radiofrequency pulses during a certain period (saturation time typically ranges from 1-3 s). To avoid direct saturation of the ligand, the frequency and length of the saturation pulses are optimized for selectively irradiating specific protons of the protein; i.e., they must be applied at a frequency vacant of any ligand signals and with an appropriate length (Figure 1). As a rule of thumb for 50 ms saturation pulses, 1 ppm difference should be kept from the saturation region to the closest ligand signals. Generally, selective saturation pulses applied on the aliphatic region of the protein provide increased saturation effects. Alternatively, aromatic protons (6-7 ppm) can also be irradiated if the ligand molecule does not contain any aromatic signals. This is very useful for naturally occurring glycans, as they do not bear aromatic groups. Once a certain region of the protein is selectively irradiated, the saturation propagates along the protein via dipolar 1H-1H cross-relaxation (spin diffusion). Eventually, the saturation reaches the protein protons at the binding site, which is then transferred to the sugar protons that are in close contact (r &#60; 5 &#197;) with the receptor via intermolecular 1H-1H NOEs. Obviously, the intensity of the signals of the saturated ligand protons decreases. After receiving the saturation, due to the binding kinetics, the transiently bound ligands (fast exchange is required) dissociate and the saturation information is accumulated in the free state. Due to this process, the NMR on-resonance spectra present diminished signals (Figure 2).
To clearly exhibit this intensity perturbation of the 1H nuclei of a binding glycan, a control proton NMR spectrum (off-resonance) is acquired in which the saturation is applied far away from any receptor or carbohydrate signal (usually between 40-100 ppm), under the same conditions. The subtracted 1D spectrum between the off-resonance and on-resonance exclusively shows the signals of the 1H nuclei of the ligand that have modified intensities: those that were close enough to the receptor binding site to receive the magnetization (Figure 2).
Nevertheless, not all the 1H nuclei of the bound carbohydrate receive the same amount of saturation. Theoretically, the magnetization transfer from the receptor to the bound ligand is distance-dependent (1/r6). This means that the intensities of transferred saturation among the glycan 1H nuclei contain information on the spatial proximities between the protons of the ligand and those of the receptor, and the STD NMR intensities are larger for those protons that are closer to the receptor. Accordingly, the STD NMR experiment also allows determining the binding epitope of the carbohydrate (Figure 2 and Figure 3) since protons of the ligand sitting closer to the protein surface show higher intensities than those that do not directly participate in the binding.
The experiment can be applied to systems with weak-medium affinity, rarely to systems with strong affinities in the low &#181;M or nM range. Indeed, it requires that the dissociation rate be fast in the relaxation time scale. Otherwise, the saturation transfer information is lost through relaxation before the ligand dissociates.
On the other hand, protein-based NMR experiments are unique to unraveling ligand-protein interaction with amino acid level accuracy without solving the atomic resolution structures. It directly examines molecular recognition phenomena in solution without the need for co-crystallization. CSP analysis mapping is exceptionally powerful for discovering ligands and mapping the protein binding site (Figure 4 and Figure 5). This method is applicable to any range of affinities between the mM and nM range, even for systems where the exchange rate is slow in the chemical shift time scale21.
Nevertheless, this approach will probably not work for proteins with molecular weights above 30-40 kDa due to relaxation issues. The TROSY alternative18 can then be used, being particularly powerful when coupled with protein deuteration. Moreover, the protein should be uniformly labeled with 15N (and another sample double labeled with 13C and 15N to be able to complete the required backbone assignment). Therefore, protein expression conditions, including the corresponding expression system should be optimized to be able to obtain milligram amounts of protein. Proteins that display a tendency to oligomerize or aggregate are also not suitable for this analysis. The instrument used herein to record the NMR data is a Bruker 800 MHz spectrometer equipped with a TCI cryoprobe. It would be highly challenging to use this methodology using instruments below 600 MHz or without a cryogenic probe.

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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mm Shigemi microtube set mat</td>
			<td>CortecNet SAS</td>
			<td>S30BMS-005B</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha-Lactose-Agarose</td>
			<td>Sigma-Aldrich Qu&#237;mica S.L.</td>
			<td>7634-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride (15 N, 99%)</td>
			<td>LC-0179-N-50G</td>
			<td>Tracer Tecnolog&#237;as Anal&#237;ticas S.L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin (Sodium Salt)&#160;</td>
			<td>Melford Laboratories LTD</td>
			<td>A40040</td>
			<td></td>
		</tr>
		<tr>
			<td>BIOVIA Discovery studio&#160;</td>
			<td>BIOVIA, Dassault Syst&#232;mes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3) Chemically Competent Cells&#160;</td>
			<td>Merck Life Science, S.L.U.</td>
			<td>CMC0014-40X40UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-22R</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O&#160;</td>
			<td>Cambridge Isotope Laboratories, Inc.</td>
			<td>DLM-4-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Eppendorf</td>
			<td>Innova 42</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (Isopropyl &#223;-D-1-thiogalactopyranoside)</td>
			<td>VWR International Eurolab S.L.</td>
			<td>VW437144N</td>
			<td></td>
		</tr>
		<tr>
			<td>LacNAc</td>
			<td>Elicityl</td>
			<td>GLY008</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani (LB) Broth</td>
			<td>Merck Life Science, S.L.U.</td>
			<td>3397-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Matraz Erlenmeyer B N 5000 CC&#160;</td>
			<td>VWR International Eurolab S.L.</td>
			<td>214-1137</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>Bio-Rad</td>
			<td>1610780</td>
			<td></td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>PyMOL Molecular Graphics System</td>
			<td>Version 2.0 Schr&#246;dinger</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics &#38; Materials, Inc.</td>
			<td>VC 505</td>
			<td></td>
		</tr>
		<tr>
			<td>Superconducting NMR magnet</td>
			<td>Bruker</td>
			<td>600 MHz AVANCE III</td>
			<td></td>
		</tr>
		<tr>
			<td>Superconducting NMR magnet</td>
			<td>Bruker</td>
			<td>800 MHz AVANCE III</td>
			<td></td>
		</tr>
	</tbody>
</table>

disentangling glycan-protein interactions: nuclear magnetic resonance (nmr) to the rescue

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.

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.

Herein, we present a protocol for the exploitation of 1H-STD NMR and 1H-15N HSQC experiments to unravel the details of the binding interaction between lectins and small oligosaccharides. The results obtained in the analysis of the molecular recognition of LacNAc by hGalectin-7 (hGal-7) are included, serving as an illustrative example of the successful implementation of the protocol and the effectiveness of these NMR methodologies to study the fine details of the molecular recognition process. Figure 3 shows the 1H-STD NMR spectrum for the interaction of LacNAc with hGal-7. The existence of STD NMR signals indicates binding (Figure 3A). Moreover, only those signals belonging to protons in close contact with the protein show up, allowing the delineation of the binding epitope (Figure 3B). Figure 4 highlights how the&#160;1H-15N HSQC spectrum of a protein can be used as its fingerprint, and Figure 5 illustrates the application of 1H-15N heteronuclear single quantum coherence (HSQC) titration experiments to define the chemical shift perturbation of hGalectin-7 backbone amide groups upon LacNAc binding. These data not only reveal the existence of interaction but also delineate the lectin&#39;s binding interface. Figure 6 demonstrates how the analysis of the titration data enables the estimation of the binding affinity of LacNAc by hGalectin-7, which falls in the high micromolar range. This finding is consistent with results obtained using alternative techniques.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66530/66530fig01.jpg" /> 
Figure 1: The selection of the on-resonance frequency. 1H-NMR spectrum of LacNAc:hGal-7 50:1 ratio in deuterated phosphate-buffered saline at pH 7.4 is shown. Signals of the ligand (LacNAc) are confined in the region between 2.0-5.2 ppm. The saturation frequency is carefully selected to ensure the absence of ligand protons within a 1-2 ppm range, allowing the selective irradiation of the protein&#39;s protons. <a href="https://www.jove.com/files/ftp_upload/66530/66530fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66530/66530fig02.jpg" /> 
Figure 2: The STD NMR experiment. Schematic representation of the STD experiment: the first spectrum (off-resonance) serves as a reference while in the second (on-resonance), protein saturation is performed. The saturation is efficiently propagated across the entire protein and transferred to the ligand protons in close contact with the protein. The resulting difference spectrum (STD spectrum) yields only those resonances that have experienced saturation. The analysis of the STD experiment allows the epitope mapping of the binding sugar. <a href="https://www.jove.com/files/ftp_upload/66530/66530fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66530/66530fig03.jpg" /> 
Figure 3: Binding analysis from the ligand&#39;s perspective. (A) Superimposition of the off-resonance and 1H STD-NMR spectra for the interaction of LacNAc with hGal-7. In the STD spectrum, only those signals belonging to protons in close contact with the protein show up. The annotation of the 1H resonances of the ligand is reported in the off-resonance spectrum. (B) The relative STD intensities were colored-mapped into the chemical structure of LacNAc. <a href="https://www.jove.com/files/ftp_upload/66530/66530fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66530/66530fig04.jpg" /> 
Figure 4: The 1H-15N HSQC spectrum of a protein represents its fingerprint. (A) 1H-15N HSQC spectrum of 100 &#181;M of hGal-7 in the apo form. The spectrum was recorded at 25 &#176;C. Some NH cross-peaks were annotated with the label of their corresponding amino acid. (B) Each NH pair displays a unique chemical shift that depends on the chemical environment and consequently, on the 3D structure of the protein. <a href="https://www.jove.com/files/ftp_upload/66530/66530fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66530/66530fig05.jpg" /> 
Figure 5: Binding analysis from the protein&#39;s perspective. (A) Superimposition of the 1H-15N HSQC spectra recorded for the titration of LacNAc into hGal-7 solution is shown. Inspection of the spectra, where several cross-peaks experience chemical shift changes, clearly indicates interaction. (B) The plot of the maximum chemical shift perturbations (maxCSP) of the backbone amide signals deduced from the titration of LacNAc (15 equivalents) with hGal-7. (C) The most perturbed amino acids of hGal-7, according to the CSP analysis are mapped into the 5gal PDB structure. In the 3D model, the red coloration refers to CSP value over 2&#963;, whereas the pink ones to values between 1&#963; and 2&#963;. The colored region likely represents the binding site. <a href="https://www.jove.com/files/ftp_upload/66530/66530fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66530/66530fig06.jpg" /> 
Figure 6: KD determination based on 1H-15N HSQC titration experiments. (A) Representation of the pattern of 1H-15N HSQC-based titration depending on the chemical exchange rate in the NMR time scale of the system in the study (fast, intermediate, or slow). A fast exchange regime was observed in the case of LacNAc/hGal-7 interaction. (B) Fitting curve and KD estimation obtained from the CSP analysis at varying ligand concentrations for the model system of hGal-7 and LacNAc disaccharide. The estimated KD is reported with the corresponding error as an average of the data for 20 different amino acids; (C) Snippets of the 1H,15N-HSQC spectra displaying the shift of selected cross-peaks during the titration. <a href="https://www.jove.com/files/ftp_upload/66530/66530fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

disentangling-glycan-protein-interactions-nuclear-magnetic-resonance

Research

JoVE Journal

Chemistry

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.

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

1.0K Views.  CIC bioGUNE, Basque Research and Technology Alliance (BRTA).  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. 

Advanced NMR Techniques for Glycan-Protein Interaction Analysis

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