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Umemoto, Kimiko; Leffler, Hakon

doi:10.1023/a:1011269008175

Cited by 24 publications

(25 citation statements)

References 6 publications

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“…3A) is in agreement with the already published assignment of this protein [13]. The 1 H- 15 N HSQC of the free N-terminal domain (Fig.…”

Section: Resultssupporting

confidence: 90%

“…CRD chemical shift assignments were obtained from [13] and two sets of heteronuclear NMR experiments (HNCA and HNCOCA, and HNCO and HNCACO) were performed on the 15 N and 13 C-labeled CRD sample at 400 mM concentration in the presence and the absence of lactose. 1 H- 15 N-HSQC titrations were performed on 15 N-labeled proteins at 40 µM concentration for CRD and 30 µM for full length protein (FL), in the absence and the presence of various concentrations of ligands (lactose or LNnT).…”

Section: Methodsmentioning

confidence: 99%

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Glycan Dependence of Galectin-3 Self-Association Properties

et al. 2014

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Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.

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“…3A) is in agreement with the already published assignment of this protein [13]. The 1 H- 15 N HSQC of the free N-terminal domain (Fig.…”

Section: Resultssupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Glycan Dependence of Galectin-3 Self-Association Properties

et al. 2014

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“…The third cross peak, A 142 , because of similarities in exchange rates, must be correlated with either A6 or A4. These assignments agree with those previously made using triple resonance experiments (A 142 is assigned to A6) 26. While there is some ambiguity in assignment, the effort required for assignment is minimal.…”

Section: Discussionsupporting

confidence: 86%

“…This is a 15 kDa carbohydrate-binding protein; its resonances have been previously assigned using conventional triple resonance methods. (BioMagResBank accession # 4909 and PDB # 1A3K) 26. For the present study the protein has been labeled with 15 N alanine producing a set of six HSQC cross-peaks for assignment.…”

Section: Introductionmentioning

confidence: 99%

NMR Resonance Assignments of Sparsely Labeled Proteins: Amide Proton Exchange Correlations in Native and Denatured States

Nkari

Prestegard

2009

J. Am. Chem. Soc.

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Protein NMR assignments of large proteins using traditional triple resonance techniques depends on double or triple labeling of samples with 15 N, 13 C, and 2 H. This is not always practical with proteins that require expression in non-bacterial hosts. Labeling with isotopically labeled versions of single amino acids (sparse labeling) often is possible, however resonance assignment then requires a new strategy. Here a procedure for the assignment of cross peaks in 15 N-1 H correlation spectra of sparsely labeled proteins is presented. It relies on the correlation of proton-deuterium amide exchange rates in native and denatured spectra of the intact protein, followed by correlation of chemical shifts in the spectra of the denatured protein with chemical shifts of sequenced peptides derived from the protein.The procedure is successfully demonstrated on a sample of a protein, Galectin-3, selectively labeled with 15 N at all alanine residues.

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“…71,72 Backbone chemical shifts of L2- and L3-bound Gal3C were assigned using HNCACB spectra. (73) Arginine side-chains were assigned using CCC-TOCSY-NEHE(74) and CCONH.…”

Section: Methodsmentioning

confidence: 99%

Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3

et al. 2010

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Rational drug design is predicated on knowledge of the three-dimensional structure of the protein−ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein−carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

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Cited by 24 publications

References 6 publications

Glycan Dependence of Galectin-3 Self-Association Properties

Glycan Dependence of Galectin-3 Self-Association Properties

NMR Resonance Assignments of Sparsely Labeled Proteins: Amide Proton Exchange Correlations in Native and Denatured States

Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3

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