Dataset Information

Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3.

ABSTRACT: Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free-state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin-3, and report enhanced RDCs that prove consistent with known bound-ligand geometries for this protein.

SUBMITTER: Zhuang T

PROVIDER: S-EPMC2242564 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3.

Zhuang Tiandi T Leffler Hakon H Prestegard James H JH

Protein science : a publication of the Protein Society 20060602 7

Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements ...[more]

PMID: 16751604

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Project description:The solution conformation behavior of a dermatan-derived tetrasaccharide--Delta HexA-(1-->3)-GalNAc4S-beta-(1-->4)-IdoA-alpha-(1-->3)-red-GalNAc4S (S is a sulfate group)--has been explored by means of NMR spectroscopy, especially by NOE-based conformational analysis. The tetrasaccharide was present as four species, two of which are chemically different in the anomeric orientation of the reducing 2-deoxy-2-acetamido-galactose (red-GalNAc) residue, while the other two are the result of different conformations of the iduronic acid (IdoA) unit. The two alpha-beta-interconverting anomers were present in a 0.6:1 ratio. Ring conformations have been defined by analysis of (3)J(H,H) coupling constants and interresidual NOE contacts. Both 2-deoxy-2-acetamido-galactose (GalNAc) residues were found in the (4)C(1) chair conformation, the unsaturated uronic acid (Delta-Hex A) adopts a strongly predominant half-chair (1)H(2) conformation, while the IdoA residue exists either in the (1)C(4) chair or in the (2)S(0) skewed boat geometries, in a 4:1 ratio. There is a moderate flexibility of Phi and Psi torsions as suggested by nuclear Overhauser effects (NOEs), molecular modeling (MM), and molecular dynamics (MD) studies. This was further investigated by residual dipolar couplings (RDCs). One-bond C--H RDCs ((1)D(C,H)) and long-range H-H ((3)D(H,H)) RDCs were measured for the tetrasaccharide in a phage solution and interpreted in combination with restrained MD simulation. The RDC-derived data substantially confirmed the validity of the conformer distribution resulting from the NOE-derived simulations, but allowed an improved definition of the conformational behavior of the oligosaccharides in solution. In summary, the data show a moderate flexibility of the four tetrasaccharide species at the central glycosidic linkage. Differences in the shapes of species with the IdoA in skew and in chair conformations and in the distribution of the sulfate groups have also been highlighted.

Dataset Information

Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3.

Publications

Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3.

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