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13C-NMR relation study of heparin-disaccharide interactions with tripeptides GRG and GKG.

ABSTRACT: Heparin is a polydisperse sulphated copolymer consisting mostly of 1-->4 linked glucosamine and uronic acid residues, i.e. 2-deoxy-2-sulphamido-D-glucopyranose 6-sulphate and L-idopyranosyluronic acid 2-sulphate. 13C NMR has been used to study the interactions of heparinase-derived and purified heparin disaccharide with N- and C-terminally-blocked tripeptides GRG and GKG. Titration of the disaccharide with peptide indicates that GRG binds the disaccharide more strongly than does GKG, with interactions in either case being stronger at uronate ring positions. In the presence of GRG, a carboxylate pKa depression suggests electrostatic interactions between the arginine guanidinium group and the uronate carboxylate group. 13C relaxation data have been acquired for all disaccharide and peptide carbons in the presence and absence of GRG and GKG. 13C relaxation rates for the disaccharide are significantly faster in the presence of peptide, especially with GRG. Analysis of these relaxation data has been done in terms of molecular diffusion constants, D [symbol: see text] and D parallel, and an angle alpha between D parallel and a molecular frame defined by the moment of inertia tensor calculated for an internally rigid disaccharide. Disaccharide conformational space in these calculations has been sampled for both uronate half-chair forms (2H1 and 1H2) and over a range of glycosidic bond angles defined by motional order parameters and inter-residue nuclear Overhauser effects (+/- 30 degree from the average). In the absence of peptide, the ratio D [symbol: see text] /D parallel falls between 0.4 and 0.7; therefore molecular diffusion occurs preferentially about D parallel, which runs through both disaccharide rings. In the presence of peptide, D [symbol: see text] /D parallel is decreased, indicating that GRG is oriented along D parallel and proximal to the uronic acid ring. A model for this is shown.

SUBMITTER: Mikhailov D

PROVIDER: S-EPMC1217216 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Project description:1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.

Project description:The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly (13)C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular (13)C-(13)C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D (1)H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for (13)C T1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ~200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks, which are often difficult to resolve in the parent 2D spectra.

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13C-NMR relation study of heparin-disaccharide interactions with tripeptides GRG and GKG.

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