Exploiting the carboxylate chemical shift to resolve degenerate resonances in spectra of 13C-labelled glycosaminoglycans.

Glycosaminoglycans (GAG) are important vertebrate extracellular matrix polysaccharides that comprise repeated units of an acidic and an N-acetylated sugar. The constituent acidic sugars are central to their biological functions, but have been largely inaccessible to NMR because the (1)H resonances overlap with those from other residues. Here, pulse sequences that address this failure are developed using (13)C-enriched oligosaccharides of the glycosaminoglycan, hyaluronan, as model systems. Two pulse sequences are presented that exploit the unique chemical shifts and scalar couplings present at the carboxylate moiety to filter out coherences from the N-acetylated sugars and produce simple spectra containing only resonances from the acidic sugars. The first sequence uses one-bond couplings to correlate the carboxylate carbon with the adjacent carbon and its directly attached proton, while the second sequence exploits a long-range coupling to correlate the carboxylate carbon with the anomeric proton and carbon of the same residue. In addition, inclusion of an isotropic mixing block into these sequences allows resonances from the otherwise degenerate ring protons to be resolved. Spectra from the hyaluronan tetra- and hexasaccharides show that all glucuronic acid (GlcA) residues can be resolved from one another, allowing nuclei to be assigned in a sequence-specific manner. However, in some spectra, resonances are observed at positions not predicted by spin-operator analysis, and simulations reveal that these additional magnetisation transfers result from strong-coupling. These experiments represent a foundation from which new structural and biochemical information can be obtained in a sequence-specific manner for the acidic sugar residues in hyaluronan and other glycosaminoglycans

Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.

We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a cell-free translation system. Here this method is combined with the conformational sensitivity of 19F NMR to monitor the folded state of the expressed protein. This report describes the optimisation of 6-fluorotryptophan incorporation in a His-tagged human serum retinol-binding protein (RBP), a disulphide bonded beta-barrel protein. Appropriate reagent concentrations for producing fluorine labelled RBP in a cell-free translation system are described. It is shown that 19F NMR is a suitable method for monitoring the production of correctly folded protein from a high-throughput expression system

Activation of H-2 by halogenocarbonylbis(phosphine)rhodium(I) complexes. The use of parahydrogen induced polarisation to detect species present at low concentration

Complexes of the form RhX(CO)(PR3)2 [X = Cl, Br or I; R = Me or Ph] reacted with H2 to form a series of binuclear complexes of the type (PR3)2H2Rh(μ-X)2Rh(CO)(PR 3) [X = Cl, Br or I, R = Ph; X = I, R = Me] and (PMe3)2(X)HRh(μ-H)(μ-X)Rh(CO)(PMe3) [X = Cl, Br or I] according to parahydrogen sensitised 1H, 13C, 31P and 103Rh NMR spectroscopy. Analogous complexes containing mixed halide bridges (PPh3)2H2Rh(μ-X)(μ-Y)Rh(CO)(PPh 3) [X, Y = Cl, Br or I; X ≠ Y] are detected when RhX(CO)(PPh3)2 and RhY(CO)(PPh3)2 are warmed together with p-H2. In these reactions only one isomer of the products (PPh3)2H2Rh(μ-I)(μ-Cl)Rh(CO)(PPh 3) and (PPh3)2H2Rh(μ-I)(μ-Br)Rh(CO)(PPh 3) is formed in which the μ-iodide is trans to the CO ligand of the rhodium(I) centre. When (PPh3)2H2Rh(μ-Cl)(μ-Br)Rh(CO)(PPh 3) is produced in the same way two isomers are observed. The mechanism of the hydrogen addition reaction is complex and involves initial formation of RhH2X(CO)(PR3)2 [R = Ph or Me], followed by CO loss to yield RhH2X(PR3)2. This intermediate is then attacked by the halide of a precursor complex to form a binuclear species which yields the final product after PR3 loss. The (PPh3)2H2Rh(μ-X)2Rh(CO)(PPh 3) systems are shown to undergo hydride self exchange by exchange spectroscopy with rates of 13.7 s-1 for the (μ-Cl)2 complex and 2.5 s-1 for the (μ-I)2 complex at 313 K. Activation parameters indicate that ordering dominates up to the rate determining step; for the (μ-Cl)2 system ΔH‡ = 52 ± 9 kJ mol-1 and ΔS‡ = -61 ± 27 J K-1 mol-1. This process most likely proceeds via halide bridge opening at the rhodium(III) centre, rotation of the rhodium(III) fragment around the remaining halide bond and bridge re-establishment. If the triphenylphosphine ligands are replaced by trimethylphosphine distinctly different reactivity is observed. When RhX(CO)(PMe3)2 [X = Cl or Br] is warmed with p-H2 the complex (PMe3)2(X)HRh(μ-H)(μ-X)Rh(CO)(PMe3) [X = Cl or Br] is detected which contains a bridging hydride trans to the rhodium(I) PMe3 ligand. However, when X = I, the situation is far more complex, with (PMe3)2H2Rh(μ-I)2Rh(CO)(PMe 3) observed preferentially at low temperatures and (PMe3)2(I)HRh(μ-H)(μ-I)Rh(CO)(PMe3) at higher temperatures. Additional binuclear products corresponding to a second isomer of (PMe3)2(I)HRh(μ-H)(μ-I)Rh(CO)(PMe3), in which the bridging hydride is trans to the rhodium(I) CO ligand, and (PMe3)2HRh(μ-H)(μ-I)2Rh(CO)(PMe 3) are also observed in this reaction. The relative stabilities of related systems containing the phosphine PH3 have been calculated using approximate density functional theory. In each case, the (μ-X)2 complex is found to be the most stable, followed by the (μ-H)(μ-X) species with hydride trans to PH3. © The Royal Society of Chemistry 1999

Activation of H-2 by halogenocarbonylbis(phosphine)rhodium(I) complexes. The use of parahydrogen induced polarisation to detect species present at low concentration

Complexes of the form RhX(CO)(PR3)2 [X = Cl, Br or I; R = Me or Ph] reacted with H2 to form a series of binuclear complexes of the type (PR3)2H2Rh(μ-X)2Rh(CO)(PR 3) [X = Cl, Br or I, R = Ph; X = I, R = Me] and (PMe3)2(X)HRh(μ-H)(μ-X)Rh(CO)(PMe3) [X = Cl, Br or I] according to parahydrogen sensitised 1H, 13C, 31P and 103Rh NMR spectroscopy. Analogous complexes containing mixed halide bridges (PPh3)2H2Rh(μ-X)(μ-Y)Rh(CO)(PPh 3) [X, Y = Cl, Br or I; X ≠ Y] are detected when RhX(CO)(PPh3)2 and RhY(CO)(PPh3)2 are warmed together with p-H2. In these reactions only one isomer of the products (PPh3)2H2Rh(μ-I)(μ-Cl)Rh(CO)(PPh 3) and (PPh3)2H2Rh(μ-I)(μ-Br)Rh(CO)(PPh 3) is formed in which the μ-iodide is trans to the CO ligand of the rhodium(I) centre. When (PPh3)2H2Rh(μ-Cl)(μ-Br)Rh(CO)(PPh 3) is produced in the same way two isomers are observed. The mechanism of the hydrogen addition reaction is complex and involves initial formation of RhH2X(CO)(PR3)2 [R = Ph or Me], followed by CO loss to yield RhH2X(PR3)2. This intermediate is then attacked by the halide of a precursor complex to form a binuclear species which yields the final product after PR3 loss. The (PPh3)2H2Rh(μ-X)2Rh(CO)(PPh 3) systems are shown to undergo hydride self exchange by exchange spectroscopy with rates of 13.7 s-1 for the (μ-Cl)2 complex and 2.5 s-1 for the (μ-I)2 complex at 313 K. Activation parameters indicate that ordering dominates up to the rate determining step; for the (μ-Cl)2 system ΔH‡ = 52 ± 9 kJ mol-1 and ΔS‡ = -61 ± 27 J K-1 mol-1. This process most likely proceeds via halide bridge opening at the rhodium(III) centre, rotation of the rhodium(III) fragment around the remaining halide bond and bridge re-establishment. If the triphenylphosphine ligands are replaced by trimethylphosphine distinctly different reactivity is observed. When RhX(CO)(PMe3)2 [X = Cl or Br] is warmed with p-H2 the complex (PMe3)2(X)HRh(μ-H)(μ-X)Rh(CO)(PMe3) [X = Cl or Br] is detected which contains a bridging hydride trans to the rhodium(I) PMe3 ligand. However, when X = I, the situation is far more complex, with (PMe3)2H2Rh(μ-I)2Rh(CO)(PMe 3) observed preferentially at low temperatures and (PMe3)2(I)HRh(μ-H)(μ-I)Rh(CO)(PMe3) at higher temperatures. Additional binuclear products corresponding to a second isomer of (PMe3)2(I)HRh(μ-H)(μ-I)Rh(CO)(PMe3), in which the bridging hydride is trans to the rhodium(I) CO ligand, and (PMe3)2HRh(μ-H)(μ-I)2Rh(CO)(PMe 3) are also observed in this reaction. The relative stabilities of related systems containing the phosphine PH3 have been calculated using approximate density functional theory. In each case, the (μ-X)2 complex is found to be the most stable, followed by the (μ-H)(μ-X) species with hydride trans to PH3. © The Royal Society of Chemistry 1999

NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1.

EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated (15)N, (13)C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded beta sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor alpha4beta1

The solution structure of EMILIN1 globular C1q domain reveals a disordered insertion necessary for interaction with the alpha4beta1 integrin.

The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded beta-sandwich fold of the gC1q domain is reduced to nine beta strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the alpha4beta1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of alpha4beta1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu(933)). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-alpha4beta1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site

The solution structure of EMILIN1 globular C1q domain reveals a disordered insertion necessary for interaction with the alpha4beta1 integrin

The extracellular matrix protein EMILIN1 (elastin microfi- bril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfi- bril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded \u2424-sandwich fold of the gC1q domain is reduced to nine \u2424 strands with a consequent increase in the size of the cen- tral cavity lumen. An unstructured loop, resulting from an inser- tion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, spe- cifically engages the \u24234\u24241 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of \u24234\u24241 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu933). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-\u24234\u24241 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site