Jonathan Boyd

The three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions.

The solution structure of the tenth type III module of fibronectin has been determined using nuclear magnetic resonance techniques. The molecule has a fold similar to that of immunoglobulin domains, with seven beta strands forming two antiparallel beta sheets, which pack against each other. Both beta sheets contribute conserved hydrophobic residues to a compact core. The topology is more similar to that of domain 2 of CD4, PapD, and the extracellular domain of the human growth hormone receptor than to that of immunoglobulin C domains. The module contains an Arg-Gly-Asp sequence known to be involved in cell adhesion. This tripeptide is solvent exposed and lies on a conformationally mobile loop between strands F and G, consistent with its cell adhesion function.

Chiroptical and stoichiometric evidence of a specific, primary dimerisation process in alginate gelation

Abstract The stoichiometry of calcium-ion chelation to alginate chains has been investigated by circular dichroism (c.d.), and by equilibrium dialysis in the presence of various concentrations of sodium chloride. C.d. intensity in the carboxylate π → π * spectral region increases linearly with calcium-ion concentration up to a level equivalent to half the total poly- L -guluronate stoichiometric requirement, and thereafter shows little further change. Similarly, the level of bound calcium resistant to displacement by swamping concentrations of sodium ions is equivalent to half the stoichiometric requirement of poly- L -guluronate chain-sequences alone. In terms of the previously developed “egg-box” model of co-operative junction-zone formation in alginate gelation, these results are interpreted as showing that the primary mechanism of interchain association is by dimerisation of poly- L -guluronate chain-segments in a regular, buckled, two-fold conformation related to that characterized for the free acid in the solid state, with tight interchain chelation of calcium to the carboxylate groups on the interior faces of the dimer (i.e., half the carboxylate residues of the participating chain-sequences). This interpretation is entirely consistent with previous evidence from electron microscopy, and offers a simple rationalisation of experimental results from competitive-ion binding studies.

Comparison of techniques for 1H-detected heteronuclear 1H15N Spectroscopy☆

Abstract It has previously been demonstrated that 1 H-detected heteronuclear 1 H 15 N correlation techniques increase the number of proteins that can be usefully studied by NMR. All previous studies of this type have utilized 1 H 15 N heteronuclear multiple-quantum coherence to encode 15 N chemical shift. In this paper we show that 15 N single-quantum coherence has several significant advantages including substantially better 15 N resolution (and a reduction in the transverse relaxation rate of coherence evolving during the t 1 evolution period). Experiments based upon the two techniques are demonstrated and compared using a sample of the c subunit of the Escherichia coli F 1 -F 0 ATP-synthase.

Solution structure and ligand-binding site of the SH3 domain of the p85α subunit of phosphatidylinositol 3-kinase

SH3 domains are found in proteins associated with receptor tyrosine kinase signal transduction complexes. The solution structure of the SH3 domain of the 85 kd regulatory subunit of phosphatidylinositol 3-kinase is shown to be a compact beta barrel consisting of five beta strands arranged in two beta sheets of three and two strands. The structure is similar to that of chicken brain alpha spectrin but represents a distinct class of SH3 domain, with an insertion between the second and third beta strands that may influence binding specificity. 1H chemical shift changes induced by complex formation with a synthetic peptide derived from the SH3-binding protein dynamin, together with amino acid sequence comparisons, suggest that the ligand-binding site consists of a hydrophobic surface flanked by two charged loops.

Influence of cross-correlation between dipolar and anisotropic chemical shift relaxation mechanisms upon longitudinal relaxation rates of 15N in macromolecules

Abstract The intramolecular dipole—dipole and chemical shift anisotropy relaxation mechanisms for the 15 N nucleus of the peptide bond have comparable magnitudes. The cross-correlation (or interference) between these two relaxation mechanisms is analysed using the protein human epidermal growth factor (hEGF). It is demonstrated that the longitudinal 15 N relaxation recovery is not defined by a unique time constant. A two-dimensional experiment to monitor specifically the cross-correlation between the two relaxation mechanisms is discussed. It is also shown that a simple procedure, the inclusion of 1 H broadband decoupling, removes the effects of the cross-correlation upon the 15 N longitudinal relaxation and leads to exponential 15 N recovery.

A refined solution structure of hen lysozyme determined using residual dipolar coupling data.

A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H–15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low‐energy calculated structures has an average backbone RMSD of 0.50±0.13Å to the mean structure and of 1.49±0.10Å to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation‐dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior.

Structural Studies of Alginic acid, using a bacterial poly-α-L-guluronate lyase☆

Abstract An extracellular poly-α- L -guluronate lyase from Klebsiella aerogenes degrades those blocks from alginate which contain both mannuronic and guluronic acid residues (poly-MG blocks) to a mixture of oligosaccharides. From an analysis of these products, it is concluded that poly-MG blocks do not have a strictly alternating sequence of the two uronic acid residues. Enzymic degradation of various samples of algal alginate to leave the poly-M blocks intact has shown that these blocks have a uniform chain-length, estimated at 24 residues.

Isolation of a poly-α-l-guluronate lyase from Klebsiella aerogenes

Abstract The bacterium Klebsiella aerogenes type 25 produces an extracellular alginolyase which has been partly purified. The enzyme is specific for the α- l -guluronosyl linkages in whole alginate and fractions therefrom. The end products of its action on polyguluronic acid blocks are mainly the unsaturated di- and tri-saccharides, with a smaller proportion of the homologous tetrasaccharide. Some general properties of the enzyme are reported.

The hairpin structure of the 6F11F22F2 fragment from human fibronectin enhances gelatin binding

The solution structure of the 6F11F22F2 fragment from the gelatin‐binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non‐contiguous 6F1 and 2F2 modules. The buried surface area between 6F1 and 2F2 (∼870 Å2) is the largest intermodule interface seen in fibronectin to date. The dissection of 6F11F22F2 into the 6F11F2 pair and 2F2 results in near‐complete loss of gelatin‐binding activity. The hairpin topology of 6F11F22F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin‐binding domain. This is the first high‐resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear ‘string of beads’.

Dynamic studies of a fibronectin type I module pair at three frequencies: Anisotropic modelling and direct determination of conformational exchange.

SummaryA detailed analysis of the 15N relaxation of a pair of modules from fibronectin is presented. The overall dimensions of the protein structure can be approximated by a cylinder with an axial ratio D∥/D⊥ of 1.9. T1, T2 and NOE data, collected at three 15M frequencies (50.6, 60.8 and 76 MHz), can be fitted satisfactorily to a Lipari-Szabo model, taking anisotropy into account. A method for analysing the exchange contribution to relaxation is presented. This contribution depends upon the predicted Binf0sup2frequency dependence in the fast exchange limit of these exchange terms. Using this analysis, relatively slow conformational exchange contributions are detected around one of the disulphide bonds in the first module of the pair.