A monodisperse transmembrane α-helical peptide barrel

The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

A general method for co-crystallisation of concanavalin A with carbohydrates.

A small grid of conditions has been developed for co-crystallization of the plant lectin concanavalin A (conA) and polysaccharides. Crystals have been obtained of complexes of conA with alpha 1-2 mannobiose, 1-methyl alpha 1-2 mannobiose, fructose, a trisaccharide and a pentasaccharide. The crystals diffract to resolutions of 1.75-2.7 Angstrom using a copper rotating-anode source. The crystals are grown in the presence of polyethylene glycol 6K [10=20%(w/v)] at around pH 6.0. Optimization for each particular carbohydrate requires small adjustments in the conditions; however, all complexes give some crystalline precipitate in this limited grid. The alpha 1-2 mannobiose complex crystals diffract to 1.75 Angstrom with space group I222 and cell dimensions a = 91.7, b = 86.8, c = 66.6 Angstrom . One monomer is present in the asymmetric unit. The 1-methyl alpha 1-2 mannobioside complex crystallizes in space group P2(1)2(1)2(1), cell dimensions a = 119.7, b = 119.7, c = 68.9 Angstrom and diffract to 2.75 Angstrom. One tetramer is present in the asymmetric unit. Two crystal forms of the conA-fructose complex have been obtained. The first has space group P2(1)2(1)2(1), cell dimensions a = 121.7, b = 119.9, c = 67.3 Angstrom with a tetramer in the asymmetric unit and diffracts to 2.6 Angstrom. The second crystallizes in space group C222(1), cell dimensions a = 103.3, b = 117.9, c = 254.3 Angstrom with two dimers in the asymmetric unit and diffracts to 2.42 Angstrom. Structures and crystallization of the trisaccharide-conA and pentasaccharide-conA complexes have already been reported. In all complexes, the protein is found as a tetramer, although varying combinations of non-crystallographic and crystallographic symmetry are involved in generating the tetramer. The precise packing of the tetramer varies from crystal to crystal and it is likely that this variability facilitates crystallization.</p

Concanavalin A distorts the GlcNAc b1-2 Man linkage of the pentasaccharide core upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;6)]- Man) of N-linked oligosaccharides with a K-a of 1.41 x 10(6) M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man- (1--&gt;6)]-Man to 2.7 Angstrom. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1--&gt;6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1--&gt;3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1--&gt;2)-Man glycosidic linkage PSI torsion angle on the 1--&gt;6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1--&gt;6 arm GlcNAc site.</p

Concanavalin A distorts the GlcNAc b1-2 Man linkage of the pentasaccharide core upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;6)]- Man) of N-linked oligosaccharides with a K-a of 1.41 x 10(6) M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man- (1--&gt;6)]-Man to 2.7 Angstrom. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1--&gt;6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1--&gt;3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1--&gt;2)-Man glycosidic linkage PSI torsion angle on the 1--&gt;6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1--&gt;6 arm GlcNAc site.</p

A general method for co-crystallisation of concanavalin A with carbohydrates.

A small grid of conditions has been developed for co-crystallization of the plant lectin concanavalin A (conA) and polysaccharides. Crystals have been obtained of complexes of conA with alpha 1-2 mannobiose, 1-methyl alpha 1-2 mannobiose, fructose, a trisaccharide and a pentasaccharide. The crystals diffract to resolutions of 1.75-2.7 Angstrom using a copper rotating-anode source. The crystals are grown in the presence of polyethylene glycol 6K [10=20%(w/v)] at around pH 6.0. Optimization for each particular carbohydrate requires small adjustments in the conditions; however, all complexes give some crystalline precipitate in this limited grid. The alpha 1-2 mannobiose complex crystals diffract to 1.75 Angstrom with space group I222 and cell dimensions a = 91.7, b = 86.8, c = 66.6 Angstrom . One monomer is present in the asymmetric unit. The 1-methyl alpha 1-2 mannobioside complex crystallizes in space group P2(1)2(1)2(1), cell dimensions a = 119.7, b = 119.7, c = 68.9 Angstrom and diffract to 2.75 Angstrom. One tetramer is present in the asymmetric unit. Two crystal forms of the conA-fructose complex have been obtained. The first has space group P2(1)2(1)2(1), cell dimensions a = 121.7, b = 119.9, c = 67.3 Angstrom with a tetramer in the asymmetric unit and diffracts to 2.6 Angstrom. The second crystallizes in space group C222(1), cell dimensions a = 103.3, b = 117.9, c = 254.3 Angstrom with two dimers in the asymmetric unit and diffracts to 2.42 Angstrom. Structures and crystallization of the trisaccharide-conA and pentasaccharide-conA complexes have already been reported. In all complexes, the protein is found as a tetramer, although varying combinations of non-crystallographic and crystallographic symmetry are involved in generating the tetramer. The precise packing of the tetramer varies from crystal to crystal and it is likely that this variability facilitates crystallization.</p

Man a1-2 Mana-OMe concanavalin A complex reveals balance of forces involved in carbohydrate recognition

We have determined the crystal structure of the methyl glycoside of Man alpha 1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha 1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha 1-3 and alpha 1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha 1-2 linked disaccharide, Crystals of the Man alpha 1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 Angstrom, b = 119.7 Angstrom, c = 68.9 Angstrom and diffract to 2.75 Angstrom. The final model has good geometry and an R factor of 19.6% (R-free = 22.8%), One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible, In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted,In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.</p

Man a1-2 Mana-OMe concanavalin A complex reveals balance of forces involved in carbohydrate recognition

We have determined the crystal structure of the methyl glycoside of Man alpha 1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha 1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha 1-3 and alpha 1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha 1-2 linked disaccharide, Crystals of the Man alpha 1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 Angstrom, b = 119.7 Angstrom, c = 68.9 Angstrom and diffract to 2.75 Angstrom. The final model has good geometry and an R factor of 19.6% (R-free = 22.8%), One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible, In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted,In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.</p

Crystallization of succinylated concanavalin A bound to a synthetic bivalent ligand and preliminary structural analysis

Crystals have been obtained of succinylated concanavalin A complexed to a novel bidentate synthetic ligand. The crystals are the first example of a lectin with a synthetic multivalent ligand and the first report of crystallization of succinylated concanavalin A. The crystals were obtained by sitting-drop vapour diffusion equilibrating with a solution of 20% polyethylene glycol, pH 5, 293.5 K. Crystals are orthorhombic, belonging to space group C222(1) with unit-cell dimensions of a = 99.1, b = 127.4, c = 118.9 Angstrom. The asymmetric unit contains a dimer, with over 65% of the volume occupied by water. The ligand cross links concanavalin A monomers. Succinylated concanavalin A is known to be a dimer in solution, yet it is found as the typical concanavalin A tetramer in the crystal. The contacts holding together the tetramer appear extensive and suggest that a fine balance between dimer and tetramers exists. Data to 2.65 Angstrom have been collected and the structure determined by the molecular replacement method.</p