The determination of a suitable buffer environment for a protein of interest is not an easy task. The requirements of advanced techniques, the demands on the biological material and the researcher time needed for buffer optimization, as well as personal inflexibility, lead frequently to the use of sub-optimal buffers. Here, we demonstrate the design of a 48-condition buffer screen that can be used to determine an appropriate environment for downstream studies. By the combination of several techniques (differential scanning fluorimetry, dynamic light scattering, and bio-layer interferometry), we are able to assess the protein stability, homogeneity and binding activity across the screen with less than half a milligram of protein in 1 day. The application of this screen helps to avoid unsuitable conditions, to explain problems observed upon protein analysis and to choose the most suitable buffers for further research. The screen can be routinely used as a primary screen for buffer optimization in labs and facilities.

• Keywords
• Bio-layer interferometry, Buffer, Differential scanning fluorimetry, Dynamic light scattering, Protein stability, Screening,
• MeSH
• Dynamic Light Scattering MeSH
• Fluorometry MeSH
• Proteins MeSH
• Buffers MeSH
• Protein Stability * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteins MeSH
• Buffers MeSH

Series of multivalent α-l-fucoside containing glycoclusters and variously decorated l-fucosides were synthesized to find potential inhibitors of fucose-specific lectins and study the structure-binding affinity relationships. Tri- and tetravalent fucoclusters were built using copper-mediated azide-alkyne click chemistry. Series of fucoside monomers and dimers were synthesized using various methods, namely glycosylation, an azide-alkyne click reaction, photoinduced thiol-en addition, and sulfation. The interactions between compounds with six fucolectins of bacterial or fungal origin were tested using a hemagglutination inhibition assay. As a result, a tetravalent, α-l-fucose presenting glycocluster showed to be a ligand that was orders of magnitude better than a simple monosaccharide for tested lectins in most cases, which can nominate it as a universal ligand for studied lectins. This compound was also able to inhibit the adhesion of Pseudomonas aeruginosa cells to human epithelial bronchial cells. A trivalent fucocluster with a protected amine functional group also seems to be a promising candidate for designing glycoconjugates and chimeras.

• Keywords
• cystic fibrosis, glycoclusters, hemagglutination, l-fucosides, lectins, multivalency,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Fucose chemistry   metabolism   MeSH
• Fungal Proteins chemistry   metabolism   MeSH
• Hemagglutination MeSH
• Lectins chemistry   metabolism   MeSH
• Humans MeSH
• Hemagglutination Inhibition Tests MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• fucose-binding lectin MeSH Browser
• Fucose MeSH
• Fungal Proteins MeSH
• Lectins MeSH

Protein-carbohydrate interactions are very often mediated by the stacking CH-π interactions involving the side chains of aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) or phenylalanine (Phe). Especially suitable for stacking is the Trp residue. Analysis of the PDB database shows Trp stacking for 265 carbohydrate or carbohydrate like ligands in 5 208 Trp containing motives. An appropriate model system to study such an interaction is the AAL lectin family where the stacking interactions play a crucial role and are thought to be a driving force for carbohydrate binding. In this study we present data showing a novel finding in the stacking interaction of the AAL Trp side chain with the carbohydrate. High resolution X-ray structure of the AAL lectin from Aleuria aurantia with α-methyl-l-fucoside ligand shows two possible Trp side chain conformations with the same occupation in electron density. The in silico data shows that the conformation of the Trp side chain does not influence the interaction energy despite the fact that each conformation creates interactions with different carbohydrate CH groups. Moreover, the PDB data search shows that the conformations are almost equally distributed across all Trp-carbohydrate complexes, which would suggest no substantial preference for one conformation over another.

• MeSH
• Databases, Protein MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Lectins chemistry   metabolism   MeSH
• Carbohydrate Metabolism * MeSH
• Tryptophan chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• lectin, Aleuria aurantia MeSH Browser
• Lectins MeSH
• Tryptophan MeSH

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcβ1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed β-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

• MeSH
• Bacterial Proteins genetics   immunology   MeSH
• Host-Pathogen Interactions immunology   MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Lectins chemistry   genetics   immunology   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Photorhabdus genetics   immunology   MeSH
• Surface Plasmon Resonance MeSH
• Base Sequence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• fucose-binding lectin MeSH Browser
• Lectins MeSH

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

• MeSH
• Aspergillus fumigatus immunology   pathogenicity   MeSH
• Adult MeSH
• Fluorescent Antibody Technique MeSH
• Fucose metabolism   MeSH
• Fungal Proteins immunology   metabolism   MeSH
• Lectins immunology   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Macrophages immunology   metabolism   MeSH
• Disease Models, Animal MeSH
• Mucins immunology   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Pulmonary Aspergillosis immunology   metabolism   MeSH
• Flow Cytometry MeSH
• Immunity, Mucosal immunology   MeSH
• Spores, Fungal immunology   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• fucose-binding lectin MeSH Browser
• Fucose MeSH
• Fungal Proteins MeSH
• Lectins MeSH
• Mucins MeSH