Request ID: cfg_rRequest_1224

Status : Approved
Project Description : Analysis to identify suitable ligands for the crystallization and structural determination of the Flo5 and Flo11 proteins and BabA adhesion
CFG Member : Not Consortium Member
Requester First Name : Manuel
Requester Last Name : Maestre-Reyna
Head of Lab First Name : Lars-Oliver
Head of Lab Last Name : Essen
Assigned First Name : Lars-Oliver
Assigned Last Name : Essen
Requester Email : sirverdemer@gmail.com
Requester Interest : Information not entered/not applicable.
Request Date [yyyy-mm-dd] : 0007-11-28
Institution : Philipps Universitaet Marburg (Germany)
Shipping Address : Hans-Meerwein Str. 35043 Marburg Hessen Germany Tel: 004964212825714
Comments : Information not entered/not applicable.
CFG Core : H
Resource Type : Glycoarray
Amount Requested : Information not entered/not applicable.
Date that your RNA/GBP samples will be sent to the core [yyyy-mm-dd] : 0007-12-03
Experiment to be conducted : In our lab, two groups of glycan-binding proteins are being studied. Namely Helicobacter pylory adhesins and flocculins from Saccharomyces cerevisiae. BabA is an adhesin found in the outer membrane of Helicobacter pylori. Several studies have shown that BabA contributes significantly to H. pylori adhesion to the stomach’s inner mucosa by binding the Lewis b (Leb) antigen, but no biochemical data are yet available for this interaction [Björnham O. et. al. J Biomed Opt. (2005) 10(4):44024, Bai Y.et. al. World J Gastroenterol (2004) 10(17):2560-2.]. In our lab, the BabA extracellular domain is expressed as inclusion bodies and refolded under either mildly or under strongly acidic conditions. As the native protein is initially exposed to the acid medium of the stomach until the bacterium’s urease activity is initiated and ammonia is exported to the cell surface, both refolding modi might be relevant for protein function,. It is therefore of great interest to biochemically characterize the binding to any oligosaccharides and to identify the active conformation for this protein. The Flo5 and Flo11 proteins are two of the five flocculins present in Saccharomyces cerevisiae. They are expressed and presented on the cell-surface for example in starvation states and cause the yeast to aggregate. This is important in biotechnical processes like industrial fermentation, but also medically relevant in mating and biofilm formation. We have overexpressed and purified the A-domains of these flocculins which are supposed to mediate aggregation. The A-domain of Flo5 shows significant sequence identity to the all-beta PA14 domain [Rigden et al., Trends Biochem Sci 29, 335–339]. This domain is present in many bacterial toxins but also in eukaryotic proteins and is assumed to bind mannose, which, as a constituent of the core-glycans, is ubiquitous on the cell-surface of yeast. The Flo11 protein shows a similar domain structure to the other flocculins [Verstrepen et al., Nature Reviews Microbiology 2, 533-540], but no significant sequence identity. It is found to be important in biofilm formation, [Verstrepen et al., Molecular Microbiology (2006) 60 (1), 5–15], but it’s binding partners are not determined yet. Amongst the aims of our lab, first and foremost comes the crystallization and structural determination of flocculins as well as of both BabA conformations, with and without the ligand. A necessary first step to achieve co-crystals is to identify suitable ligands, a task that would be best performed through the Consortium for Functional Glycomics glycan array technology. Furthermore, the identification of ligands is critical in order to undertake binding studies, which are also inside the scope of our projects. Data collected with the help of the Consortium for Functional Glycomics could ultimately be used to develop optimized industrial methods of yeast-biofermentation and ways to interfere with bacterial and fungal infections. In the following table, we show the probes we intend to send you: Protein Labelling method pH Flo 11 Alexafluor fluorophore 9 Flo 11 His-tag (immunofluorescence) 8 Flo 5 Alexafluor fluorophore 9 Flo 5 His-tag (immunofluorescence) 8 BabA 235 Alexafluor fluorophore 5.8 BabA 235 His-tag (immunofluorescence) 5.8 BabA 235 Alexafluor fluorophore 2.5 BabA 235 His-tag (immunofluorescence) 2.5
Within Scope of Consortium : Y
If yes, indicate the person responsible for inputing data into core B : David Smith
GBP being Addressed : Protein Labelling method pH Flo 11 Alexafluor fluorophore 9 Flo 11 His-tag (immunofluorescence) 8 Flo 5 Alexafluor fluorophore 9 Flo 5 His-tag (immunofluorescence) 8 BabA 235 Alexafluor fluorophore 5.8 BabA 235 His-tag (immunofluorescence) 5.8 BabA 235 Alexafluor fluorophore 2.5 BabA 235 His-tag (immunofluorescence) 2.5
Specifc aims being addressed : Define the specificity and affinity for carbohydrate ligands. Identify the ligand(s) that mediate GBP binding. Determine the structures of selected GBPs.