Projects

Central Service Projects

Synthetic chitin/chitosan oligomers

In this service project, we will prepare chemically defined oligomers of chitin. Using automated glycan assembly (AGA) we can obtained oligomers with full control over the sequence, length, degree and pattern of acetylation. Representative structures can be found in the references below. Each oligomer is provided on a mg scale (1-5 μmols), for interest in bigger quantities please contact the responsible person. Upon request, the oligomer can be prepared with an amino-linker at the reducing end for further conjugation. Additional modifications can be discussed upon request.

References:

Tyrikos-Ergas, T.; Bordoni, V.; Fittolani, G.; Chaube, M.; Grafmüller, A.; Seeberger, P. H.; Delbianco, M.: Systematic structural characterization of chitooligosaccharides enabled by Automated Glycan Assembly. Chemistry – A European Journal 27 (7), pp. 2321 – 2325 (2021)

Fittolani, G.; Tyrikos-Ergas, T.; Vargová, D.; Chaube, M.; Delbianco, M.: Progress and challenges in the synthesis of sequence controlled polysaccharides. Beilstein Journal of Organic Chemistry 17, pp. 1981 – 2025 (2021)

Huang, J.-Y.; Delbianco, M.: Recent developments in solid-phase glycan synthesis. Synthesis (2022), DOI: 10.1055/a-1938-2293

Contact person:

Dr. Martina Delbianco

Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces

Am Mühlenberg 1, 14476 Potsdam, Germany

E-mail: martina.delbianco@mpikg.mpg.de

Production and characterisation of chitosans: DP, DA, PA

In this service project, we can prepare and provide:

• a Codeχ-starter set of chitosans (mg amounts) to be used by partners interested in exploring the influence of DP, DA, and PA on any physico-chemical property or biological activity of chitosans – if this is interesting to you and you can contribute with your assay(s) to this comparative analysis, or if you can provide a special chitosan for the set, please contact Bruno Moerschbacher (moersch@uni-muenster.de)
• chitin polymers (crystalline, insoluble) from shrimp (alpha) and squid (beta), possibly also from fungi and insects (less pure)
• chitosan polymers with different degrees of acetylation (DA) prepared by partial chemical N-acetylation of polyglucosamine and, hence, random pattern of acetylation (PA); if required, these may be prepared with different degrees of polymerisation (DP) (medium double-digit mg range)
• chitosan polymers with different DAs and possibly DPs, prepared by enzymatic N-acetylation and, depending on the enzyme used, different non-random PAs (low double-digit mg range)
• selected, fully defined, partially acetylated chitosan oligomers (e.g. all possible dimers, trimers, and tetramers, some pentamers and hexamers) prepared by partial enzymatic deacetylation of chitin oligomers or by partial enzymatic N-acetylation of glucosamine oligomers (single-digit mg range)
• mixtures of partially acetylated chitosan oligomers with known DP and DA distribution and end-defined PA prepared by enzymatic depolymerization of chitosan polymers using chitosan hydrolases with known subsite specificities (up to g range)
• chitin and chitosan affinity proteins (artificial lectins) tagged with different fluorescent tags
• recombinant chitin and chitosan degrading enzymes (different chitinases, chitosanases, and chitin deacetylases)
• chitosan beads (prepared by enzymatic surface deacetylation of chitin beads)

In terms of analysis, we can offer as a service:

• structural analysis of chitosan polymer samples regarding their average DP and their dispersity Đ, their average DA, and their PA
• sequencing of chitosan oligomers, i.e. determination of DP, DA, and PA (including in not too complex mixtures)
• structural analysis of mixtures of chitosan polymers and oligomers (as e.g. often obtained during enzymatic digestion of chitosan polymers)
• quantification and average DA analysis of chitosans in “simple” biological matrices (such as typical fungal cell walls)

In collaborations (e.g. when you send a doctoral candidate to us to perform analyses under the supervision of e.g. Dr. Stefan Cord-Landwehr or Dr. Ratna Singh), we can offer:

• quantification and average DA analysis of chitosans in more complex (or new-to-us) biological matrices
• analyses of chitin and chitosan modifying enzymes (chitinases, chitosanases, chitin deacetylases) (e.g. determination of subsite specificities and preferences, processivity, transglycosylating activity, basic kinetic parameters)
• in silico modelling of chitin/chitosan interacting proteins (chitin or chitosan binding proteins, receptors, enzymes) (e.g. 3D structure prediction, ligand docking, protein-ligand interactions, protein-protein interactions, molecular dynamics, DFT calculations for studying electronic
• properties of oligomers with varying DP, DA, and PA, structure-based docking, screening against an oligomer library with varying DP, DA, and PA)
• transcriptomics and microbiome meta-transcriptomics data analysis

Contact person

Prof. Dr. Bruno Moerschbacher

Westfälische Wilhelms-Universität Münster
Institut für Biologie und Biotechnologie der Pflanzen (IBBP)
Schlossplatz 8
48143 Münster

moersch@uni-muenster.de

Analysis of chito-protein and protein-protein and interactions

This service project provides equipment and expertise for the determination of binding properties of chito-protein and protein-protein interactions by surface-coated quart crystal microbalance-dissipation (QCM-D) chips and grating-coupled interferometry (GCI).

As chitin and chitosan (C/CS) polymers are insoluble fibrillar materials, accurate measurement of kinetic parameters and determination of equilibrium constants for  C/CS interacting proteins is challenging. We have developed a rapid QCM-D based method to determine binding constants of chitin-binding proteins on chitin-coated gold surfaces (Vogt et al, 2018, Analyst 143, 5255-5263). For this purpose, alpha-chitin was first trimethylsilylated and coated onto the sensor chips. After desilylation, regular fibril‑like structures with a typical center-to-center spacing of 85 nm were observed by atomic force microscopy. Using different setups and data evaluation methods for QCM-D measurements, we determined k_on and k_off, and calculated the K_D values for binding of a recombinant CBD from Bacillus circulans chitinase A1. This bacterial CBD has been analyzed in detail before and thus served as a well-characterized reference for determining chitin-binding properties by QCM-D measurements. The obtained K_D values were in good agreement with those measured for other bacterial CBDs usually ranging between 1 to 10 µM. We will provide this experimental approach in Service Project 3 to determine unknown binding affinities of various chitin-binding domains/proteins to crystalline alpha-chitin fibers. We further will extend this method to allow measurements for the binding to beta-chitin fibers, and to chitins and chitosans with various degrees of acetylation and polymerization.

In this service project, we will also offer measurements of protein interactions using grating coupled interferometry (GCI), which is based on the highly sensitive waveguide interferometry principle. For this purpose, the GCI device will be requested as a central device of the consortium. The system uses interchangeable, reusable chips based on a robust microfluidic system that prevents clotting and allows measurements in complex solutions, as it provides excellent signal-to-noise ratios due to an extended light-to-sample interaction length. Functionalized chips are available with different coatings to bind one of the interaction partners. Therefore, interaction measurement are possible in undiluted plasma and serum, crude membrane extracts and complex matrices. This allows high-resolution determination of binding parameters with reliable kinetics below 1pg/mm2 without the need of data averaging. In addition, the measurements of the interaction of very small ligands (>1000 Da, including COS) with large proteins or particles (e.g. liposomes) are possible. The kinetic range to be determined is very broad and ultra-fast transition times (reliable off-rates of up to 10 sec^-1). The system will be extended for high throughput measurements at single ligand concentrations using the waveRAPID Kinetics™ in the second funding period. In addition, reusable WAVEchips will be developed that are equipped with layers of different chemically defined chito-oligomers (coupled via polyethylene glycol linkers using thiol chemistry, in collaboration with SP).

In addition to determination of biding parameters , we provide the following support:

• Non-radioactive assays for measuring chitin synthesis
• Fluorescent recombinant chitin-binding proteins
• Production and pruficiation of recombinant proteins in bacterial, fungal or insect cells

References

S. Vogt, M. Kelkenberg, T Nöll, B. Steinhoff, H. Schönherr, H. Merzendorfer, G. Nöll. “Rapid determination of binding parameters of chitin binding domains using chitin-coated quartz crystal microbalance sensor chips.” Analyst 143, 5255-63 (2018)

C. Hong, N.J. Byrne, B. Zamlynny, S. Tummala, L. Xiao, J.M. Shipman, A.T. Partridge, C. Minnick, M.J. Breslin, M.T. Rudd, et al. “Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation.” Nat Commun 12, 815. DOI: https://doi.org/10.1038/s41467-021-21087-6 (2021)

H. Jankovics, B. Kovacs, A. Saftics, T. Gerecsei, E. Toth, I. Szekacs, F. Vonderviszt, R. Horvath. “Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers”. Sci Rep 10, 22253. DOI: https://doi.org/10.1038/s41598-020-79226-w (2020)

Ö. Kartal, F. Andres, M.P. Lai, R. Nehme, R & K. Cottier. ” waveRAPID- A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVE system.” SLAS DISCOVERY: Advancing the Science of Drug Discovery, 24725552211013827. DOI: https://doi.org/10.1101/2021.02.05.42987 (2021)

P. Jimenez Sandoval, J. Santiago. “In vitro analytical approaches to study plant ligand-receptor interactions”, Plant Physiology, DOI: https://doi.org/10.1104/pp.19.01396 (2020)

Contact person

Prof. Dr. Hans Merzendorfer

Department Chemistry-Biology, School of Natural Science and Technology, University of Siegen, Germany

Adolf-Reichwein-Str. 2, 57076 Siegen, Germany

hans.merzendorfer@uni-siegen.de