S2004 Methods for characterization of biomolecular interactions – classical versus modern Mgr. Josef Houser, Ph.D. houser@mail.muni.cz Biomolecular interactions on the cell level Work with cells Positives • Real environment • All components present – metabolites, protein subunits,... • (No purification) Negatives • Inhomogeneity • High background/interference • Cell culture contamination • Instrument cleaning • Safety Calorimetry – whole cells experiments • Study of energy changes during microbial growth – isothermal (not titration!) calorimetry. • Non-destructive method, sample (cells) can be used after experiment. • Monitoring for hours or days. Calorimetry – whole cells experiments • Study of the interactions – isothermal titration calorimetry. • Example: Calorimetry – whole cells experiments Results – molar enthalpies! Calorimetry – whole cells experiments • Study of the interaction – isothermal titration calorimetry • Interaction is temperature-dependent (change in membrane fluidity). • Metabolic activity of bacteria – blank experiment with inactive bacteria necessary. • Disinfection of the machine necessary. • Specialized calorimetres preferable. SPR – whole cells experiments • Bacteria and eukaryotic cells interactions. • Immobilization necessary. • Low flow rate needed – cells diffuse much more slowly • Problems: - complexity of whole cell interaction with their biological ligand (simple binding constant equations cannot be applied, arbitrary/apparent KDs ) - changes of the living cells during time - variability within population (changes in resonance signals,). SPR – whole cells experiments • Cells are large compared to SPR effective range (app 300 nm from the sensorchip surface) • Different distances of captured cells can influence the response • The instrument only detects the portion of the cell within the detection range • The signal from the binding of whole cells is lower than expected Effective plasmon range SPR – whole cells experiments • Could be used for bacteria and also eukaryotic cells interactions. • Example: Immobilization of glycans • Non-covalent non-specific adsorption • Electrostatic interactions • Covalent immobilization • Detection of bound cells • Cell-permeant fluorescent nuclei staining dyes (SYTO 62) • Live/dead assays (Calcein AM and ethidium homodimer) High throughput! Arrays Example: Glycoarray Technologies Microarrays 2016, 5, 3; doi:10.3390/microarrays5010003 • Atomic force microscopy • Scanning of (cell) surface with modified probe AFM Flow cytometry • Detection of individual cells in flow system • Fluorescence & light scattering • Individual protein labeling – co-occurence Agglutination/precipitation methods • One binding partner on the cell surface • Second binding partner multivalent • Interaction detected as cell agglutination (macroscopic in well, microscopic under microscope) • Red blood cells (hemagglutination), yeast, latex beads Microscopy/fluorescent microscopy • Labeled probe (protein, antibody,…) • Interaction and localization Some not suitable techniques • BLI – confusing interpretation, shaking • MST – cells motility, heterogeneity • AUC – cell size – too heavy = too fast sedimentation • DLS – sedimentation, low resolution • TSA, DSF, DSC – heating necessary Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz CF Head: Michaela Wimmerová • +420 549 498 166 • michaela.wimmerova@ceitec.cz bic@ceitec.cz bic.ceitec.cz Biomolecular I nteraction and Crystallization Core Facility