S2004 Methods for characterization of biomolecular interactions – classical versus modern

Přírodovědecká fakulta
podzim 2014
Rozsah
2/0. 2 kr. (plus ukončení). Doporučované ukončení: zk. Jiná možná ukončení: k.
Vyučující
Mgr. Eva Dubská (přednášející)
Mgr. Josef Houser, Ph.D. (přednášející)
Mgr. Jan Komárek, Ph.D. (přednášející)
Mgr. Lenka Malinovská, Ph.D. (přednášející)
prof. RNDr. Michaela Wimmerová, Ph.D. (přednášející)
Garance
prof. RNDr. Michaela Wimmerová, Ph.D.
Národní centrum pro výzkum biomolekul - Přírodovědecká fakulta
Dodavatelské pracoviště: Národní centrum pro výzkum biomolekul - Přírodovědecká fakulta
Omezení zápisu do předmětu
Předmět je otevřen studentům libovolného oboru.
Cíle předmětu
At the end of the course students should be able to: understand and explain principles of thermodynamics and kinetics of biomolecular interactions;
evaluate thermodynamics of binding
interpret binding curves
make reasoned decisions about selecting a proper method for particular applications
Osnova
  • 1. Introduction Biomolecular interactions (significance, biological relevance, equilibrium, association and dissociation constants, kinetics). Types of interactions (hydrophobic, coulombic, hydrogen bonds, van der Waals forces). 2. Classical versus modern methodology Classical methods for characterization of interactions (equilibrium dialysis, frontal chromatography). Direct measurement of complex formation (change in absorbance, fluorescence intensity, fluorescence polarization). Surface plasmon resonance (theoretical background, comparison with ELISA methods). 3. Thermodynamics of protein-ligand interactions Thermodynamics of binding (Gibbs free energy, enthalpy, entropy). Macroscopic and microscopic views. The pH and temperature dependence of complex formation. Entropy-enthalpy compensation. Isothermal titration calorimetry (theoretical background, comparison with surface plasmon resonance). 4. Oligomerization and protein-protein interactions Protein stoichiometry, stereochemistry. Protein folding (hydrophobic effect, hydrophilic interactions, hydrogen bonds, electrostatic forces, water molecules). Subunit-subunit interactions (electrostatic and shape complementarity). Protein-protein recognition sites. Determination of oligomerization (cross-linking, analytical ultracentrifugation). 5. Characterization of interactions on cell level Possibility of whole cell assay within SPR and ITC. Modeling of native conditions (sensor chip versus in-solution measurement). Haemagglutination and fluorescence (theory, application). In vivo living-cell imaging. Host-pathogen interactions imaging in tissues. PRACTICAL EXERCISES 1. Surface plasmon resonance (SPR) Preparation of samples. Direct and inhibition assay. Data evaluation. Demonstration of strong points and weaknesses of SPR method. 2. Isothermal titration calorimetry (ITC) Measurement of high-affinity and low-affinity interactions. Displacement measurement. Data evaluation and analysis. Limits of ITC, possible problems. Single-injection measurement. 3. Cross-linking and analytical ultracentrifugation Cross-linking with several different agents and proteins (importance of suitable experimental set-up). Determination of quaternary structure by analytical ultracentrifugation, pH dependence of oligomerization. Data evaluation. 4. Haemagglutination and fluorescence microscopy Haemagglutination with different proteins and blood types. Inhibition of haemagglutination. Fluorescence microscopy (microscopy with labeled proteins and living cells, combination with haemagglutination). Microscopy of biofilms and living nematodes.
Literatura
    doporučená literatura
  • Protein-protein interactions : methods and applications. Edited by Haian Fu. Totowa, N.J.: Humana Press, 2004. xvi, 532. ISBN 1588291200. info
  • Protein-ligand interactions : hydrodynamics and calorimetry : a practical approach. Edited by Stephen E. Harding - Babur Z. Chowdhry. 1st pub. Oxford: Oxford University Press, 2001. xxiv, 330. ISBN 0-19-963749-0. info
  • Protein-ligand interactions : structure and spectroscopy : a practical approach. Edited by S. E. Harding - Babur Z. Chowdhry. 1st pub. Oxford: Oxford University Press, 2001. xxvi, 436. ISBN 0199637474. info
Výukové metody
Intensive course covering lectures, class discussion, and practical exercises
Metody hodnocení
in-term assessment, final written test
Vyučovací jazyk
Angličtina
Další komentáře
Studijní materiály
Předmět je dovoleno ukončit i mimo zkouškové období.
Předmět je vyučován každoročně.
Výuka probíhá blokově.
Nachází se v prerekvizitách jiných předmětů
Předmět je zařazen také v obdobích podzim 2011 - akreditace, podzim 2015, podzim 2016, podzim 2017, podzim 2018, podzim 2019, podzim 2020.