S2004 Methods for characterization of biomolecular interactions – classical versus modern

Faculty of Science
Autumn 2019
Extent and Intensity
2/0/0. 2 credit(s) (plus extra credits for completion). Recommended Type of Completion: zk (examination). Other types of completion: k (colloquium).
Teacher(s)
Mgr. Jitka Holková (lecturer)
Mgr. Josef Houser, Ph.D. (lecturer)
Mgr. Jan Komárek, Ph.D. (lecturer)
prof. RNDr. Michaela Wimmerová, Ph.D. (lecturer)
Guaranteed by
prof. RNDr. Michaela Wimmerová, Ph.D.
National Centre for Biomolecular Research – Faculty of Science
Supplier department: National Centre for Biomolecular Research – Faculty of Science
Course Enrolment Limitations
The course is offered to students of any study field.
Course objectives
The students will learn about the aspects of biomolecular interactions, their significance and biological relevance, and they will get an overview of both traditional and state-of-art methods for the study of biomolecular interactions.
Learning outcomes
At the end of the course students should be able to: understand and explain principles of thermodynamics and kinetics of biomolecular interactions; understand thermodynamics of binding, interpret binding curves make reasoned decisions about selecting a proper method for particular applications
Syllabus
  • 1. Introduction to 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, measurement of high-affinity and low-affinity interactions. displacement measurement, single-injection measurement, comparison with other methods, limits of ITC, possible problems. 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. Microscale thermophoresis (MST) – theoretical background, labelling and assay development, determination of Kd and stoichiometry, applications 6. 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. 7. Importance of sample preparation („garbage in, garbage out“), methods for sample quality determination.
Literature
    recommended literature
  • 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
Teaching methods
Intensive theoretical course (lectures and discussions).
Assessment methods
in-term assessment, final written test
Language of instruction
English
Further comments (probably available only in Czech)
The course can also be completed outside the examination period.
The course is taught annually.
The course is taught: in blocks.
Note related to how often the course is taught: January 10-13, 2017.
General note: in blocks, January 10-13, 2017.
Listed among pre-requisites of other courses
The course is also listed under the following terms Autumn 2011 - acreditation, Autumn 2014, Autumn 2015, Autumn 2016, autumn 2017, Autumn 2018, Autumn 2020, autumn 2021, Autumn 2022, Autumn 2023, Autumn 2024.
  • Enrolment Statistics (Autumn 2019, recent)
  • Permalink: https://is.muni.cz/course/sci/autumn2019/S2004