772409 Instructional course IA - Spectroscopic and calorimetric analysis of proteins (in Eng.)


Art
Übung
Semesterstunden
2
Vortragende/r (Mitwirkende/r)
Pfanzagl, Vera , Hofbauer, Stefan , Furtmüller, Paul Georg
Organisation
Angeboten im Semester
Sommersemester 2021
Unterrichts-/ Lehrsprachen
Englisch

Lehrinhalt

The course focuses on techniques related to structural and thermodynamic analysis of protein unfolding by means of (bio)molecular spectroscopy and calorimetry.

Introduced techniques include electronic circular dichroism (ECD) and fluorescence and UV-Vis spectroscopy as well as differential scanning calorimetry (DSC).

The fundamentals and application of these methods is introduced as is the handling of the corresponding instruments.

In detail students will learn to record and interpret the ECD spectrum of a protein in the far-UV, near-UV and visible region.

Programme(s) that calculate the content of secondary structure elements will be introduced.

Thermal unfolding of a model protein will be performed as well as calculation of equilibrium constants at different temperatures.

The student will learn data presentation as van’t Hoff plot as well as calculation of enthalpy, entropy and free energy of unfolding.

Besides Tm value(s) the thermal stability (free energy at room temperature) is obtained.

Fluorescence and UV-Vis spectroscopy will be used to follow the chemical denaturation by either urea or guanidinium hydrochloride of a model protein by monitoring changes of the intrinsic fluorescence of tryptophan or tyrosine upon unfolding.

Finally, the advantages of DSC in structural and functional analysis of proteins are shown.

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Lehrziel

After completion of the course students will know the advantages and disadvantages of circular dichroism and fluorescence spectroscopy as well as DSC in structural and functional analysis of protein stability.
Additionally, they will know the application and experimental setup of these spectroscopic and calorimetric techniques in obtaining structural (e.g. secondary structure content, conformational change etc) and thermodynamic data (e.g. conformational and thermal stability, thermodynamic parameters of binding) of the proteins of interest
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