Fluorescence spectroscopic studies of protein conformational dynamics

Fluorescence spectroscopic studies of protein conformational dynamics

Kroehn, Phillip Gunther

Dissertation

Angenommen am: 2013-10-21

Erschienen: 2013-10-31

Betreuer: Enderlein, Jörg Prof. Dr.

Gutachter: Enderlein, Jörg Prof.

Gutachter: Grubmüller, Helmut Prof. Dr.

Zum Verlinken/Zitieren: http://hdl.handle.net/11858/00-1735-0000-0001-BC14-D

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Name: PhdThesis Phillip Kroehn1.pdf

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Zusammenfassung

Englisch

The folding of a protein into its native structure is the fundamental prerequisite for its functionality. The polypeptide chain itself contains the chemical information necessary for spontaneous folding of the protein. Nevertheless, many proteins need chaperones in order to support their folding into their native structure. It is currently possible to predict the three dimensional structure of some small proteins from their primary structure. Linking protein structure and chain composition, however, is essential for understanding of molecular mechanisms in every living organism. Detailed understanding of the protein folding process is one of the most important questions in the natural sciences. Protein folding occurs in microseconds to minutes on nanometer length scales. Therefore, a method of high accuracy is necessary in order to monitor protein folding. F orster resonance energy transfer (FRET) is a method that o ers the unique opportunity to measure distances in molecules with high precision. Application of single molecule experiments is the only way that allows monitoring folding events in detail. In this work, protein folding is measured using single molecule FRET experiments. The di usion of small molecules is very fast (around 10^-6 cm2/s) and therefore challenging to determine. In the present study, this problem is addressed by using a highly precise setup specialized for single molecule measurements that allows for determining di usion coe cients with an accuracy of 10% or better. A WW-domain protein is used as a model system for the folding of small globular proteins and therefore yields fundamental insights into the principle of protein folding in general. WW-domains are small, three-stranded, all- protein-domains (6 kDa) which appear in several di erent proteins. In general, the handling of small proteins with standard biochemical methods is very challenging. However, in this work, we show how the preparation of WW-domain FRET samples was conducted combining several protein puri cation techniques and, thus, overcoming the di culties of labeling.

Keywords: Protein folding, protein labeling, FRET, single molecule spectroscopy