Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered and Freely Diffusing Proteins

Atta, Diaa

Dissertation / PhD Thesis/Book

Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered and Freely Diffusing Proteins

Atta, D. (Corresponding author)FZJ*

2011
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-89336-763-4

Jülich : Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag, Schriften des Forschungszentrums Jülich. Schlüsseltechnologien / Key Technologies 35, IV, 126 S. (2011) = Universität Düsseldorf, Diss., 2011

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Please use a persistent id in citations: http://hdl.handle.net/2128/4556

Abstract: In my thesis I aimed to study protein folding/unfolding transitions and protein synthesis using single molecule fluorescence techniques. For this purpose I employed confocal and wide-field fluorescence microscopes. For surface immobilized biomolecules I set up and calibrated an autofocus wide-field system for time-resolved simultaneous dual color imaging. In the case of freely diffusing proteins a confocal microscope with pulsed excitation was used for time-resolved anisotropy measurements and for fluorescence correlation spectroscopy (FCS). In the first project the synthesis of the green fluorescence protein (GFP) has been monitored while the protein was tethered to surface immobilized functional ribosomes. In time resolved studies we obtained characteristic maturation times of our GFP mutant which was rather fast (5 min) compared to most other GFP mutants. In further studies we aimed to analyze the folding process in more detail by incorporating fluorescent dyes (BODIPY-TMR) into the polypeptide chain using a cell-free transcription/translation system. First results were obtained which prove a partial integration of the dye into chain and gave valuable findings how to continue in order to establish a Förster resonance energy transfer (FRET) pair for folding studies. In the second part of this work we performed protein folding studies on single molecule level with Phosphoglycerate kinase (PGK) encapsulated in polymeric vesicles as nano-containers. Some of the most interesting properties of polymeric vesicles are their remarkable stability against harsh environmental conditions and their permeability for chemical denaturants. We demonstrated in an application on protein folding, that surface-tethered polymerosomes are suitable to perform time-resolved single molecules studies with encapsulated proteins. A third project dealt with a structural and dynamical characterization of protein structures by employing FCS in order to obtain hydrodynamic radii of the protein. We studied a few multi-domain proteins under different concentration of a chemical denaturant (guanidine hydrochloride, GndHCl) and observed at low denaturant concentrations ($\le$ 1 M GndHCl) a slight compaction of the protein structures which was followed by a drastic expansion of the proteins in the unfolded state. For PGK we were also able to resolve structural parameters of the protein by analyzing a characteristic fast process which was caused by photo induced electron transfer (PET).

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