This title appears in the Scientific Report :
2017
Please use the identifier:
http://hdl.handle.net/2128/14886 in citations.
Etablierung eines Systems aus Cysteinmutanten der Phosphoglycerat-Kinase für Entfaltungsstudien mit Einzelmolekül-FRET
Etablierung eines Systems aus Cysteinmutanten der Phosphoglycerat-Kinase für Entfaltungsstudien mit Einzelmolekül-FRET
Proteins maintain life through a multitude of tasks within biological systems. To gain their functionality, it is crucial that proteins obtain their native structure which is hidden within their amino acid sequence. Deciphering how the amino acid code translates into 3D structures is the key to unde...
Saved in:
Personal Name(s): | Schöne, Antonie (Corresponding author) |
---|---|
Contributing Institute: |
Molekulare Biophysik; ICS-5 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2017
|
Physical Description: |
137 S. |
Dissertation Note: |
RWTH Aachen, Diss., 2017 |
ISBN: |
978-3-95806-237-5 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Functional Macromolecules and Complexes |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Schlüsseltechnologien / Key Technologies
143 |
Subject (ZB): | |
Link: |
OpenAccess OpenAccess |
Publikationsportal JuSER |
Proteins maintain life through a multitude of tasks within biological systems. To gain their functionality, it is crucial that proteins obtain their native structure which is hidden within their amino acid sequence. Deciphering how the amino acid code translates into 3D structures is the key to understand how proteins really work. In this context, the twodomain protein phosphoglycerate kinase (PGK) has proven to be an excellent model for multi-domain proteins. It is known that both domains of PGK interact during folding. The N-terminal domain only gains its native structure in presence of the C-terminal domain. The C-domain also folds individually but the process is facilitated by the Ndomain. In addition, at least one intermediate state is involved. A detailed picture of the folding pathway of PGK and to what extent intermediates are populated is still missing. It is challenging to unravel the mechanisms of tertiary structure formation, especially since subpopulations are hard to identify with ensemble methods. To avoid averaging over all conformations, single-molecule methods are a promising tool to distinguish and quantify intermediate states. Here, a set of three yPGK cysteine variants for site-specific labeling with fluorescent dyes for single molecule fluorescence resonance energy transfer (FRET) was established. This system is designed to follow motions in between and within the individual domains displayed by distance changes of fluorophores during unfolding transitions under denaturing conditions. It was verified that secondary and tertiary structures were not considerably affected by cysteine mutations applying circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). In addition, all PGK cysteine mutants were catalytically active. The native states of the double labeled PGK variants were thoroughly characterized by fluorescence correlation spectroscopy (FCS) and single molecule FRET. Thus, a quality test to proof the suitability of yPGK variants for single-molecule FRET studies was established. First unfolding experiments (applying GndHCl for chemical denaturation) under equilibrium conditions were performed for all variants of the system. Unfolding of the inter-domain mutant, carrying a fluorophor in each of its domains, follows the classical two-state model. In contrast, the intra-domain mutants show more complex unfolding patterns. The results indicate that the N-domain forms a compact intermediate. The C-domain seems to stay locally compact even beyond the critical range of denaturant concentrations. Further studies based on the optimized sample preparation and the established quality test will provide detailed insights into the unfolding pattern of yPGK and its domains. Promising candidates to extend the proposed system were already identified. |