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Single-molecule FRET studies of protein function and conformational dynamics. from DNA nanotechnology to viral and bacterial infections
Single-molecule FRET studies of protein function and conformational dynamics. from DNA nanotechnology to viral and bacterial infections
All fundamental cellular processes are governed by dynamic interactions of nano-sized biomolecules such as proteins and DNA. A technique well suited to resolve inter- and intramolecular distances is Förster Resonance Energy Transfer (FRET). By combining FRET with single-molecule spectroscopy, heterogeneities in the system can be detected and dynamic information becomes accessible. In this thesis, single-molecule FRET (smFRET) experiments were performed using either the small observation volume of a confocal microscope or the evanescent field of a total internal reflection (TIRF) microscope to get insights into the enzymatic activity of three proteins. To study the end-joining of DNA double strands by the T4 DNA ligase, a DNA origami platform was designed to control the stoichiometry and spatial arrangement of the interaction partners, thereby increasing their local concentration. The ligation process could be followed in real time and the same pair of DNA strands repeatedly ligated and cut, which highlights the applicability of the DNA origami platform to a wide range of multimolecular interaction studies on the single-molecule level. In a second project, the underlying catalytic mechanism of NSP2, a rotavirus protein required for genome replication and virus assembly, was investigated by comparing the RNA unwinding activity of full-length and C-terminally truncated mutants. Although the C-terminal region was less efficient in destabilizing the secondary structure of RNA, it proved to be essential for RNA release via charge repulsion and is thus a prerequisite for a full cycle of chaperone activity. In the last study, the conformational states of bacterial adhesion protein SdrG were studied. This protein initiates nosocomial infections through its stable attachment to human fibrinogen. By combining FRET-derived distances and molecular dynamics (MD) simulations, the structure of the dynamic apoprotein could be modeled, providing information inaccessible to other methods. The diverse topics researched in this thesis - ranging from DNA nanotechnology to viral and bacterial infections - emphasize the multifaceted capabilities of smFRET.
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Bartnik, Kira
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Bartnik, Kira (2020): Single-molecule FRET studies of protein function and conformational dynamics: from DNA nanotechnology to viral and bacterial infections. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

All fundamental cellular processes are governed by dynamic interactions of nano-sized biomolecules such as proteins and DNA. A technique well suited to resolve inter- and intramolecular distances is Förster Resonance Energy Transfer (FRET). By combining FRET with single-molecule spectroscopy, heterogeneities in the system can be detected and dynamic information becomes accessible. In this thesis, single-molecule FRET (smFRET) experiments were performed using either the small observation volume of a confocal microscope or the evanescent field of a total internal reflection (TIRF) microscope to get insights into the enzymatic activity of three proteins. To study the end-joining of DNA double strands by the T4 DNA ligase, a DNA origami platform was designed to control the stoichiometry and spatial arrangement of the interaction partners, thereby increasing their local concentration. The ligation process could be followed in real time and the same pair of DNA strands repeatedly ligated and cut, which highlights the applicability of the DNA origami platform to a wide range of multimolecular interaction studies on the single-molecule level. In a second project, the underlying catalytic mechanism of NSP2, a rotavirus protein required for genome replication and virus assembly, was investigated by comparing the RNA unwinding activity of full-length and C-terminally truncated mutants. Although the C-terminal region was less efficient in destabilizing the secondary structure of RNA, it proved to be essential for RNA release via charge repulsion and is thus a prerequisite for a full cycle of chaperone activity. In the last study, the conformational states of bacterial adhesion protein SdrG were studied. This protein initiates nosocomial infections through its stable attachment to human fibrinogen. By combining FRET-derived distances and molecular dynamics (MD) simulations, the structure of the dynamic apoprotein could be modeled, providing information inaccessible to other methods. The diverse topics researched in this thesis - ranging from DNA nanotechnology to viral and bacterial infections - emphasize the multifaceted capabilities of smFRET.