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Single-molecule fluorescence spectroscopy. from two to three colors and beyond
Single-molecule fluorescence spectroscopy. from two to three colors and beyond
Single-molecule fluorescence spectroscopy is a powerful tool for the study of physical and biological processes through the use of fluorescent probes. By combining the femtoliter-sized observation volume of a confocal microscope with low concentrations of analytes, single fluorescent molecules can be observed as they freely diffuse in solution. From the many parameters of the fluorescence signal, a wealth of information is obtained about the structure, dynamics and interactions of the studied system. The objective of this thesis was the development, implementation and application of quantitative single-molecule fluorescence methods. To this end, a software framework for the analysis of solution-based single-molecule measurements of Förster resonance energy transfer (FRET) has been developed as part of the PAM software package. In addition, the new method of three-color photon distribution analysis (3C-PDA) is introduced in this thesis, enabling a quantitative analysis of single-molecule three-color FRET experiments. The developed analysis framework has been applied to elucidate coordinated conformational changes in the Hsp70 chaperone protein BiP, to study the conformational dynamics of a small fragment of the cellulosome, to investigate energy transfer pathways in complex artificial dye arrangements and to quantify the nanosecond dynamics of an intrinsically disordered peptide. For several studies, molecular dynamics (MD) simulations have also been used to support and cross-validate the experimental results. Here, the focus is to provide a comprehensive overview of the used methodologies, their theoretical background and their application to the various experimental systems.
single molecule, fluorescence, biophysics, Förster resonance energy transfer
Barth, Anders
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Barth, Anders (2018): Single-molecule fluorescence spectroscopy: from two to three colors and beyond. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Single-molecule fluorescence spectroscopy is a powerful tool for the study of physical and biological processes through the use of fluorescent probes. By combining the femtoliter-sized observation volume of a confocal microscope with low concentrations of analytes, single fluorescent molecules can be observed as they freely diffuse in solution. From the many parameters of the fluorescence signal, a wealth of information is obtained about the structure, dynamics and interactions of the studied system. The objective of this thesis was the development, implementation and application of quantitative single-molecule fluorescence methods. To this end, a software framework for the analysis of solution-based single-molecule measurements of Förster resonance energy transfer (FRET) has been developed as part of the PAM software package. In addition, the new method of three-color photon distribution analysis (3C-PDA) is introduced in this thesis, enabling a quantitative analysis of single-molecule three-color FRET experiments. The developed analysis framework has been applied to elucidate coordinated conformational changes in the Hsp70 chaperone protein BiP, to study the conformational dynamics of a small fragment of the cellulosome, to investigate energy transfer pathways in complex artificial dye arrangements and to quantify the nanosecond dynamics of an intrinsically disordered peptide. For several studies, molecular dynamics (MD) simulations have also been used to support and cross-validate the experimental results. Here, the focus is to provide a comprehensive overview of the used methodologies, their theoretical background and their application to the various experimental systems.