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• W2969256494 abstract "This thesis presents developments in fluorescence techniques that allow for the study of Fluorescence Resonance Energy Transfer between multiple chromophores in single molecules in solution. Multi-parameter Fluorescence Detection (MFD) is a single-molecule technique in which several fluorescence parameters are recorded simultaneously. In this work MFD is combined to other techniques, Fluorescence Correlation Spectroscopy and Probability Distribution Analysis, to enhance their capabilities: -For FCS it is introduced a method to obtain the separate the correlation contributions of the different species in a multi-component system. -For PDA correction for multi-molecular events and brightness heterogeneities are proposed. Moreover a mathematical model for the dynamic interconversion of a two state system is implemented. The possibility to observe and characterize multiple-step FRET in single molecules was tested by investigating double stranded DNA labeled with three fluorophores, one donor (D) and two acceptors (A1 and A2). The sequence was designed so that the single FRET steps take place between D and A1 and between A1 and A2, even though D-A2 transfer was also observed. Fluorescence signal from each dye was detected, and good separation was achieved. These results suggested that, already as qualitative assay, it is possible to label multi-domain or multi-molecular systems and have information about proximity or simultaneous presence of the labeled positions. Exploiting the MFD capabilities donor quenching and A2 heterogeneity were detected. Quantitative analysis of the system was precluded due to the complex photophysics of A2. The interconversion between the stacking conformers, A/B and A/D, of a Holiday junction was studied as a function of Mg2+ concentration. At least 3 equilibria, taking place at different timescales, were individuated. The equilibria were associated to the transitions between the conformer A/B and its Mg-bound form, A/B and A/D and A/D and its Mg-bound form. Comparison between different Holliday junctions suggested that the timescales of the different equilibria are sequence independent and only the interconversion equilibrium between A/B and A/D is characteristic of each junction. The open cruciform structure was not observed, even in the absence of Mg2+ where it is supposed to be the only populated conformer. These results led to a revision of the commonly accepted interconversion model. In the model proposed in this work it is hypothesized that in absence of metal ions the Holliday junction is oscillating between the two folded conformers and only when Mg2+ is coordinated the junction is fixed in the conformation it is at the moment. The disassembly of mononucleosomes was studied by quantitative single-molecule FRET with high spatial resolution. Reversible dissociation was induced by increasing NaCl concentration. At least three species with different FRET efficiencies were identified: a high-FRET species corresponding to the intact nucleosome, a mid-FRET species that was attributed to a first intermediate with a partially unwrapped DNA, and a low-FRET species characterized by a very broad FRET distribution, representing a highly unwrapped structure. FCS analysis indicated that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 A measured for the high-FRET species is consistent with the known crystallographic structure. A geometric model of the nucleosome disassembly predicts exactly the presence of the observed FRET species and confirms their assignment to two populations in the unwrapping pathway." @default.
• W2969256494 created "2019-08-29" @default.
• W2969256494 creator A5026655832 @default.
• W2969256494 date "2010-01-01" @default.
• W2969256494 modified "2023-09-27" @default.
• W2969256494 title "Fluorescence resonance energy transfer between multiple chromophores studied by single-molecule spectroscopy" @default.
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• W2969256494 hasPublicationYear "2010" @default.
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