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Towards a single-molecule FRET study of Frauenfelder's nonexponential rebinding of CO in myoglobin

Early time-resolved experiments by Frauenfelder on the ensemble of the kinetic rebinding of CO to myoglobin molecules resulted in a stretched exponential relaxa-tion due to a very large spread of the reaction rates of individual molecules.

Eskandari Alughare, Z.
23 June 2022
Thesis in Leiden Repository

These results were assigned to the heterogeneity in this system originated from different conformations of different single-molecule proteins and from the widely different re-action rates associated with each of these conformations. The work presented in this thesis contains two lines of research. On the one hand, we investigate the Förster Resonance Energy Transfer (FRET) of dye labeled-carboxymyoglobin (MbCO) in the ensemble to show the feasibility of performing single molecule-FRET experiments to study the kinetic rebinding of CO to myoglobin (Chapter 3,4). On the other hand, we study the Förster theory about a stretched-exponential fluorescence intensity decay under ensemble conditions for a distribution of acceptors in the vicinity of each donor; This non-exponential kinetics arise from a distribution of the exponential steps originated from different single molecules. Using single-molecule microscopy, we study the histograms of the decay rates of single fluorophore molecules (Azaoxa-triangulenium, ADOTA dye) as donor in the presence of acceptors (ATTO575Q dye) both doped in thin polymeric layers as exponential which average out as non-exponential decay in the ensemble (Chapter 5).

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