Single molecule dynamics

University dissertation from Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Abstract: Properties found in an ensemble of molecules may not be understood if it is not possible to study each molecule one by one. This thesis is concerned with the study of the dynamic properties of individual complex biological molecules as observed by confocal single-molecule fluorescence spectroscopy. Single molecules are studied under conditions that are biologically relevant; in aqueous solution at room temperature. Single DNA molecules upon which a fluorescence sensor is attached (tetrahethylrhodmine, TMR) are detected freely diffusing in solution as well as immobilized in solution. Conformational movements of single species of DNA-TMR induce intramolecular spectroscopic fluctuations. The dynamics of the spectroscopic fluctuations of single DNA-TMR molecules are not ergodic on the observed time scale. The histogram of the transition rates of many single molecules is similar to the distribution of exponential functions leading to stretched exponential transition kinetics found in the ensemble. These results imply that the phase space of the DNA-TMR may be divided into components in which each DNA-TMR molecule becomes trapped on the observed time scale. The enzyme-product complex of single horseradish peroxidase enzyme molecules catalysing the oxidation of dihydrorhodamine 6G (substrate) into rhodamine 6G (product) is observed. While the dissociation of the enzyme-product complex show exponential kinetics, the rate at which the enzyme-product complex is formed show a broad distribution. The data and analysis indicate that substrate interaction with the enzyme select a set of conformational substates (CS) for which the enzyme is active. The quantity of information (combinations of different methodologies to measure a single molecule, for example) as well as the quality in the analysis of the information (higher order correlation analysis, for example) set the limits of future analyses of single molecule dynamics.

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