Analysis of Fluorescence Flicker as a Tool to Monitor Proton Transport and Biomolecular Interactions

Sammanfattning: The overall focus of this thesis is on fluorescence flicker processes of fluorescent molecules, e.g. protonation-deprotonation or singlet-triplet electronic state transitions, intrinsic or generated by their interaction with their environment, monitored by fluorescence spectroscopy.Understanding proton migration along membranes and membrane proteins in cells is essential for understanding energy metabolism. It has been seen that certain membrane-spanning proton-transporter proteins in the respiratory chain in the mitochondrial inner membrane take up protons faster than the rate limited by diffusion. To explain these observations it has been suggested that there is a proton-collecting antenna, consisting of negatively and protonatable residues on the surface of these proteins, which increases the rate of uptake. Using fluorescence correlation spectroscopy and artificial biological membranes the proton collecting antenna effect is verified, as well as the proton migration properties on these membranes at various surface buffer concentrations.Fluorescence flicker due to singlet-triplet electronic state transitions in a fluorescent molecule is interesting because of the long transition time between the two states. This means that the molecule has a long time to interact with the local environment, and can therefore be used as a microenvironmental sensor. A novel method for monitoring photo-induced, transient, long-lived, non- or weakly fluorescent states, e.g. the triplet state, was developed. With this method, only the time averaged intensity is detected and used for determining the triplet state kinetics. This method has several advantages, in particular it lends itself well for parallelization, over traditional methods including fluorescence correlation spectroscopy.