A Random Walk Towards the Golden Fleece

Abstract

In this thesis we used single-molecule FRET to investigate the kinetic properties of a protein called Argonaute. While traditionally one uses bulk methods to investigate the molecular properties of proteins, bulk methods do not confer information that is transient, since that is inherently averaged out. Single-molecule methods, as their name imply, allow one to observe interactions between individual molecules and their substrates. This allows one to see fast kinetics which may otherwise be missed. Furthermore, while the bulk methods give one only the average kinetics, single molecule methods also give the distribution of probabilities to access certain bound or conformational states, which in turn can give the observer information of the nature of the stochastic process. We rely in this thesis on single-molecule FRET, an abbreviation for Förster Resonance Energy Transfer: It’s a process where energy is transferred through dipole-dipole interaction from a donor fluorophore to an acceptor fluorophore. The distance between fluorophores determines the efficiency of transfer on a length scale of » 10 nm. Since many biological processes take place on this length scale, this technique is exquisitely suitable to study biological processes real-time on the smallest scale, whether it is conformational changes, protein-protein interactions, or in this case, protein target search studies.