Single molecule measurements on lipase enzymes to quantify dynamic and static disorder

The modern scientist wields a plethora of powerful imaging and spectroscopic techniques, such as atomic force microscopy, scanning tunneling microscopy, transmission electron microscopy and even optical detectors sensitive enough to measure the signal from a single photon. These techniques are all able to dissect the secrets of nature down to the single molecule level; information so far inaccessible in conventional ensemble experiments. Contrary to ensemble-based measurements single-molecule experiments have the potential to unveil heterogeneous behavior in seemingly identical molecules. In the context of enzymes it is well established, that two major heterogeneities exists; (i) differences in the catalytic rate of individual molecules and (ii) temporal variations in the catalytic rate of the same molecule. The former is known as static disorder, whereas the latter has been dubbed dynamic disorder. In this Ph. D. project I will describe how I applied fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence (TIRF) microscopy to directly measure and quantify static and dynamic disorder of single lipase molecules.