Fluorescence-based analysis of enzymes at the single-molecule level

Enzymes, and proteins in general, consist of a dynamic ensemble of different conformations, which fluctuate around an average structure. Single-molecule experiments are a powerful tool to obtain information about these conformations and their contributions to the catalytic reaction. In contrast to classical ensemble measurements, which average over the whole population, singlemolecule experiments are able to detect conformational heterogeneities, to identify transient or rare conformations, to follow the time series of conformational changes and to reveal parallel reaction pathways. A number of single-molecule studies with enzymes have proven this potential showing that the activity of individual enzymes varies between different molecules and that the catalytic rate constants fluctuate over time. From a practical point of view this review focuses on fluorescence-based methods that have been used to study enzymes at the single-molecule level. Since the first proof-of-principle experiments a wide range of different methods have been developed over the last 10 years and the methodology now needs to be applied to answer questions of biological relevance, for example about conformational changes induced by allosteric effectors or mutations.