title: Fluorescence-based analysis of enzymes at the single-molecule level.
abstract: 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, single-molecule 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.
authors.affiliation: Katholieke Universiteit Leuven, Department of Chemistry, Belgium. K.Blank@science.ru.nl
authors.forename: Kerstin
authors.initials: K
authors.lastname: Blank
authors.forename: Gert
authors.initials: G
authors.lastname: De Cremer
authors.forename: Johan
authors.initials: J
authors.lastname: Hofkens
mesh_terms: D002384:Catalysis; D004798:Enzymes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D005544:Forecasting; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D004798:Enzymes; D005456:Fluorescent Dyes