Enzymes are an everyday part of modern life. They can be found in applications ranging from washing powders to the making of beer. The mechanism of action of enzymes has been investigated for almost a century and to date all studies have been carried out in bulk, ensemble experiments. Recent single molecule measurements revealed that enzymes actually ‘sleep’, ‘breathe’ and ‘work’. A single enzyme is a fluctuating species and ‘walks’ across a conformational landscape changing it activities as it goes.
To understand how an enzyme really works, we are developing a generic method for immobilizing enzymes, so that the effect of external factors such as pH, temperatures and solvent on the activity can be studied. To date we have studied non-specific absorption, the use of a protein-foot, encapsulation in a virus more recently immobilization in a sol gel, the properties of which (pore size, hydrophilic character) can be readily tuned. Once immobilized the activities of single enzymes are studied by confocal fluorescence microscopy. Every time the single enzyme carries out a turnover, a fluorescent product is detected and then disappears into the bulk, or is quickly bleached. By following and recording the flashes of fluorescence intensity, the properties of the enzymatic process can be determined.
This approach allows for the first time the study of how an enzyme really works.
Subprojects:
Victor I. Claessen
| Single Enzyme kinetics |
Nolte group for physical-organic and supramolecular chemistry
