Credit: © 2009 ACS

The atomic force microscope (AFM) is used to study a variety of problems from protein–ligand interactions to the folding behaviour of proteins, but not complex catalytic reactions of enzymes. By integrating an AFM with total internal reflection fluorescence microscopy, researchers at the Radboud University in Nijmegen and research centres in Germany now show that it is possible to probe the influence of force on enzymatic activity at the single-molecule level1.

To apply force on the enzyme, Kerstin Blank and colleagues connected the enzyme to the AFM by means of a reversible antibody–antigen interaction. Lipase B enzyme (which converts a substrate to a measurable fluorescent product) was covalently bound onto a glass cover slip on one end and a fusion protein, which acts as the antigen, on the other. As the cantilever approaches the enzyme, the fusion protein interacts with the antibody, which is bound to an agarose bead on the AFM cantilever. When the cantilever retracts, the enzyme is stretched until the antibody–antigen interaction ruptures, causing the enzyme to relax. Enzymatic activity was measured by the fluorescence correlated with rupture events, and analysis revealed that the highest probability of detecting enzymatic activity occurred about 1.7 seconds after a rupture event.

Further theoretical analysis offered an insight on the energy landscape of the process, and it is suggested that this tool offers a new approach to study the role of force in biocatalytic reactions.