Biomolecular applications of single-molecule measurements : Kinetics and dynamics of a single enzyme reaction

In this work we describe preliminary experiments in which we have used ultra-sensitive fluorescence microscopy to observe the dynamics of individual enzyme molecules acting upon a substrate. The enzyme, P-galactosidase from E.coli, is specifically immobilized onto a glass substrate while maintaining its functionality. The immobilized protein degrades a fluorogenic substrate to produce a fluorescent product, whose generation can be observed in real time. Individual copies of beta-galactosidase can be observed for many minutes, allowing the measurement of a large number of successive substrate turnover events. A rudimentary analysis of these turnovers using autocorrelation functions is presented, and a strong heterogeneity in reaction rates between different molecules is observed. In addition, the challenges inherent in successful surface immobilization of proteins for single-molecule experiments are discussed.