Capillary electrophoresis laser-induced fluorescence investigations of individual molecules of Escherichia coli beta-galactosidase.

摘要

Fifteen years following the first demonstration of single molecule fluorescence detection, single molecule studies of enzymes have revealed that nominally identical individual enzyme molecules are functionally heterogeneous. Different individual molecules exhibit different catalytic rates under identical conditions, and individual enzyme molecules show fluctuating rates over broad timescales. The structural basis and the biological sources for such heterogeneity remains poorly understood. Herein, studies are presented of the beta-galactosidase from Escherichia coli, using capillary electrophoresis with laser-induced fluorescence (CE-LIF), to investigate the sources of catalytic heterogeneity at the single molecule level. Two new single molecule CE-LIF assays for beta-galactosidase were developed based upon the synthetic fluorogenic substrates 9H-(1,3-dichloro- 9,9-dimethyacridin-2-one-7-yl)-beta-D-galactopyranoside (DDAO-gal) and fluorescein-∃-D-digalactopyranoside (FDG). Limited proteolysis as a possible source for single molecule heterogeneity, and for the changes in activity of a population of individual molecules over time, was investigated by inducing enzyme expression in two E. coli strains in the presence of a broad spectrum of protease inhibitors. The effect of protease inhibitors was found to be limited. beta-galactosidase was expressed from a lacZ linear template from two different E. coli strains using an in vitro protein expression system to determine if in vitro synthesized enzyme was identical to its in vivo counterpart. In vitro synthesized enzyme was found to be less active than in vivo sources. The differences were attributed to deficient N-terminal methionine removal and the higher rates of translation error associated with in vitro protein synthesis. Single molecule separations revealed that individual molecules of beta-galactosidase were electrophoretically distinct, and that the electrophoretic heterogeneity was independent of source of enzyme, method of measurement or of capillary coating. Electrophoretic modeling indicated that slight variations of shape and charge could account for the observed range. The magnitude of these differences, and published rates for translation error suggested that translation error could be a possible source of the variations. The extent of single molecule catalytic variation was reduced in a mutant with a hyperaccurate translation phenotype implying that translation error is a source of the heterogeneity. Streptomycin-induced translation error reduced average activity, but did not lead to an increase in catalytic heterogeneity. No relationship between translation error and electrophoretic heterogeneity was observed. A novel CE-LIF assay was developed for the continuous monitoring of the catalytic activity and electrophoretic mobility of individual beta-galactosidase molecules. Thermally-induced catalytic fluctuations were observed suggesting that individual enzyme molecules were capable of conformational fluctuations that supported different catalytic rates.