title: Conformational change in individual enzyme molecules.
abstract: Single β-galactosidase molecule assays were performed using a capillary electrophoresis based protocol, employing post-column laser-induced fluorescence detection. In a first set of experiments, the distribution of single β-galactosidase molecule catalytic rates and electrophoretic mobilities were determined from lysates of Escherichia coli strains containing deletions for different heat shock proteins and grown under normal and heat shock conditions. There was no clear observed pattern of effect of heat shock protein expression on these distributions. In a second set of experiments, individual enzyme molecule catalytic rates were determined at 21 °C before and after 2 sequential brief periods of incubation at 50, 28, and 10 °C. The brief incubations at 50 °C caused a change in the enzyme molecules resulting in a different catalytic rate. Any given molecule was just as likely to show an increase in rate as a decrease, resulting in no significant difference in the average rate of the population. The average change in individual molecule rate was dependent upon the temperature of the brief incubation period, with a lesser average change occurring at 28 °C and negligible change at 10 °C. A third set of experiments was similar to that of the second with the exception that it was electrophoretic mobility that was considered. This provided a similar result. Incubation at higher temperature resulted in a change in electrophoretic mobility. The probability of an individual molecules switching to a higher mobility was approximately equal to that of switching to a lower mobility, resulting in no net average change in the population. The magnitude of the changes in electrophoretic mobilities suggest that the associated conformational changes are subtle.
authors.affiliation: a Department of Chemistry, 360 Parker Building, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Jeremie J
authors.initials: JJ
authors.lastname: Crawford
authors.affiliation: b Department of Chemistry, 599 Portage Avenue, University of Winnipeg, Winnipeg, MB R3B 2G3, Canada.
authors.forename: Frannie
authors.initials: F
authors.lastname: Itzkow
authors.affiliation: b Department of Chemistry, 599 Portage Avenue, University of Winnipeg, Winnipeg, MB R3B 2G3, Canada.
authors.forename: Joanna
authors.initials: J
authors.lastname: MacLean
authors.affiliation: b Department of Chemistry, 599 Portage Avenue, University of Winnipeg, Winnipeg, MB R3B 2G3, Canada.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Craig
mesh_terms: D000166:Acridines / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary; D024202:Electrophoretic Mobility Shift Assay; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response; D006358:Hot Temperature / Q000009:adverse effects; D007763:Lac Operon; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013269:Stochastic Processes; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000166:Acridines; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005697:Galactosides; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D005690:Galactose
keywords: capillary electrophoresis; catalytic heterogeneity; changement conformationnel; chauffage; conformational change; electrophoretic heterogeneity; heat shock proteins; heating; hétérogénéité catalytique; hétérogénéité électrophorétique; protéines de choc thermique; électrophorèse capillaire; β-galactosidase