Effect of size, quaternary structure and translational error on the static and dynamic heterogeneity of beta-galactosidase and measurement of electrophoretic dynamic heterogeneity.
Single enzyme molecule assays were performed using capillary electrophoresis-based protocols on beta-galactosidase from Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus helveticus and Bacillus circulans. The enzyme was found to show static heterogeneity with respect to catalytic rate and the variance in rate increased with protein size. This is consistent with the proposal that random errors in translation may be an important underlying component of enzyme heterogeneity. Additionally these enzymes were found to show static heterogeneity with respect to electrophoretic mobility. Comparison of wild-type and rpsL E. coli beta-galactosidase expressed in the presence and absence of streptomycin suggested that increases in error do not result in detectable increases in the dynamic heterogeneity of activity with increasing temperature. Finally, a method was developed to measure the dynamic heterogeneity in electrophoretic mobility.
Craig
Douglas B
DB
Chemistry Department, University of Winnipeg, Winnipeg, Manitoba R3B2E9, Canada. d.craig@uwinnipeg.ca
Haslam
Allison M
AM
Silverstein
Harlyn J
HJ
Chikamatsu
Miki
M
Shadabi
Elnaz
E
Nichols
Ellert R
ER
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics; D008968:Molecular Conformation; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013307:Streptomycin / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C506766:RpsL protein, E coli; D001616:beta-Galactosidase; D013307:Streptomycin