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Bottom-up single-molecule strategy for understanding subunit function of tetrameric ?-galactosidase.

ABSTRACT: In this paper, we report an example of the engineered expression of tetrameric ?-galactosidase (?-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric ?-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10-4 per base). In addition, we studied the kinetics of the mistranslated ?-gal at the single-molecule level.

SUBMITTER: Li X

PROVIDER: S-EPMC6099853 | biostudies-other | 2018 Aug

REPOSITORIES: biostudies-other

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Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase.

Li Xiang X Jiang Yu Y Chong Shaorong S Walt David R DR

Proceedings of the National Academy of Sciences of the United States of America 20180730 33

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme ...[more]

PMID: 30061400

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