Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
Edited by Susan Marqusee, University of California, Berkeley, CA, and approved July 6, 2018 (received for review April 2, 2018)
Significance
β-Galactosidase (β-gal) is one of the most commonly used enzymes in molecular biology. There is widespread interest in understanding the details of how the different monomeric units in the tetrameric structure of β-gal influence the kinetics and affect the enzyme activity. In this work, we present a bottom-up strategy to synthesize β-gal containing varying numbers of active monomers in vitro and profile the single-enzyme activity for each specific configuration. We were able to identify four distinct configurations of the engineered β-gal by measuring the activities of individual β-gal molecules. Based on the resulting correlation between enzyme activity and configuration, we studied rare occurrences of mistranslation using a microwell array platform and determined an accurate in vitro mistranslation frequency.
Abstract
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 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 β-gal at the single-molecule level.
Footnotes
↵1X.L. and Y.J. contributed equally to this work.
↵2Present address: Arbor Biotechnologies, Cambridge, MA 02139.
- ↵3To whom correspondence should be addressed. Email: dwalt{at}bwh.harvard.edu.
Author contributions: D.R.W. designed research; X.L. and Y.J. performed research; S.C. contributed new reagents/analytic tools; X.L. and Y.J. analyzed data; and X.L., Y.J., and D.R.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1805690115/-/DCSupplemental.
Published under the PNAS license.
Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase
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- Biochemistry
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