A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen
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Tikhonova-2015-Transcription Blocker.pdf
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Author(s)
Tikhonova, Elena B.
Ethayathulla, Abdul S.
Su, Yue
Hariharan, Parameswaran
Xie, Shicong
Guan, Lan
Date Issued
January 2015
Journal
Scientific Reports
Publisher
Nature Publishing Group
Citation
Tikhonova, Elena B., Abdul S. Ethayathulla, Yue Su, Parameswaran Hariharan, Shicong Xie, and Lan Guan. “A Transcription Blocker Isolated from a Designed Repeat Protein Combinatorial Library by in Vivo Functional Screen.” Scientific Reports 5 (January 28, 2015): 8070.
Version
Final published version
Abstract
A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.
MIT Department
Massachusetts Institute of Technology. Computational and Systems Biology Program
Massachusetts Institute of Technology. Department of Biology
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DOI of Published Version
https://doi.org/10.1038/srep08070
