title: A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.
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.
authors: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Elena B
authors: EB
authors: Tikhonova
authors: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Abdul S
authors: AS
authors: Ethayathulla
authors: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Yue
authors: Y
authors: Su
authors: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Parameswaran
authors: P
authors: Hariharan
authors: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Shicong
authors: S
authors: Xie
authors: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Lan
authors: L
authors: Guan
mesh_terms: D000595:Amino Acid Sequence; D017487:Ankyrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D017487:Ankyrins; D029968:Escherichia coli Proteins; D019151:Peptide Library; D027981:Symporters; C021548:melibiose permease; D000519:alpha-Galactosidase
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