Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.
Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-he...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2008-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC2568859?pdf=render |
| _version_ | 1818217640925069312 |
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| author | Hiu-Kwan Chow Jing Xu S Hesam Shahravan Antonia T De Jong Gang Chen Jumi A Shin |
| author_facet | Hiu-Kwan Chow Jing Xu S Hesam Shahravan Antonia T De Jong Gang Chen Jumi A Shin |
| author_sort | Hiu-Kwan Chow |
| collection | DOAJ |
| description | Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d) 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions. |
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| format | Article |
| id | doaj.art-40fcce8149c34c6a9d479b0883d38312 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-12T07:11:05Z |
| publishDate | 2008-01-01 |
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| series | PLoS ONE |
| spelling | doaj.art-40fcce8149c34c6a9d479b0883d383122022-12-22T00:33:38ZengPublic Library of Science (PLoS)PLoS ONE1932-62032008-01-01310e351410.1371/journal.pone.0003514Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.Hiu-Kwan ChowJing XuS Hesam ShahravanAntonia T De JongGang ChenJumi A ShinMinimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d) 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.http://europepmc.org/articles/PMC2568859?pdf=render |
| spellingShingle | Hiu-Kwan Chow Jing Xu S Hesam Shahravan Antonia T De Jong Gang Chen Jumi A Shin Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro. PLoS ONE |
| title | Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro. |
| title_full | Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro. |
| title_fullStr | Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro. |
| title_full_unstemmed | Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro. |
| title_short | Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro. |
| title_sort | hybrids of the bhlh and bzip protein motifs display different dna binding activities in vivo vs in vitro |
| url | http://europepmc.org/articles/PMC2568859?pdf=render |
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