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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...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2568859/ https://www.ncbi.nlm.nih.gov/pubmed/18949049 http://dx.doi.org/10.1371/journal.pone.0003514 |
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author | Chow, Hiu-Kwan Xu, Jing Shahravan, S. Hesam De Jong, Antonia T. Chen, Gang Shin, Jumi A. |
author_facet | Chow, Hiu-Kwan Xu, Jing Shahravan, S. Hesam De Jong, Antonia T. Chen, Gang Shin, Jumi A. |
author_sort | Chow, Hiu-Kwan |
collection | PubMed |
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 α-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. |
format | Text |
id | pubmed-2568859 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-25688592008-10-24 Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro Chow, Hiu-Kwan Xu, Jing Shahravan, S. Hesam De Jong, Antonia T. Chen, Gang Shin, Jumi A. PLoS One Research Article 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 α-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. Public Library of Science 2008-10-24 /pmc/articles/PMC2568859/ /pubmed/18949049 http://dx.doi.org/10.1371/journal.pone.0003514 Text en Chow et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Chow, Hiu-Kwan Xu, Jing Shahravan, S. Hesam De Jong, Antonia T. Chen, Gang Shin, Jumi A. Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro |
title | Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro
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title_full | Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro
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title_fullStr | Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro
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title_full_unstemmed | Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro
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title_short | Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro
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title_sort | hybrids of the bhlh and bzip protein motifs display different dna-binding activities in vivo vs. in vitro |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2568859/ https://www.ncbi.nlm.nih.gov/pubmed/18949049 http://dx.doi.org/10.1371/journal.pone.0003514 |
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