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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...

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Autores principales: Chow, Hiu-Kwan, Xu, Jing, Shahravan, S. Hesam, De Jong, Antonia T., Chen, Gang, Shin, Jumi A.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2008
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.
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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
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
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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