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-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 (Kd 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

The basic helix-loop-helix region of human neurogenin 1 is a monomeric natively unfolded protein which forms a

Neuronal specification is regulated by the activity of transcription factors containing the basic helix-loop-helix motif (bHLH); these regulating proteins include, among others, the neurogenin (Ngn) family, related to the atonal family of genes. Neurogenin 1 (NGN1) is a 237-residue protein that contains a bHLH domain and is involved in neuronal differentiation. In this work, we synthesized the bHLH region of NGN1 (bHLHN) comprising residues 90-150 of the full-length NGN1. The domain is a monomeric natively unfolded protein with a pH-dependent premolten globule conformation, as shown by several spectroscopic techniques (namely, NMR, fluorescence, FTIR, and circular dichroism). The unfolded character of the domain also explains, first, the impossibility of its overexpression in several Escherichia coli strains and, second, its insolubility in aqueous buffers. To the best of our knowledge, this is the first extensive study of the conformational preferences of a bHLH domain under different solution conditions. Upon binding to two DNA E-boxes, the protein forms "fuzzy" complexes (that is, the complexes were not fully folded). The affinities of bHLHN for both DNA boxes were smaller than those of other bHLH domains, which might explain why the protein-DNA complexes were not fully folded.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe