The Basic Helix-Loop-Helix Region of Human Neurogenin 1 Is a Monomeric Natively Unfolded Protein Which Forms a "Fuzzy" Complex upon DNA Binding

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.