Fragment-Based NMR Study of the Conformational Dynamics in the bHLH Transcription Factor Ascl1.

The Achaete-scute homolog 1 (Ascl1) protein regulates a large subset of genes that leads neuronal progenitor cells to distinctive differentiation pathways during human brain development. Although it is well known that Ascl1 binds DNA as a homo- or heterodimer via its basic helix-loop-helix (bHLH) motif, little is known about the conformational sampling properties of the DNA-free full-length protein, and in particular about the bHLH domain-flanking N- and C-terminal segments, which are predicted to be highly disordered in solution. The structural heterogeneity, low solubility, and high aggregation propensity of Ascl1 in aqueous buffer solutions make high-resolution studies of this protein a challenging task. Here, we have adopted a fragment-based strategy that allowed us to obtain high-quality NMR data providing, to our knowledge, the first comprehensive high-resolution information on the structural propensities and conformational dynamics of Ascl1. The emerging picture is that of an overall extended and highly dynamic polypeptide chain comprising three helical segments and lacking persistent long-range interactions. We also show that the C-terminal helix of the bHLH domain is involved in intermolecular interactions, even in the absence of DNA. Our results contribute to a better understanding of the mechanisms of action that govern the regulation of proneural transcription factors.

Just a Flexible Linker? The Structural and Dynamic Properties of CBP-ID4 Revealed by NMR Spectroscopy.

Here, we present a structural and dynamic description of CBP-ID4 at atomic resolution. ID4 is the fourth intrinsically disordered linker of CREB-binding protein (CBP). In spite of the largely disordered nature of CBP-ID4, NMR chemical shifts and relaxation measurements show a significant degree of α-helix sampling in the protein regions encompassing residues 2-25 and 101-128 (1852-1875 and 1951-1978 in full-length CBP). Proline residues are uniformly distributed along the polypeptide, except for the two α-helical regions, indicating that they play an active role in modulating the structural features of this CBP fragment. The two helical regions are lacking known functional motifs, suggesting that they represent thus-far uncharacterized functional modules of CBP. This work provides insights into the functions of this protein linker that may exploit its plasticity to modulate the relative orientations of neighboring folded domains of CBP and fine-tune its interactions with a multitude of partners.

Structural and Dynamic Characterization of the Molecular Hub Early Region 1A (E1A) from Human Adenovirus.

The small-DNA human adenovirus encodes one of the most versatile molecular hubs, the E1A protein. This protein is essential for productive viral infection in human cells and a vast amount of biologically relevant data are available on its interactions with host proteins. Up to now, however, no high-resolution structural and dynamic information on E1A is available despite its important biological role. Among the different spliced variants of E1A, two are expressed at high level in the early stage of infection. These are 243 and 289 residues isoforms. Herein, we present their NMR characterization, showing that they are both highly disordered, but also demonstrate a certain heterogeneous behavior in terms of structural and dynamic properties. Furthermore, we present the characterization of the isolated domain of the longer variant, known as CR3. This study opens the way to understanding at the molecular level how E1A functions.

Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks.

The growing recognition of the several roles that intrinsically disordered proteins play in biology places an increasing importance on protein sample availability to allow the characterization of their structural and dynamic properties. The sample preparation is therefore the limiting step to allow any biophysical method being able to characterize the properties of an intrinsically disordered protein and to clarify the links between these properties and the associated biological functions. An increasing array of tools has been recruited to help prepare and characterize the structural and dynamic properties of disordered proteins. This chapter describes their sample preparation, covering the most common drawbacks/barriers usually found working in the laboratory bench. We want this chapter to be the bedside book of any scientist interested in preparing intrinsically disordered protein samples for further biophysical analysis.

Nonredundant roles for cytochrome c2 and two high-potential iron-sulfur proteins in the photoferrotroph Rhodopseudomonas palustris TIE-1.

The purple bacterium Rhodopseudomonas palustris TIE-1 expresses multiple small high-potential redox proteins during photoautotrophic growth, including two high-potential iron-sulfur proteins (HiPIPs) (PioC and Rpal_4085) and a cytochrome c2. We evaluated the role of these proteins in TIE-1 through genetic, physiological, and biochemical analyses. Deleting the gene encoding cytochrome c2 resulted in a loss of photosynthetic ability by TIE-1, indicating that this protein cannot be replaced by either HiPIP in cyclic electron flow. PioC was previously implicated in photoferrotrophy, an unusual form of photosynthesis in which reducing power is provided through ferrous iron oxidation. Using cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and flash-induced spectrometry, we show that PioC has a midpoint potential of 450 mV, contains all the typical features of a HiPIP, and can reduce the reaction centers of membrane suspensions in a light-dependent manner at a much lower rate than cytochrome c2. These data support the hypothesis that PioC linearly transfers electrons from iron, while cytochrome c2 is required for cyclic electron flow. Rpal_4085, despite having spectroscopic characteristics and a reduction potential similar to those of PioC, is unable to reduce the reaction center. Rpal_4085 is upregulated by the divalent metals Fe(II), Ni(II), and Co(II), suggesting that it might play a role in sensing or oxidizing metals in the periplasm. Taken together, our results suggest that these three small electron transfer proteins perform different functions in the cell.

The heterogeneous structural behavior of E7 from HPV16 revealed by NMR spectroscopy.

The E7 protein from human papillomavirus (HPV) plays a key role in oncogenesis; for this reason, it is a target of great biomedical interest. To date, no high resolution information is available for the full protein. We present here the NMR characterization of the entire E7 from HPV16, one of the most oncogenic variants of the virus. The protein is very heterogeneous in terms of structural and dynamic properties with a highly flexible N-terminal module and a more structured C terminus. This opens possibilities for studies of molecular-level interactions and post-translational modifications of the protein to unravel functional details that might be linked to its highly oncogenic potential.

Monitoring HPV-16 E7 phosphorylation events.

HPV-16 E7 is one of the key proteins that, by interfering with the host metabolism through many protein-protein interactions, hijacks cell regulation and contributes to malignancy. Here we report the high resolution investigation of the CR3 region of HPV-16 E7, both as an isolated domain and in the full-length protein. This opens the way to the atomic level study of the many interactions in which HPV-16 E7 is involved. Along these lines we show here the effect of one of the key post-translational modifications of HPV-16 E7, the phosphorylation by casein kinase II.