Magdalena Wojtas

Intrinsic disorder of Drosophila melanogaster hormone receptor 38 N-terminal domain

Drosophila hormone receptor 38 (dHR38), an ortholog of the vertebrate NR4A subclass of nuclear receptors, responds to ecdysteroids, which mediate developmental transitions during the Drosophila life cycle. However, this response is independent of the ecdysteroid receptor, and it does not involve binding of ecdysteroids to dHR38. It has been suggested that ecdysteroids may indirectly activate dHR38, perhaps by recruiting specific proteins. There have been recent reports pointing out the decisive role that nuclear receptor N‐terminal domains (NTDs) have in protein–protein interactions that are important for regulation of gene expression. It is reasonable to assume that dHR38‐NTD may also be involved in some protein–protein interactions that are critical for the ecdysteroid signaling pathway. To facilitate the exploration of the molecular basis of these interactions, we developed and optimized a protocol for the efficient expression and purification of the recombinant dHR38‐NTD. Using a diverse array of biochemical and biophysical methods, we carried out the first structural characterization of dHR38‐NTD. The results of our study indicate that dHR38‐NTD exhibits a characteristic reminiscent of pre‐molten globule‐like intrinsically disordered proteins existing in a partially unfolded conformation with regions of secondary structures. The dHR38‐NTD structure, which apparently comprises some local, ordered, tertiary structure clusters, is pliable and can adopt more ordered conformations in response to changes in environmental conditions. Thus, dHR38‐NTD, which exhibits the structural and functional characteristic of a pre‐molten globule‐like intrinsically disordered protein, could serve as a platform for multiple protein–protein interactions, possibly including interactions with proteins involved in an unusual ecdysteroid signaling pathway. Proteins 2011.© 2010 Wiley‐Liss, Inc.

Single-molecule electrometry

Mass and electrical charge are fundamental properties of biological macromolecules. Although molecular mass has long been determined with atomic precision, a direct and precise determination of molecular charge remains an outstanding challenge. Here we report high-precision (<1e) measurements of the electrical charge of molecules such as nucleic acids, and globular and disordered proteins in solution. The measurement is based on parallel external field-free trapping of single macromolecules, permits the estimation of a dielectric coefficient of the molecular interior and can be performed in real time. Further, we demonstrate the direct detection of single amino acid substitution and chemical modifications in proteins. As the electrical charge of a macromolecule strongly depends on its three-dimensional conformation, this kind of high-precision electrometry offers an approach to probe the structure, fluctuations and interactions of a single molecule in solution.

Calcium Ion Binding Properties and the Effect of Phosphorylation on the Intrinsically Disordered Starmaker Protein

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stms affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Intrinsically disordered and pliable Starmaker-like protein from medaka (Oryzias latipes) controls the formation of calcium carbonate crystals.

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

Calponin-Like Chd64 Is Partly Disordered

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function.

The Effect of Counter Ions on the Conformation of Intrinsically Disordered Proteins Studied by Size-Exclusion Chromatography

Counter ions are able to change the conformation of intrinsically disordered proteins (IDPs) to a more compact structure via the reduction of electrostatic repulsion. When the extended IDP conformation is transformed into a more ordered one, the value of the Stokes radius should decrease. Size-exclusion chromatography is a simple method for the determination of the Stokes radius, which describes the hydrodynamic properties of protein molecules. In our paper size-exclusion chromatography experiments of Starmaker (a highly acidic IDP), in the presence of various counter ions, are presented as an example of a simple experimental method, which provides valuable information about subtle counter ions-induced conformational changes in IDP.