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Int J Biol Macromol ; 188: 391-403, 2021 Oct 01.
Article in English | MEDLINE | ID: covidwho-1347646


One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/isolation & purification , Liquid-Liquid Extraction/methods , SARS-CoV-2/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/isolation & purification , Intrinsically Disordered Proteins/metabolism , Nucleic Acids/chemistry , Nucleic Acids/metabolism , Protein Aggregates , Protein Structure, Quaternary , Protein Structure, Secondary
Cells ; 10(3)2021 03 05.
Article in English | MEDLINE | ID: covidwho-1129686


The superfamily of nuclear receptors (NRs), composed of ligand-activated transcription factors, is responsible for gene expression as a reaction to physiological and environmental changes. Transcriptional machinery may require phase separation to fulfil its role. Although NRs have a similar canonical structure, their C-terminal domains (F domains) are considered the least conserved and known regions. This article focuses on the peculiar molecular properties of the intrinsically disordered F domain of the ecdysteroid receptor from the Aedes aegypti mosquito (AaFEcR), the vector of the world's most devastating human diseases such as dengue and Zika. The His-Pro-rich segment of AaFEcR was recently shown to form the unique poly-proline helix II (PPII) in the presence of Cu2+. Here, using widefield microscopy of fluorescently labeled AaFEcR, Zn2+- and Cu2+-induced liquid-liquid phase separation (LLPS) was observed for the first time for the members of NRs. The perspectives of this finding on future research on the F domain are discussed, especially in relation to other NR members.

Ions/metabolism , Mosquito Vectors/pathogenicity , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Aedes , Animals , Humans