Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein.
Papaya mosaic virus (PapMV) is a flexuous rod shape virus made of 1400 subunits that assemble around a plus sense genomic RNA. The structure determination of PapMV and of flexuous viruses in general is a major challenge for both NMR and X-ray crystallography. In this report, we present the characterization of a truncated version of the PapMV coat protein (CP) that is suitable for NMR study. The deletion of the N-terminal 26 amino acids of the PapMV CP (CP27-215) generates a monomer that can be expressed to high level and easily purified for production of an adequate NMR sample. The RNA gel shift assay showed that CP27-215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6x His tag at the C-terminus improved the solubility of the monomer and allowed its concentration to 0.2 mM. The CD spectra of the truncated and the wild-type proteins were similar, suggesting that both proteins are well ordered and have a similar secondary structure. CP27-215 was 15N labeled for NMR studies and a 2D 1H-15N-HSQC spectrum confirmed the presence of a well-ordered structure and the monomeric form of the protein. These results show that CP27-215 is amenable to a complete and exhaustive NMR study that should lead to the first three-dimensional structure determination of a flexuous rod shape virus.
Lecours
Katia
K
Centre de Recherche sur la fonction, la structure et l'ingénierie des protéines CREPSIP, Department of Biochemistry and Microbiology, Laval University, Pavillon C-E Marchand, Que., PQ, Canada G1K 7P4.
Tremblay
Marie-Hélène
MH
Gagné
Marie-Eve Laliberté
ME
Gagné
Stéphane M
SM
Leclerc
Denis
D
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D029441:Carica / Q000821:virology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D008969:Molecular Sequence Data; D017863:Potexvirus / Q000737:chemistry* / Q000235:genetics
D036022:Capsid Proteins