Solid phase synthesis of the retroviral nucleocapsid protein NCp10 of Moloney murine leukaemia virus and related "zinc-fingers" in free SH forms. Influence of zinc chelation on structural and biochemical properties.
The core of retroviruses contains a highly conserved, low molecular weight, basic protein that binds nucleic acids and is essential for genomic RNA packaging. The 56 amino acid protein, NCp10, of Moloney Murine Leukaemia virus (MoMuLV) has the CysX2 CysX4 HisX4 Cys zinc finger-like motif shared by all retrovirus nucleocapsid proteins. The native protein and five modified peptides containing the zinc binding domain were synthesized by solid phase in order to investigate the structural and biochemical role of Zn2+ chelation in MoMuLV NCp10 activity. The purity of the synthetic molecules was verified by HPLC and their sequences were confirmed by amino acid analysis and sequencing in the case of NCp10. Thiol dosage agreed with the theoretical value of free cysteine for all these molecules. Fluorescence measurements performed on synthetic NCp10 and zinc finger fragments showed that the tryptophan quantum yield was Zn2(+)-dependent, allowing a 1:1 stoichiometry for the complex to be determined. The apparent affinity constant of NCp10 for the metal was estimated to be superior to 10(6) M-1. The synthetic protein, in the presence of Zn2+ ions, possesses all the biological properties of NCp10 isolated from virions. It catalyzes both the MoMuLV RNA dimerization and the annealing of the replication primer tRNA(Pro) onto MoMuLV RNA.
Cornille
F
F
Department of Organic Chemistry, INSERM U266, CNRS UA498, UFR of Pharmaceutical and Biological Sciences, Paris, France.
Mely
Y
Y
Ficheux
D
D
Savignol
I
I
Gerard
D
D
Darlix
J L
JL
Fournie-Zaluski
M C
MC
Roques
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D015683:Gene Products, gag / Q000138:chemical synthesis* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D012313:RNA / Q000235:genetics; D012361:RNA, Transfer, Pro / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers
D015683:Gene Products, gag; D010446:Peptide Fragments; D012361:RNA, Transfer, Pro; D013438:Sulfhydryl Compounds; D014758:Viral Core Proteins; C068662:retroviral nucleocapsid protein NCp10, Moloney murine leukemia virus; D012313:RNA; D015032:Zinc