Purification and characterization of the assembly factor P17 of the lipid-containing bacteriophage PRD1.
Assembly factors, proteins assisting the formation of viral structures, have been found in many viral systems. The gene encoding the assembly factor P17 of bacteriophage PRD1 has been cloned and expressed in Escherichia coli. P17 acts late in phage assembly, after capsid protein folding and multimerization, and sorting of membrane proteins has occurred. P17 has been purified to near homogeneity. It is a tetrameric protein displaying a rather high heat stability. The protein is largely in an alpha-helical conformation and possesses a putative leucine zipper which is not essential for protein function, as judged by in vitro mutagenesis and complementation analysis. Although heating does not cause structural changes in the conformation of the protein, the dissociation of the tetramer into smaller units is evident as diminished self-quenching of the fluorescently labeled P17. Similarly, dissociation of the tetramer is also obtained by dialysis of the protein against 6-M guanidine hydrochloride (GdnHCl) or 1% SDS. The reassembly of these smaller units upon cooling is evident from resonance energy transfer.
Caldentey
J
J
Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland. javier.caldentey@helsinki.fi
Hänninen
A L
AL
Holopainen
J M
JM
Bamford
J K
JK
Kinnunen
P K
PK
Bamford
D H
DH
D001435:Bacteriophages / Q000737:chemistry / Q000502:physiology*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005454:Fluorescence Polarization; D006146:Guanidines; D016350:Leucine Zippers; D008055:Lipids / Q000032:analysis; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000302:isolation & purification*; D019065:Virus Assembly
D004279:DNA, Viral; D003902:Detergents; D006146:Guanidines; D008055:Lipids; D014759:Viral Envelope Proteins; C118469:assembly factor P17; D012967:Sodium Dodecyl Sulfate