Subunit fusion confers tolerance to peptide insertions in a virus coat protein.
An octapeptide sequence called Flag was inserted into the bacteriophage MS2 coat protein at two different locations and its effects on protein folding and virus assembly were determined. Assays of the translational repressor and capsid assembly functions of the recombinants show that when the peptide is inserted at its N-terminus coat protein folds properly into the form that binds RNA (i.e., the dimer), but is defective for capsid assembly. On the other hand, a recombinant protein which is expected to display the Flag insertion as a surface loop does not fold correctly and, as a consequence, is proteolytically degraded. Genetic fusion of the two subunits of the coat dimer results in a protein considerably more tolerant of these structural perturbations and mostly corrects the defects accompanying Flag peptide insertion. Increased resistance of the single-chain coat protein to urea denaturation indicates that the fused dimer is substantially more stable than wild type. Covalent joining of subunits of oligomers probably represents a general strategy for engineering increased protein stability.
Peabody
D S
DS
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine and, Albuquerque, New Mexico, 87131, USA.
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D002847:Chromatography, Agarose; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D017909:Levivirus / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014508:Urea; D019065:Virus Assembly
D036022:Capsid Proteins; D009842:Oligopeptides; D010455:Peptides; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014508:Urea; C085706:FLAG peptide