Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis.
A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The alpha A-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E. coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed.
Pushko
P
P
Institute of Molecular Biology, Latvian Academy of Sciences, Riga.
Kozlovskaya
T
T
Sominskaya
I
I
Brede
A
A
Stankevica
E
E
Ose
V
V
Pumpens
P
P
Grens
E
E
D000595:Amino Acid Sequence; D000914:Antibodies, Viral; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure; D036022:Capsid Proteins; D003090:Coliphages / Q000235:genetics* / Q000254:growth & development / Q000276:immunology / Q000648:ultrastructure; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D017433:Protein Structure, Secondary; D012317:RNA Phages / Q000235:genetics* / Q000254:growth & development / Q000276:immunology / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D000914:Antibodies, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C085782:coat protein, Enterobacteria phage fr