Polymerization of bacteriophage T4 tail sheath protein mutants truncated at the C-termini.
Gene 18 of bacteriophage T4 encodes the contractile protein of the tail sheath. Previous work has shown that the full-length recombinant gene product (gp) 18 of 658 amino acid residues assembles in Escherichia coli cells into a long polysheath structure. However, the gp18 mutants truncated at the N-termini form insoluble aggregates similar to inclusion bodies. In this study, six plasmid vectors expressing the recombinant gp18 proteins truncated at the C-termini have been constructed. The CDelta58, CDelta129, CDelta152, C[g1]72, CDelta248, and CDelta287 proteins contain 600, 529, 506, 486, 410, and 371 residues of the full-length gp18 molecule, respectively. All the recombinant proteins were soluble and, except for the CDelta287 mutant, were assembled into polysheath-related structures. Electron microscopy of negatively stained purified proteins was performed and the resulting images were analyzed by computing their Fourier transforms. The CDelta58 and CDelta129 mutants, in addition to forming common contracted-type polysheath structures, assembled into thinner filaments that we called "noncontracted polysheaths" (NCP). The CDelta152, CDelta172, and CDelta248 proteins assembled into the NCP type only. Image processing showed that the NCP filaments significantly differ from both extended sheaths of T4 particle and polysheaths. The structure of the NCP filaments might correspond to the transitional helices postulated by Moody (J. Mol. Biol., 1973, 80, 613-636) that appeared during the process of tail contraction. Our results suggest that a short region at the C-terminus of the CDelta129 protein determines the contractile properties of the gp18 molecule. The shortest, the CDelta287 protein, does not assemble into regular structures, thus indicating that a sequence's stretch at the C-end of the CDelta248 mutant might be responsible for polymerization of gp18.
Poglazov
B F
BF
Bach Institute of Biochemistry, 33 Leninskii Prospect, Moscow, 117071, Russia.
Efimov
A V
AV
Marco
S
S
Carrascosa
J
J
Kuznetsova
T A
TA
Aijrich
L G
LG
Kurochkina
L P
LP
Mesyanzhinov
V V
VV
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D017384:Sequence Deletion; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
D017931:DNA Primers; D011994:Recombinant Proteins; D017376:Viral Tail Proteins; C402835:glycoprotein 18, bacteriophage T4