The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage

Abstract

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.

FIG. 1

Overexpression and isolation of the tail lysozyme, gp 5. After SDS-PAGE, overexpressed proteins were transferred onto a polyvinylidene difluoride membrane and stained with Coomassie brilliant blue R250 (lanes 1 to 3) or subjected to immunoblotting (lane 4 and 5). Lanes: 1, molecular mass standard; 2 and 4, overexpressed proteins after Ni-NTA column elution; 3 and 5, tail proteins. Arrow, gp 5* (mature gp 5). The proportional staining density of each band reflects the efficiency of transfer to PVDF membrane and does not necessarily represent the stoichemistry.

FIG. 2

Analysis of the C-terminal fragment of gp 5. After electrophoresis, proteins were transferred onto a polyvinylidene difluoride membrane and stained with Coomassie brilliant blue R250 (lanes 5 and 6) or subjected to immunoblotting (lanes 1 to 4, 7, and 8). Anti-gp 18 antiserum was used in lanes 1 and 2, anti C-terminal peptide (QYTIDGSRIDIG) antiserum was used in lanes 3, 4, 7, and 8. Lanes: 1, 3, 5, and 8, tail proteins; 2 and 4, 5am phage particle; 6, molecular mass standard; 7, pre-gp5-his complemented 5am phage.

FIG. 3

Turbidity assay of lysozyme activity. The lysozyme activity of gp 5* (□), gp 5* mixed with isolated C-term-his (○), hen egg lysozyme standard (▵), and nicked pre-gp5-his (◊) are shown. The activities of 1 μg of protein for the lysozyme proteins were 0.13, 0.12, 0.11, and 0.014, respectively. The assay was carried out as described in Materials and Methods. OD₄₅₀, optical density at 450 nm.

FIG. 4

Far-UV CD spectrum of gp 5* (---) and C-term-his (—). The secondary structures of gp 5* and C-term-his, as estimated by CONTIN, were 27% α-helix, 31% β-sheet, and 24% β-turn and 11% α-helix, 58% β-sheet, and 0% β-turn, respectively.

FIG. 5

Sedimentation equilibrium of pre-gp5-his (A) and gp 5* (B). Molecular weights of 194,400 ± 10,410 and 40,700 ± 4,500 for pre-gp5-his and gp 5*, respectively, were obtained, which indicated that the former is a trimer, and the latter is a monomer. Residuals are the difference between the experimental data and the fitted curve and have to be evenly scattered throughout the cell.