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 β-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.

Construction of a Ball‐and‐Spike Protein Supramolecule

“…From the CD spectra of gp5C, LisDps, and BSPS (Figure 4), the negative peaks of gp5C (216–218 nm) and LisDps (208 nm and 222 nm) suggest that the gp5C is predominantly in the β‐sheet conformation, whereas LisDps is predominantly α‐helical in conformation (Figure 4 A). These spectra agree with previous data12, 13 and are consistent with the X‐ray crystal structures 5. 8 The CD spectrum of BSPS is similar to that calculated by combining the spectrum of LisDps with that of gp5C (Figure 4 B).…”

Molecular assembly and structure of the bacteriophage T4 tail

“…The main structural component of the hub, (gp27) 3 (gp5) 3 , was determined by Kanamaru et al (2002). Kanamaru et al had found that gp5 undergoes maturational processing in which the peptide bond between Ser351 and Ala352 is cleaved to generate gp5* and (gp5C) 3 (Kanamaru et al 1999).…”

Bionanotube Tetrapod Assembly by In Situ Synthesis of a Gold Nanocluster with (Gp5–His6)3 from Bacteriophage T4

“…The expression and purification of proteins containing His 6 fragments were performed as reported previously 21. 22 The proteins were checked by SDS PAGE and MALDI‐TOF MS (Voyager instrument, PE Biosystems). All reactions were carried out at 4 °C.…”

“…The complexes of (gp27–gp5) 3 , (gp5) 3 , and (gp5C) 3 were overexpressed and purified with the aid of a His 6 fragment introduced onto the C terminus of gp5 as described in previous reports 21. 22 We then studied the self‐assembly of trimeric gp5 coupled with the in situ synthesis of Au nanoclusters (NCs). The three histidine‐rich regions form a triad at the C termini of (gp5–His 6 ) 3 (Figure 1 a).…”