Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein

Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 Angstrom. which perform a chaperone function during virus assembly. The short C-terminal region has beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements, The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (DeltaH = 1390 kJ.mol trimer(-1)), at 336 K (DeltaH = 7600 kJ.mol trimer(-1)), and at 345 K (DeltaH = 515 kJ.mol trimer(-1)). These transitions were assigned to the N-terniinal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy DeltaH(res) = 21 kJ.mol residue(-1). The ratio of DeltaH(VH)/DeltaH(cal) for the coiled-coil part of the 120, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13. respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.