title: Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.
abstract: 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 A, which perform a chaperone function during virus assembly. The short C-terminal region has a 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 (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, 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 Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal 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.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Sergei P
authors.initials: SP
authors.lastname: Boudko
authors.forename: Yuri Y
authors.initials: YY
authors.lastname: Londer
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Letarov
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Sernova
authors.forename: Juergen
authors.initials: J
authors.lastname: Engel
authors.forename: Vadim V
authors.initials: VV
authors.lastname: Mesyanzhinov
mesh_terms: D017122:Bacteriophage T4 / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006358:Hot Temperature; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D014764:Viral Proteins; C057839:fibritin protein, Enterobacteria phage T4