A HELICAL COAT PROTEIN RECOGNITION DOMAIN OF THE BACTERIOPHAGE-P22 SCAFFOLDING PROTEIN

The scaffolding protein of bacteriophage P22 directs the assembly of a n icosahedral procapsid, a metastable shell that is the precursor for DNA packaging. The full-length protein has been shown previously to ex ist in a monomer-dimer-tetramer equilibrium of elongated and predomina ntly alpha-helical molecules. Two deletion-mutant fragments of the sca ffolding protein, comprising amino acid residues 141 to 303 and 141 to 292, respectively, have been constructed, overexpressed in Escherichi a coli, and purified. Removal of residues 1 to 140 yields a protein th at is assembly-active both in vitro and in vivo, while the removal of the C-terminal 11 residues (293 to 303) leads to complete loss of scaf folding activity. Sedimentation analysis reveals that both scaffolding fragments exist in a monomer-dimer equilibrium governed by apparent d issociation constants Kd(141-303) = 640 mu M and Kd(141-292) = 880 mu M. Tetramer formation is not observed for either fragment; thus, the t etramerization domain of the scaffolding subunit resides in the N-term inal portion of the poly-peptide chain. Examination of both fragments by circular dichroism, Raman and NMR spectroscopies indicates a highly alpha-helical fold in each case. Nonetheless, pronounced differences are observed between spectral signatures of the two fragments. Notably , Raman spectra of fragments 141-292 and 141-303 indicate that elimina tion of residues 293 to 303 results in unfolding of an a-helical coat protein ''recognition'' domain encompassing about 20 to 30 residues. T he thermostability of fragment 141-303, monitored over a wide concentr ation range by circular dichroism and Raman spectroscopy, indicates a broad denaturation transition for the monomeric (low concentration) fo rm, while more cooperative unfolding is observed for the dimeric (high concentration) form. A lesser increase in cooperativity upon dimeriza tion is obtained for fragment 141-292. Additionally, the C-terminal re cognition domain constitutes the most stable and cooperative unit in t he 141-303 fragment. Measurement of hydrogen-isotope exchange kinetics in scaffolding fragments by time-resolved Raman spectroscopy shows th at the C terminus is the only protected segment of the polypeptide cha in. On the basis of the measured hydrodynamic and spectroscopic proper ties, a domain structure is proposed for the scaffolding subunit. The roles of these domains in P22 procapsid assembly are discussed. (C) 19 98 Academic Press