The scaffolding protein of bacteriophage P22 directs the assembly of an icosahedral procapsid, a metastable shell that is the precursor for DNA packaging. The full-length protein has been shown previously to exist in a monomer-dimer-tetramer equilibrium of elongated and predominantly α-helical molecules. Two deletion-mutant fragments of the scaffolding protein, comprising amino acid residues 141 to 303 and 141 to 292, respectively, have been constructed, overexpressed in Escherichia coli, and purified. Removal of residues 1 to 140 yields a protein that 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 scaffolding activity. Sedimentation analysis reveals that both scaffolding fragments exist in a monomer-dimer equilibrium governed by apparent dissociation constants K(d(141-303)) = 640 μM and K(d(141-292)) = 880 μM. Tetramer formation is not observed for either fragment; thus, the tetramerization domain of the scaffolding subunit resides in the N-terminal portion of the polypeptide chain. Examination of both fragments by circular dichroism, Raman and NMR spectroscopies indicates a highly α-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 elimination of residues 293 to 303 results in unfolding of an α-helical coat protein 'recognition' domain encompassing about 20 to 30 residues. The thermostability of fragment 141-303, monitored over a wide concentration range by circular dichroism and Raman spectroscopy, indicates a broad denaturation transition for the monomeric (low concentration) form, while more cooperative unfolding is observed for the dimeric (high concentration) form. A lesser increase in cooperativity upon dimerization is obtained for fragment 141-292. Additionally, the C-terminal recognition domain constitutes the most stable and cooperative unit in the 141-303 fragment. Measurement of hydrogen-isotope exchange kinetics in scaffolding fragments by time-resolved Raman spectroscopy shows that the C terminus is the only protected segment of the polypeptide chain. On the basis of the measured hydrodynamic and spectroscopic properties, a domain structure is proposed for the scaffolding subunit. The roles of these domains in P22 procapsid assembly are discussed.