The formation of closed icosahedral capsids from a single species of coat protein subunit requires that the subunits assume different conformations at different lattice positions. In the double-stranded DNA bacteriophage P22, formation of correctly dimensioned capsids is mediated by interaction between coat protein subunits and scaffolding protein. Raman spectroscopy has been employed to compare the conformations of coat protein subunits which have been polymerized to form capsids in the presence and absence of the scaffolding protein. Coat protein subunits polymerized into closed procapsid shells (PS) in the presence of scaffolding protein display a Raman spectrum characterized by a broad amide I band centered at 1665 cm−1 with a discernible shoulder near 1653 cm−1, and a broad amide III profile centered at 1238 cm−1 but asymmetrically skewed to higher frequency. These spectral features indicate that the protein conformation in procapsid shells is rich in β-sheet secondary structure but contains also a significant distribution of α-helix. When biologically active, purified subunits assemble in the absence of scaffolding protein, they form polydisperse muitimers lacking the proper dimensions of procapsid closed shells. We designate these multimers as “associated subunits” (AS). The Raman spectrum of associated subunits indicates a narrower distribution of secondary structure. The associated subunits are characterized by a sharper and more intense Raman amide I band at 1666 cm−1, with no prominent amide I shoulder of lower frequency. An analogous narrowing of the Raman amide III profile is also observed for AS particles, with an accompanying shift of the amide III band center to 1235 cm−1. These findings indicate a clear-cut conversion of α-helical secondary structure in subunits of PS particles to the β-sheet conformation in subunits of AS particles. Band intensity analysis shows that this conformation switch involves 4.5% (±0.4%) of peptide residue conformational states. Specifically, Raman difference spectroscopy applied in combination with signal averaging to probe small changes in protein secondary structure reveals that the converted α-helical domain of PS constitutes 2.3% (±0.2%) of the polypeptide chain, or 10 (±1) of 430 residues in the gp5 subunit. These residues adopt the β-sheet structure in AS particles. The PS → AS difference is accompanied by pronounced changes in Raman bands associated with specific amino acid side chains. In particular, the hydrogen bonding states of tyrosine and tryptophan residues are altered significantly by the PS → AS transition, as are the conformations of aliphatic side chains. The changes observed in the Raman bands of specific amino acid residues are generally indicative of a greater variety of side chain environments for the AS state, including more solvent exposure of tyrosine and tryptophan residues. The data are thus compatible with a more β-rich secondary structure and less compact tertiary structure for the associated subunits. © 1990, American Chemical Society. All rights reserved.