Bacteriophage P22 is a double-stranded DNA containing phage. Its morphogenetic pathway requires the formation of a precursor procapsid that subsequently matures to the capsid. The stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure has been examined previously [Prevelige, P. E., King, J., & Silva, J. L. (1994) Biophys. J. 66, 1631 - 1641], The monomeric protein is very unstable to pressure and undergoes denaturation at pressures below 1.5 kbar, whereas the procapsid shell is very stable to applied pressure and does not dissociate with pressure to 2.5 kbar. However, under applied pressure the procapsid shells are cold labile, suggesting they are entropically stabilized. We have analyzed the pressure stability of mutant procapsid shells having either of two single amino acid substitutions in the coat protein (G232D and W48Q) using light-scattering and fluorescence emission methods. While the wild-type shells were stable under 2.2 kbar of pressure at room temperature (22 °C), the G232D mutant shells showed time-dependent dissociation under these conditions. Decreasing the temperature to 1 °C dramatically accelerated the dissociation of G232D mutant under applied pressure. On the other hand, the W48Q mutant shells could be dissociated easily by pressure at room temperature and displayed little dependence on temperature, suggesting a smaller entropic contribution to the stability of this mutant. The unpolymerized mutant subunits displayed a pressure stability similar to that of the wild type. These data indicate that the single-site substitutions markedly affect the stability of the assembled shell and yet have little effect on the stability of the coat protein subunit itself, suggesting that the substitutions are marking residues involved in inter-subunit interactions, either directly or through local conformational changes. The replacement of a single nonpolar amino acid (Trp48) by a polar residue (Gin) results in loss of the entropic stabilization, suggesting the importance of burial of Trp48 in a nonpolar core to stabilize entropically the icosahedral shells. Our results with the single-mutation shells dissect the protein interactions important for assembly at the level of “protein cavities” (related to volume) and “internal motion” (related to entropy). © 1995, American Chemical Society. All rights reserved.