ROLE OF ENTROPIC INTERACTIONS IN VIRAL CAPSIDS - SINGLE AMINO-ACID SUBSTITUTIONS IN P22-BACTERIOPHAGE COAT PROTEIN RESULTING IN LOSS OF CAPSID STABILITY

Bacteriophage P22 is a double-stranded DNA containing phage, Its morph ogenetic 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., and 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, u nder applied pressure the procapsid shells are cold labile, suggesting they are entropically stabilized. We have analyzed the pressure stabi lity of mutant procapsid shells having either of two single amino acid substitutions in the coat protein (G232D and W48Q) using light-scatte ring and fluorescence emission methods. While the wild-type shells wer e stable under 2.2 kbar of pressure at room temperature (22 degrees C) , the G232D mutant shells showed time-dependent dissociation under the se conditions. Decreasing the temperature to 1 degrees C dramatically accelerated the dissociation of G232D mutant under applied pressure. O n the other hand, the W48Q mutant shells could be dissociated easily b y pressure at room temperature and displayed little dependence on temp erature, suggesting a smaller entropic contribution to the stability o f this mutant. The unpolymerized mutant subunits displayed a pressure stability similar to that of the wild type. These data indicate that t he single-site substitutions markedly affect the stability of the asse mbled shell and yet have little effect on the stability of the coat pr otein subunit itself, suggesting that the substitutions are marking re sidues involved in inter-subunit interactions, either directly or thro ugh local conformational changes. The replacement of a single nonpolar amino acid (Trp48) by a polar residue (Gln) results in loss of the en tropic 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 interactio ns important for assembly at the level of ''protein cavities'' (relate d to volume) and ''internal motion'' (related to entropy).