STRUCTURE, INTERACTIONS AND DYNAMICS OF PRD1 VIRUS .1. COUPLING OF SUBUNIT FOLDING AND CAPSID ASSEMBLY

Bacteriophage PRD1, which infects Escherichia coli and Salmonella typh imurium, consists of an icosahedral capsid enclosing a membrane-packag ed double-stranded DNA genome. The viral shell has been investigated u sing time and temperature resolved Raman and ultraviolet-resonance Ram an spectroscopy to reveal novel features of the capsid structure and i ts pathway of assembly from P3 subunits. Raman spectra show that the s hell is thermostable to 50 degrees C, and disassembles between 50 and 70 degrees C with only a small change in P3 conformation. However, the products of thermal disassembly depend sensitively upon total protein concentration. Characterization by analytical ultracentrifugation ind icates that below 8 mg/ml, the purified shell dissassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed. Guanidine hydrochloride (GuHCl) dissociation of the P3 shell y ields similar results. Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degr ees C. Thus, shell disassembly diminishes P3 thermostability. Both the lower temperature transition (30 degrees C to 50 degrees C) of the tr imer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximate to 5% of the P3 p eptide backbone from alpha-helix to beta-strand. Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell. tha n in the trimer, consistent with the observed non-specific polymerizat ion of trimers at high concentration. Conversely, the exchange of indo le 1NH groups shows that approximate to 65% of tryptophan residues are protected against exchange in the assembled shell. The results sugges t a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chain s from solvent access. We propose a capsid assembly model which couple s P3 shell formation with the final step in folding of the P3 subunit. (C) 1996 Academic Press Limited