The phage lambda terminase enzyme: 1. Reconstitution of the holoenzyme from the individual subunits enhances the thermal stability of the small subunit

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The terminase enzyme from bacteriophage lambda is a hetero-trimeric complex composed of the viral gpA and gpNu1 proteins (gpA 1·gpNu1 2) and is responsible for packaging a single genome within the viral capsid. Current expression systems for these proteins require thermal induction which may be responsible for the formation of insoluble aggregates observed in E. coli. We report the re-cloning of the terminase subunits into vectors which allow low temperature induction. While this has resulted in increased solubility of the large gpA subunit of the enzyme, the small gpNu1 subunit remains insoluble under all conditions examined. This paper describes the solublization of gpNu1 with guanidinium hydrochloride and purification of the protein to homogeneity. Reconstitution of the enzyme from the individually purified subunits yields a catalytically-competent complex which exhibits activity identical to wild-type enzyme. Thermal denaturation of the proteins was monitored by circular dichroism (CD) spectroscopy and demonstrates that while unfolding of gpA is irreversible, the gpNu1 subunit refolds into a conformation which is essentially identical to the pre-heated protein. Moreover, while denaturation of gpA is highly cooperative, the small subunit unfolds over a wide temperature range and with thermodynamic parameters lower than expected for a small globular protein. Thermally-induced denaturation of the enzyme reconstituted from the individual subunits is highly cooperative with no evidence of multiple transitions. Our data demonstrate that the terminase subunits directly interact in solution, and that this interaction alters the thermal stability of the smaller gpNu1 subunit. The implication of these results with respect to assembly of a catalytically competent enzyme complex are discussed.