THE PHAGE-LAMBDA TERMINASE ENZYME - 1 - RECONSTITUTION OF THE HOLOENZYME FROM THE INDIVIDUAL SUBUNITS ENHANCES THE THERMAL-STABILITY OF THESMALL-SUBUNIT

The terminase enzyme from bacteriophage lambda is a hetero-trimeric co mplex composed of the viral gpA and gpNu1 proteins (gpA(1).gpNu1(2)) a nd is responsible for packaging a single genome within the viral capsi d. Current expression systems for these proteins require thermal induc tion which may be responsible for the formation of insoluble aggregate s observed in E. coil. We report the re-cloning of the terminase subun its into vectors which allow low temperature induction. While this has resulted in increased solubility of the large gpA subunit of the enzy me, the small gpNu1 subunit remains insoluble under all conditions exa mined. This paper describes the solublization of gpNu1 with guanidiniu m hydrochloride and purification of the protein to homogeneity. Recons titution 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 un folding of gpA is irreversible, the gpNu1 subunit refolds into a confo rmation which is essentially identical to the pre-heated protein. More over, while denaturation of gpa-is highly cooperative, the small subun it unfolds over a wide temperature range and with thermodynamic parame ters lower than expected for a small globular protein. Thermally-induc ed denaturation of the enzyme reconstituted from the individual subuni ts is highly cooperative with no evidence of multiple transitions. Our data demonstrate that the terminase subunits directly interact in sol ution, and that this interaction alters the thermal stability of the s maller gpNu1 subunit. The implication of these results with respect to assembly of a catalytically competent enzyme complex are discussed. ( C) 1998 Elsevier Science B.V. All rights reserved.